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Sample records for mouse spermatogonia involving

  1. ETV5 regulates sertoli cell chemokines involved in mouse stem/progenitor spermatogonia maintenance.

    PubMed

    Simon, Liz; Ekman, Gail C; Garcia, Thomas; Carnes, Kay; Zhang, Zhen; Murphy, Theresa; Murphy, Kenneth M; Hess, Rex A; Cooke, Paul S; Hofmann, Marie-Claude

    2010-10-01

    Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to offspring. Although growth factors responsible for self-renewal of these cells are known, the factors and mechanisms that attract and physically maintain these cells within their microenvironment are poorly understood. Mice with targeted disruption of Ets variant gene 5 (Etv5) show total loss of stem/progenitor spermatogonia following the first wave of spermatogenesis, resulting in a Sertoli cell-only phenotype and aspermia. Microarray analysis of primary Sertoli cells from Etv5 knockout (Etv5(-/-)) versus wild-type (WT) mice revealed significant decreases in expression of several chemokines. Chemotaxis assays demonstrated that migration of stem/progenitor spermatogonia toward Etv5(-/-) Sertoli cells was significantly decreased compared to migration toward WT Sertoli cells. Interestingly, differentiating spermatogonia, spermatocytes, and round spermatids were not chemoattracted by WT Sertoli cells, whereas stem/progenitor spermatogonia showed a high and significant chemotactic index. Rescue assays using recombinant chemokines indicated that C-C-motif ligand 9 (CCL9) facilitates Sertoli cell chemoattraction of stem/progenitor spermatogonia, which express C-C-receptor type 1 (CCR1). In addition, there is protein-DNA interaction between ETV5 and Ccl9, suggesting that ETV5 might be a direct regulator of Ccl9 expression. Taken together, our data show for the first time that Sertoli cells are chemoattractive for stem/progenitor spermatogonia, and that production of specific chemokines is regulated by ETV5. Therefore, changes in chemokine production and consequent decreases in chemoattraction by Etv5(-/-) Sertoli cells helps to explain stem/progenitor spermatogonia loss in Etv5(-/-) mice.

  2. ETV5 Regulates Sertoli Cell Chemokines Involved in Mouse Stem/Progenitor Spermatogonia Maintenance

    PubMed Central

    Simon, Liz; Ekman, Gail C; Garcia, Thomas; Carnes, Kay; Zhang, Zhen; Murphy, Theresa; Murphy, Kenneth M; Hess, Rex A; Cooke, Paul S; Hofmann, Marie–Claude

    2010-01-01

    Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to offspring. Although growth factors responsible for self–renewal of these cells are known, the factors and mechanisms that attract and physically maintain these cells within their microenvironment are poorly understood. Mice with targeted disruption of Ets variant gene 5 (Etv5) show total loss of stem/progenitor spermatogonia following the first wave of spermatogenesis, resulting in a Sertoli cell–only phenotype and aspermia. Microarray analysis of primary Sertoli cells from Etv5 knockout (Etv5−/−) versus wild–type (WT) mice revealed significant decreases in expression of several chemokines. Chemotaxis assays demonstrated that migration of stem/progenitor spermatogonia toward Etv5−/− Sertoli cells was significantly decreased compared to migration toward WT Sertoli cells. Interestingly, differentiating spermatogonia, spermatocytes, and round spermatids were not chemoattracted by WT Sertoli cells, whereas stem/progenitor spermatogonia showed a high and significant chemotactic index. Rescue assays using recombinant chemokines indicated that C-C-motif ligand 9 (CCL9) facilitates Sertoli cell chemoattraction of stem/progenitor spermatogonia, which express C-C-receptor type 1 (CCR1). In addition, there is protein–DNA interaction between ETV5 and Ccl9, suggesting that ETV5 might be a direct regulator of Ccl9 expression. Taken together, our data show for the first time that Sertoli cells are chemoattractive for stem/progenitor spermatogonia, and that production of specific chemokines is regulated by ETV5. Therefore, changes in chemokine production and consequent decreases in chemoattraction by Etv5−/− Sertoli cells helps to explain stem/progenitor spermatogonia loss in Etv5−/− mice. PMID:20799334

  3. Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    PubMed Central

    Mundt, Nadine; Bruentgens, Felicitas; Geilenkirchen, Petra; Machado, Patricia A.; Veitinger, Thomas; Veitinger, Sophie; Lipartowski, Susanne M.; Engelhardt, Corinna H.; Oldiges, Marco; Spehr, Jennifer

    2016-01-01

    Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP—a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts—activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca2+-activated large-conductance K+ channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis. PMID:27574293

  4. Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia.

    PubMed

    Schrans-Stassen, B H; van de Kant, H J; de Rooij, D G; van Pelt, A M

    1999-12-01

    The proto-oncogene c-kit is encoded at the white-spotting locus and in the mouse mutations at this locus affect the precursor cells of melanocytes, hematopoietic cells, and germ cells. c-kit is expressed in type A spermatogonia, but whether or not c-kit is present both in undifferentiated and differentiating type A spermatogonia or only in the latter cell type is still a matter of debate. Using the vitamin A-deficient mouse model, we studied messenger RNA (mRNA) and protein expression in undifferentiated and differentiating type A spermatogonia. Furthermore, we quantified the immuno-positive type A spermatogonia in the epithelial stages VI, VII, IX/X, and XII in normal mice to correlate c-kit expression in type A spermatogonia with the differentiation of these cells. Our results show that in the VAD situation undifferentiated type A spermatogonia express little c-kit mRNA. The A spermatogonia with a larger nucleus expressed c-Kit protein, whereas the A spermatogonia with a smaller one did not. After induction of differentiation of these cells into type A1 spermatogonia, c-kit mRNA was enhanced. The percentage of A spermatogonia expressing c-Kit protein did not change during this process, suggesting that A spermatogonia, which are committed to differentiate express c-kit. Under normal circumstances in epithelial stage VI 16%+/-2% (mean +/- SD), in VII 45%+/-15%, in IX/X 78%+/-14% and in XII 90%+/-1.9% of the type A spermatogonia were c-kit positive, suggesting that Aaligned spermatogonia gradually change from c-Kit negative to c-Kit positive cells before their differentiation into A1 spermatogonia. It is concluded that c-kit can be used as a marker for differentiation of undifferentiated into differentiating type A spermatogonia.

  5. GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

    PubMed Central

    Wang, Yong; Song, Wei; Li, Shuchun; Guan, Xin; Miao, Shiying; Zong, Shudong; Koide, SS; Wang, Linfang

    2009-01-01

    Background Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules. Results Stable mRHBDD1 knockdown GC-1 cells were sensitive to apoptotic stimuli, PS341 and UV irradiation. In vitro, they survived and proliferated normally. However, they lost the ability to survive and differentiate in mouse seminiferous tubules. Conclusion Our findings suggest that mRHBDD1 may be associated with mammalian spermatogenesis. PMID:19358743

  6. BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

    PubMed Central

    Liu, Wenbo; Wang, Fengchao; Xu, Qianhua; Shi, Junchao; Zhang, Xiaoxin; Lu, Xukun; Zhao, Zhen-Ao; Gao, Zheng; Ma, Huaixiao; Duan, Enkui; Gao, Fei; Gao, Shaorong; Yi, Zhaohong; Li, Lei

    2017-01-01

    Breast cancer amplified sequence 2 (BCAS2) is involved in multiple biological processes, including pre-mRNA splicing. However, the physiological roles of BCAS2 are still largely unclear. Here we report that BCAS2 is specifically enriched in spermatogonia of mouse testes. Conditional disruption of Bcas2 in male germ cells impairs spermatogenesis and leads to male mouse infertility. Although the spermatogonia appear grossly normal, spermatocytes in meiosis prophase I and meiosis events (recombination and synapsis) are rarely observed in the BCAS2-depleted testis. In BCAS2 null testis, 245 genes are altered in alternative splicing forms; at least three spermatogenesis-related genes (Dazl, Ehmt2 and Hmga1) can be verified. In addition, disruption of Bcas2 results in a significant decrease of the full-length form and an increase of the short form (lacking exon 8) of DAZL protein. Altogether, our results suggest that BCAS2 regulates alternative splicing in spermatogonia and the transition to meiosis initiation, and male fertility. PMID:28128212

  7. Data on in vivo phenotypes of GFRα1-positive spermatogonia stimulated by interstitial GDNF signals in mouse testes.

    PubMed

    Uchida, Aya; Kanai, Yoshiakira

    2016-09-01

    This article contains the data related to the research article "in vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay" (Uchida et al., 2016) [1]. A novel transplantation assay of growth factor-soaked beads into the mammalian testicular interstitium was developed, in order to examine the effects of various soluble factors on in vivo dynamics of the spermatogonia including spermatogonial stem cells (SSC). Here we provide the image data of GFRα1-positive stem/progenitor spermatogonia in mouse seminiferous tubules near the beads soaked in GDNF (glial cell-derived neurotrophic factor), one of the SSC niche factors. The data provide various phenotypes of GFRα1-positive spermatogonia induced by bead-derived GDNF signals, which are useful to understand the active state of GFRα1-positive stem/progenitor spermatogonia in vivo.

  8. Benzo(a)pyrene Is Mutagenic in Mouse Spermatogonial Stem Cells and Dividing Spermatogonia

    PubMed Central

    O’Brien, Jason M.; Beal, Marc A.; Yauk, Carole L.; Marchetti, Francesco

    2016-01-01

    Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number of mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). MutaMouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia, and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, P < .01) and spermatogonial stem cells (2.1-fold at highest dose, P < .01). No significant increases in MF were detected in cauda sperm or seminiferous tubule cells collected 3 days post-exposure. Dose-response modelling suggested that the mutational response in male germ cells to BaP is sub-linear at low doses. Our results demonstrate that oral exposure to BaP causes spermatogonial stem cell mutations, that different phases of spermatogenesis exhibit varying sensitivities to BaP, with dividing spermatogonia representing a window of peak sensitivity, and that sampling spermatogenic cells from the seminiferous tubules at earlier time-points may underestimate germ cell mutagenicity. This information is critical to optimize the use of the international test guideline for transgenic rodent mutation assays for detecting germ cell mutagens. PMID:27208087

  9. The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.

    PubMed

    Yoshida, Shosei; Sukeno, Mamiko; Nakagawa, Toshinori; Ohbo, Kazuyuki; Nagamatsu, Go; Suda, Toshio; Nabeshima, Yo-ichi

    2006-04-01

    Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this study, we have investigated this issue by using the unique expression of Ngn3, which is expressed specifically in the undifferentiated spermatogonia, but not in the differentiating spermatogonia or their progenitors, the gonocytes. Our lineage analyses demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3-expressing stage (Ngn3- lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3-positive undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function (Ngn3+ lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated spermatogonia (Ngn3+ lineage) or differentiating spermatogonia (Ngn3- lineage) is topographically related to the establishment of the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.

  10. Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation.

    PubMed

    Pellegrini, Manuela; Grimaldi, Paola; Rossi, Pellegrino; Geremia, Raffaele; Dolci, Susanna

    2003-08-15

    It is well established that the c-kit gene plays an essential role in the proliferation of differentiating spermatogonia in prepuberal mice. However, the mechanisms that regulate the onset of spermatogenesis, i.e. differentiation of spermatogonial stem cells and c-kit expression, are poorly understood. Here we identify a novel signal transduction system in mouse prepuberal testis regulating this developmental event, involving bone morphogenetic protein 4 (BMP4) and its transduction machinery. BMP4 is produced by Sertoli cells very early in the postnatal life and is successively down regulated in peri-puberal Sertoli cells. Its receptor Alk3 and the R-Smad Smad5 are specifically expressed both in proliferating primordial germ cells and in postnatal spermatogonia. BMP4 stimulation of cultured spermatogonia induces Smad4/5 nuclear translocation and the formation of a DNA-binding complex with the transcriptional coactivator p300/CBP. In vitro exposure of undifferentiated spermatogonia to BMP4 exerts both mitogenic and differentiative effects, inducing [3H]thymidine incorporation and Kit expression. As a result of the latter event, Kit-negative spermatogonia acquire sensitivity to Stem Cell Factor.

  11. Mouse differentiating spermatogonia can generate germinal stem cells in vivo.

    PubMed

    Barroca, Vilma; Lassalle, Bruno; Coureuil, Mathieu; Louis, Jean Paul; Le Page, Florence; Testart, Jacques; Allemand, Isabelle; Riou, Lydia; Fouchet, Pierre

    2009-02-01

    In adults, stem cells are responsible for the maintenance of many actively renewing tissues, such as haematopoietic, skin, gut and germinal tissues. These stem cells can self-renew or be committed to becoming progenitors. Stem-cell commitment is thought to be irreversible but in male and female Drosophila melanogaster, it was shown recently that differentiating germ cells can revert to functional stem cells that can restore germinal lineage. Whether progenitors are also able to generate stem cells in mammals remains unknown. Here we show that purified mouse spermatogonial progenitors committed to differentiation can generate functional germinal stem cells that can repopulate germ-cell-depleted testes when transplanted into adult mice. We found that GDNF, a key regulator of the stem-cell niche, and FGF2 are able to reprogram in vitro spermatogonial progenitors for reverse differentiation. This study supports the emerging concept that the stem-cell identity is not restricted in adults to a definite pool of cells that self-renew, but that stemness could be acquired by differentiating progenitors after tissue injury and throughout life.

  12. Stability of DNA methylation patterns in mouse spermatogonia under conditions of MTHFR deficiency and methionine supplementation.

    PubMed

    Garner, Justine L; Niles, Kirsten M; McGraw, Serge; Yeh, Jonathan R; Cushnie, Duncan W; Hermo, Louis; Nagano, Makoto C; Trasler, Jacquetta M

    2013-11-01

    Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome

  13. BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia.

    PubMed

    Yang, Yongguang; Feng, Yanmin; Feng, Xue; Liao, Shangying; Wang, Xiuxia; Gan, Haiyun; Wang, Lixian; Lin, Xiwen; Han, Chunsheng

    2016-01-01

    Spermatogenesis is sustained by the proliferation and differentiation of spermatogonial stem cells (SSCs). However, the molecules controlling these processes remain largely unknown. Here, we developed a simplified high concentration serum-containing system for the culture of mouse SSCs. Analysis of SSCs markers and transplantation results revealed that the cultured spermatogonia retained stem cell characteristics after long-term in vitro propagation. Using this culture system, the expression and function of bone morphogenetic protein 4 (BMP4) were explored. Immunostaining showed that BMP4 was predominantly expressed in germ cells and that its level increased as spermatogenesis progresses. BMP4 receptors BMPR1A and BMPRII were present in spermatogonia, spermatocytes, and round spermatids. Moreover, despite the mRNAs of these two genes being present in mouse Sertoli cells, only BMPRII was detected by using Western blotting assays. While exogenous BMP4 by itself did not induce the expression of Stra8 and c-Kit, two marker genes of differentiating spermatogonia, a significant cooperative effect of BMP4 and retinoic acid (RA) was observed. Moreover, pretreatment of cultured spermatogonia with the BMP4 antagonist Noggin could inhibit RA-induced expression of these two marker genes. In conclusion, BMP4 may exert autocrine effects and act cooperatively with RA to induce the differentiation of spermatogonia in vivo.

  14. BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

    PubMed Central

    Feng, Yanmin; Feng, Xue; Wang, Xiuxia; Gan, Haiyun; Wang, Lixian; Lin, Xiwen

    2016-01-01

    Spermatogenesis is sustained by the proliferation and differentiation of spermatogonial stem cells (SSCs). However, the molecules controlling these processes remain largely unknown. Here, we developed a simplified high concentration serum-containing system for the culture of mouse SSCs. Analysis of SSCs markers and transplantation results revealed that the cultured spermatogonia retained stem cell characteristics after long-term in vitro propagation. Using this culture system, the expression and function of bone morphogenetic protein 4 (BMP4) were explored. Immunostaining showed that BMP4 was predominantly expressed in germ cells and that its level increased as spermatogenesis progresses. BMP4 receptors BMPR1A and BMPRII were present in spermatogonia, spermatocytes, and round spermatids. Moreover, despite the mRNAs of these two genes being present in mouse Sertoli cells, only BMPRII was detected by using Western blotting assays. While exogenous BMP4 by itself did not induce the expression of Stra8 and c-Kit, two marker genes of differentiating spermatogonia, a significant cooperative effect of BMP4 and retinoic acid (RA) was observed. Moreover, pretreatment of cultured spermatogonia with the BMP4 antagonist Noggin could inhibit RA-induced expression of these two marker genes. In conclusion, BMP4 may exert autocrine effects and act cooperatively with RA to induce the differentiation of spermatogonia in vivo. PMID:27795714

  15. Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis.

    PubMed

    Niedenberger, Bryan A; Busada, Jonathan T; Geyer, Christopher B

    2015-04-01

    Prospermatogonia transition to type A spermatogonia, which provide the source for the spermatogonial stem cell (SSC) pool. A percentage of these type A spermatogonia then differentiate to enter meiosis as spermatocytes by ∼P10. It is currently unclear as to when these distinct populations are initially formed in the neonatal testis, and when the expression of markers both characteristic of and required for the adult undifferentiated and differentiating states is established. In this study, we compared expression of known spermatogonial cell fate markers during normal development and in response to the differentiation signal provided by retinoic acid (RA). We found that some markers for the undifferentiated state (ZBTB16/PLZF and CDH1) were expressed in nearly all spermatogonia from P1 through P7. In contrast, differentiation markers (STRA8 and KIT) appeared in a subset of spermatogonia at P4, coincident with the onset of RA signaling. GFRA1, which was present in nearly all prospermatogonia at P1, was only retained in STRA8/KIT- spermatogonia. From P4 through P10, there was a great deal of heterogeneity in the male germ cell population in terms of expression of markers, as markers characteristic of the undifferentiated (except GFRA1) and differentiating states were co-expressed through this interval. After P10, these fate markers diverged to mark distinct populations of undifferentiated and differentiating spermatogonia, and this pattern was maintained in juvenile (P18) and adult (P>60) testes. Taken together, these results reveal that the spermatogonia population is heterogeneous during the first wave of spermatogenesis, and indicate that neonatal spermatogonia may not serve as an ideal substitute for studying the function of adult spermatogonia.

  16. Retinoic acid induces multiple hallmarks of the prospermatogonia-to-spermatogonia transition in the neonatal mouse.

    PubMed

    Busada, Jonathan T; Kaye, Evelyn P; Renegar, Randall H; Geyer, Christopher B

    2014-03-01

    In mammals, most neonatal male germ cells (prospermatogonia) are quiescent and located in the center of the testis cords. In response to an unknown signal, prospermatogonia transition into spermatogonia, reenter the cell cycle, divide, and move to the periphery of the testis cords. In mice, these events occur by 3-4 days postpartum (dpp), which temporally coincides with the onset of retinoic acid (RA) signaling in the neonatal testis. RA has a pivotal role in initiating germ cell entry into meiosis in both sexes, yet little is known about the mechanisms and about cellular changes downstream of RA signaling. We examined the role of RA in mediating the prospermatogonia-to-spermatogonia transition in vivo and found 24 h of precocious RA exposure-induced germ cell changes mimicking those that occur during the endogenous transition at 3-4 dpp. These changes included: 1) spermatogonia proliferation; 2) maturation of cellular organelles; and 3), expression of markers characteristic of differentiating spermatogonia. We found that germ cell exposure to RA did not lead to cellular loss from apoptosis but rather resulted in a delay of ∼2 days in their entry into meiosis. Taken together, our results indicate that exogenous RA induces multiple hallmarks of the transition of prospermatogonia to spermatogonia prior to their entry into meiosis.

  17. Bleomycin, unlike other male-mouse mutagens, is most effective in spermatogonia, inducing primarily deletions.

    PubMed

    Russell, L B; Hunsicker, P R; Kerley, M K; Johnson, D K; Shelby, M D

    2000-08-21

    Dominant-lethal tests [P.D. Sudman, J.C. Rutledge, J.B. Bishop, W.M. Generoso, Bleomycin: female-specific dominant lethal effects in mice, Mutat. Res. 296 (1992) 205-217] had suggested that Bleomycin sulfate (Blenoxane), BLM, might be a female-specific mutagen. While confirming that BLM is indeed a powerful inducer of dominant-lethal mutations in females that fails to induce such mutations in postspermatogonial stages of males, we have shown in a specific-locus test that BLM is, in fact, mutagenic in males. This mutagenicity, however, is restricted to spermatogonia (stem-cell and differentiating stages), for which the specific-locus mutation rate differed significantly (P<0.008) from the historical control rate. In treated groups, dominant mutations, also, originated only in spermatogonia. With regard to mutation frequencies, this germ-cell-stage pattern is different from that for radiation and for any other chemical studied to date, except ethylnitrosourea (ENU). However, the nature of the spermatogonial specific-locus mutations differentiates BLM from ENU as well, because BLM induced primarily (or, perhaps, exclusively) multilocus deletions. Heretofore, no chemical that induced specific-locus mutations in spermatogonia did not also induce specific-locus as well as dominant-lethal mutations in postspermatogonial stages, making the dominant lethal test, up till now, predictive of male mutagenicity in general. The BLM results now demonstrate that there are chemicals that can induce specific-locus mutations in spermatogonia without testing positive in postspermatogonial stages. Thus, BLM, while not female-specific, is unique, (a) in its germ-cell-stage specificity in males, and (b) in inducing a type of mutation (deletions) that is atypical for the responding germ-cell stages (spermatogonia).

  18. A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis.

    PubMed

    Yoshida, Shosei; Sukeno, Mamiko; Nabeshima, Yo-Ichi

    2007-09-21

    Mammalian spermatogenesis produces numerous sperm for a long period based on a highly potent stem cell system, which relies on a special microenvironment, or niche, that has not yet been identified. In this study, using time-lapse imaging of green fluorescent protein-labeled undifferentiated spermatogonia (A(undiff)) and three-dimensional reconstitution, we revealed a biased localization of A(undiff) to the vascular network and accompanying Leydig and other interstitial cells, in intact testes. Differentiating spermatogonia left these niche regions and dispersed throughout the basal compartment of the seminiferous epithelium. Moreover, rearrangement of A(undiff) accompanied the vasculature alteration. We propose that the mammalian germline niche is established as a consequence of vasculature pattern formation. This is different from what is observed in Drosophila or Caenorhabditis elegans, which display developmentally specified niche structures within polarized gonads.

  19. GDNF-expressing STO feeder layer supports the long-term propagation of undifferentiated mouse spermatogonia with stem cell properties

    PubMed Central

    Wei, Xiang; Jia, Yuanyuan; Xue, Yuanyuan; Geng, Lei; Wang, Min; Li, Lufan; Wang, Mei; Zhang, Xuemei; Wu, Xin

    2016-01-01

    The development of a stem cell culture system would expedite our understanding of the biology of tissue regeneration. Spermatogonial stem cell (SSC) is the foundation for lifelong male spermatogenesis and the SSC culture has been optimized continuously in recent years. However, there have been many inconveniences to reconstruct SSC self-renewal and proliferation in vitro, such as the frequent refreshment of recombinant cytokines, including GDNF, the essential growth factor for SSC maintenance. In the present study, we observed that both STO and MEF cells, which were previously used as feeders for SSC growth, did not express GDNF, but a GDNF-expressing STO feeder could support undifferentiated mouse spermatogonia propagation in vitro for three months without the refreshment of recombinant growth factor GDNF. The cell morphology, growth rate and SSC-associated gene expression remained identical to the SSCs cultured using previous methods. The transplantation of SSCs growing on these GDNF-expressing STO feeders could generate extensive colonies of spermatogenesis in recipient testes, functionally validating the stemness of these cells. Collectively, our data indicated that the further modification of feeder cells might facilitate the self-renewal and propagation of SSCs in vitro. PMID:27827452

  20. Estimation of mutation induction rates in AT-rich sequences using a genome scanning approach after X irradiation of mouse spermatogonia.

    PubMed

    Asakawa, Jun-ichi; Nakamura, Nori; Katayama, Hiroaki; Cullings, Harry M

    2007-08-01

    We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 x 10(-5) per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (+/- 1.9) x 10(-5) and 2.3 (+/- 1.0) x 10(-5) per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (+/- 1.2) x 10(-5) per locus per Gy. The induction rate would be reduced to 0.9 x 10(-5) per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 x 10(-5) per locus per Gy or less, which is close to 1.08 x 10(-5) per locus per Gy, the current estimate for the mean mutation induction rate in mice.

  1. Effect of X-ray and ethylnitrosourea exposures separated by 24 h on specific-locus mutation frequency in mouse stem-cell spermatogonia.

    PubMed

    Russell, W L; Carpenter, D A; Hitotsumachi, S

    1988-04-01

    Specific-locus mutation frequencies in mouse stem-cell spermatogonia were determined in 3 experiments in which mature male mice were exposed to 100,m 300, or 500 R of X-rays followed, 24 h later, by intraperitoneal injection of 100 mg/kg of ethylnitrosourea (ENU). The purpose was to find out if the mutation frequencies would be augmented over those expected on the basis of additivity of the effects of the separate treatments. Such augmentation had been observed in earlier work in which exposure to 100 or 500 R of X-rays was followed 24 h later by a second exposure of 500 R. No augmentation was observed for X-rays followed by ENU. The mutation frequencies in all 3 experiments actually fell below those expected on the basis of additivity, although the reductions were not statistically significant.

  2. The effect of dose rate on the frequency of specific-locus mutations induced in mouse spermatogonia is restricted to larger lesions; a retrospective analysis of historical data.

    PubMed

    Russell, Liane B; Hunsicker, Patricia R

    2012-05-01

    A series of 19 large-scale germ-cell mutagenesis experiments conducted several decades ago led to the conclusion that low-LET radiation delivered to mouse spermatogonia at dose rates of 0.8 R/min and below induced only about one-third as many specific-locus mutations as did single, acute exposures at 24 R/min and above. A two-hit origin of the mutations was deemed unlikely in view of the then prevailing evidence for the small size of genetic lesions in spermatogonia. Instead, the dose-rate effect was hypothesized to be the result of a repair system that exists in spermatogonia, but not in more mature male reproductive cells. More recent genetic and molecular studies on the marker genes have identified the phenotypes associated with specific states of the mutant chromosomes, and it is now possible retrospectively to classify individual past mutations as "large lesions" or "other lesions". The mutation-frequency difference between high and low dose rates is restricted to the large lesion mutations, for which the dose-curve slopes differ by a factor exceeding 3.4. For other lesion mutations, there is essentially no difference between the slopes for protracted and acute irradiations; induced other lesions frequencies per unit dose remain similar for dose rates ranging over more than 7 orders of magnitude. For large lesions, these values rise sharply at dose rates >0.8 R/min, though they remain similar within the whole range of protracted doses, failing to provide evidence for a threshold dose rate. The downward bend at high doses that had been noted for X-ray-induced specific-locus mutations as a whole and ascribed to a positive correlation between spermatogonial death and mutation load is now found to be restricted to large lesion mutations. There is a marked difference between the mutation spectra (distributions among the seven loci) for large lesions and other lesions. Within each class, however, the spectra are similar for acute and protracted irradiation.

  3. The Effect of Dose Rate on the Frequency of Specific-Locus Mutations Induced in Mouse Spermatogonia is Restricted to Larger Lesions; a Retrospective Analysis of Historical Data

    SciTech Connect

    Russell, Liane B; Hunsicker, Patricia R

    2012-01-01

    A series of 19 large-scale germ-cell mutagenesis experiments conducted several decades ago led to the conclusion that low-LET radiation delivered to mouse spermatogonia at dose rates of 0.8 R/min and below induced only about one-third as many specific-locus mutations as did single, acute exposures at 24 R/min and above. A two-hit origin of the mutations was deemed unlikely in view of the then prevailing evidence for the small size of genetic lesions in spermatogonia. Instead, the dose-rate effect was hypothesized to be the result of a repair system that exists in spermatogonia, but not in more mature male reproductive cells. More recent genetic and molecular studies on the marker genes have identified the phenotypes associated with specific states of the mutant chromosomes, and it is now possible retrospectively to classify individual past mutations as "large lesions" or "other lesions". The mutation-frequency difference between high and low dose rates is restricted to the large lesion mutations, for which the dose-curve slopes differ by a factor exceeding 3.4. For other lesion mutations, there is essentially no difference between the slopes for protracted and acute irradiations; induced other lesions frequencies per unit dose remain similar for dose rates ranging over more than 7 orders of magnitude. For large lesions, these values rise sharply at dose rates >0.8 R/min, though they remain similar within the whole range of protracted doses, failing to provide evidence for a threshold dose rate. The downward bend at high doses that had been noted for X-ray-induced specific-locus mutations as a whole and ascribed to a positive correlation between spermatogonial death and mutation load is now found to be restricted to large lesion mutations. There is a marked difference between the mutation spectra (distributions among the seven loci) for large lesions and other lesions. Within each class, however, the spectra are similar for acute and protracted irradiation.

  4. Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro.

    PubMed

    Zhou, Qing; Li, Ying; Nie, Rong; Friel, Patrick; Mitchell, Debra; Evanoff, Ryan M; Pouchnik, Derek; Banasik, Brent; McCarrey, John R; Small, Christopher; Griswold, Michael D

    2008-03-01

    Vitamin A deficiency in the mouse results in an arrest in the progression of undifferentiated spermatogonia to differentiating spermatogonia. The supplement of retinol to vitamin-A-deficient mice reinitiates spermatogenesis in a synchronous manner throughout the testes. It is unclear whether the effects of retinoids are the result of a direct action on germ cells or are indirectly mediated through Sertoli cells. The expression of Stimulated by retinoic acid gene 8 (Stra8), which is required for spermatogenesis, is directly related to the availability of retinoic acid (RA). Analysis of gene expression by microarrays revealed moderate levels of Stra8 transcript in gonocytes and high levels in A and B spermatogonia. Stra8 mRNA levels were greatly reduced or absent in germ cells once they entered meiosis. This study examined the effect of retinoic acid on cultured neonatal testes and isolated gonocytes/spermatogonia in vitro. THY1(+) and KIT(+) germ cells were isolated by magnetic-activated cell sorting from the testes of mice of different ages. Isolated germ cells were cultured and treated with either vehicle (ethanol) or RA without feeder cells. We found that 1) Stra8 is predominantly expressed in premeiotic germ cells, 2) RA stimulates gonocyte DNA replication and differentiation in cultured neonatal testes, 3) in the absence of feeder cells, RA directly induces the transition of undifferentiated spermatogonia to differentiating spermatogonia by stimulating Stra8 and Kit gene expression, 4) RA dramatically stimulates Stra8 expression in undifferentiated spermatogonia but has a lesser impact in differentiating spermatogonia, 5) endogenous Stra8 gene expression is higher in differentiating spermatogonia than in undifferentiated spermatogonia and could mediate the RA effects on spermatogonial maturation, and 6) RA stimulates a group of genes involved in the metabolism, storage, transport, and signaling of retinoids.

  5. The effect of the interval between dose applications on the observed specific-locus mutation rate in the mouse following fractionated treatments of spermatogonia with ethylnitrosourea.

    PubMed

    Favor, J; Neuhäuser-Klaus, A; Ehling, U H; Wulff, A; van Zeeland, A A

    1997-03-21

    Our earlier analyses have suggested an apparent threshold dose-response for ethylnitrosourea-induced specific-locus mutations in treated spermatogonia of the mouse to be due to a saturable repair process. In the current study a series of fractionated-treatment experiments was carried out in which male (102 x C3H)F1 mice were exposed to 4 x 10, 2 x 40. 4 x 20 or 4 x 40 mg ethylnitrosourea per kg body weight with 24 h between applications; 4 x 40 mg ethylnitrosourea per kg body weight with 72 h between dose applications; and 2 x 40, 4 x 20 and 4 x 40 mg ethylnitrosourea per kg body weight with 168 h between dose applications. For all experiments with 24-h intervals between dose applications, there was no effect due to dose fractionation on the observed mutation rates, indicating the time interval between dose applications to be shorter than the recovery time of the repair processes acting on ethylnitrosourea-induced DNA adducts. In contrast, a fractionation interval of 168 h was associated with a significant reduction in the observed mutation rate due to recovery of the repair process. However, although reduced, the observed mutation rates for fractionation intervals of 168 h were higher than the spontaneous specific-locus mutation rate. These observations contradict the expectation for a true threshold dose response. We interpret this discrepancy to be due to the differences in the predictions of a mathematical abstraction of experimental data and the complexities of the biological system being studied. Biologically plausible explanations of the discrepancy are presented.

  6. Genome-Wide Deletion Screening with the Array CGH Method in Mouse Offspring Derived from Irradiated Spermatogonia Indicates that Mutagenic Responses are Highly Variable among Genes.

    PubMed

    Asakawa, Jun-Ichi; Kodaira, Mieko; Miura, Akiko; Tsuji, Takahiro; Nakamoto, Yoshiko; Imanaka, Masaaki; Kitamura, Jun; Cullings, Harry; Nishimura, Mayumi; Shimada, Yoshiya; Nakamura, Nori

    2016-12-01

    Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10(-2)/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10(-5)/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current

  7. Rad9a is required for spermatogonia differentiation in mice

    PubMed Central

    Huang, Lin; Wang, Zhen-Bo; Qi, Shu-Tao; Ma, Xue-Shan; Liang, Qiu-Xia; Lei, Guo; Meng, Tie-Gang; Liang, Li-Feng; Xian, Ye-Xin; Hou, Yi; Sun, Xiao-Fang; Zhao, Yong; Wang, Wei-Hua; Sun, Qing-Yuan

    2016-01-01

    Spermatogenesis in testes requires precise spermatogonia differentiation. Spermatocytes lacking the Rad9a gene are arrested in pachytene prophase, implying a possible role for RAD9A in spermatogonia differentiation. However, numerous RAD9A-positive pachytene spermatocytes are still observed in mouse testes following Rad9a excision using the Stra8-Cre system, and it is unclear whether Rad9a deletion in spermatogonia interrupts differentiation. Here, we generated a mouse model in which Rad9a was specifically deleted in spermatogonial stem cells (SSCs) using Cre recombinase expression driven by the germ cell-specific Vasa promoter. Adult Rad9a-null male mice were infertile as a result of completely blocked spermatogonia differentiation. No early spermatocytes were detected in mutant testicular cords of 9-day-old mice. Mutant spermatogonia were prone to apoptosis, although proliferation rates were unaffected. Rad9a deletion also resulted in malformation of seminiferous tubules, in which cells assembled irregularly into clusters, and malformation led to testicular cord disruption. Our findings suggest that Rad9a is indispensable for spermatogonia differentiation and testicular development in mice. PMID:27861152

  8. The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3.

    PubMed

    Suzuki, Hitomi; Sada, Aiko; Yoshida, Shosei; Saga, Yumiko

    2009-12-15

    Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markers of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A(s) to A(pr) cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A(s) to A(al)) and differentiating A(1) spermatogonia. In our present study, we find that A(s) and A(pr) can be basically classified into three categories: (1) GFRalpha1(+)Nanos2(+)Nanos3(-)Ngn3(-), (2) GFRalpha1(+)Nanos2(+)Nanos3(+)Ngn3(-), and (3) GFRalpha1(-)Nanos2(+/-)Nanos3(+)Ngn3(+). We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells.

  9. Sohlh2 affects differentiation of KIT positive oocytes and spermatogonia.

    PubMed

    Toyoda, Shuichi; Miyazaki, Tatsushi; Miyazaki, Satsuki; Yoshimura, Takuji; Yamamoto, Mayu; Tashiro, Fumi; Yamato, Eiji; Miyazaki, Jun-ichi

    2009-01-01

    The differentiation programs of spermatogenesis and oogenesis are largely independent. In the early stages, however, the mechanisms partly overlap. Here we demonstrated that a germ-cell-specific basic helix-loop-helix (bHLH) transcription factor gene, Sohlh2, is required for early spermatogenesis and oogenesis. SOHLH2 was expressed in mouse spermatogonia from the undifferentiated stage through differentiation and in primordial-to-primary oocytes. Sohlh2-null mice, produced by gene targeting, showed both male and female sterility, owing to the disrupted differentiation of mature (KIT(+)) spermatogonia and oocytes. The Sohlh2-null mice also showed the downregulation of genes involved in spermatogenesis and oogenesis, including the Sohlh1 gene, which is essential for these processes. Furthermore, we showed that SOHLH2 and SOHLH1 could form heterodimers. These observations suggested that SOHLH2 might coordinate with SOHLH1 to control spermatogonial and oocyte genes, including Sohlh1, to promote the differentiation of KIT(+) germ cells in vivo. This study lays the foundation for further dissection of the bHLH network that regulates early spermatogenesis and oogenesis.

  10. Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand.

    PubMed

    Rossi, Pellegrino; Lolicato, Francesca; Grimaldi, Paola; Dolci, Susanna; Di Sauro, Annarita; Filipponi, Doria; Geremia, Raffaele

    2008-01-01

    Kit ligand (KL) is a survival factor and a mitogenic stimulus for differentiating spermatogonia. However, it is not known whether KL also plays a role in the differentiative events that lead to meiotic entry of these cells. We performed a wide genome analysis of difference in gene expression induced by treatment with KL of spermatogonia from 7-day-old mice, using gene chips spanning the whole mouse genome. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia, i.e. the progressive lengthening of the S phase and the shortening of the G2/M transition. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes (for instance: Lhx8, Nek1, Rnf141, Xrcc3, Tpo1, Tbca, Xrcc2, Mesp1, Phf7, Rtel1), whereas it down-regulates typical spermatogonial markers (for instance: Pole, Ptgs2, Zfpm2, Egr2, Egr3, Gsk3b, Hnrpa1, Fst, Ptch2). Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/kit interaction in the induction of their meiotic differentiation.

  11. Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population.

    PubMed

    Lolicato, Francesca; Marino, Rita; Paronetto, Maria Paola; Pellegrini, Manuela; Dolci, Susanna; Geremia, Raffaele; Grimaldi, Paola

    2008-01-15

    Nanos gene encodes for zinc-finger protein with putative RNA-binding activity which shows an evolutionary conserved function in germ cell development. In the mouse, three Nanos homologs have been identified: Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes; however the role of Nanos3 in the testis during the postnatal period has not been explored yet. In this study, we show that, in prepuberal testis, Nanos3 is expressed in undifferentiated spermatogonia and that its up-regulation causes accumulation of cells in the G1 phase, indicating that this protein is able to delay the cell cycle progression of spermatogonial cells. This is in line with the observation that the cell cycle length of the undifferentiated germ cells is longer than in differentiating spermatogonia. We also demonstrate a conserved mechanism of action of Nanos3, involving the interaction with the murine RNA-binding protein Pumilio2 and consisting of a potential translational repressor activity. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, we show that treatment with the differentiating factor all-trans retinoic acid induces a dramatic down-regulation of its expression. These results allow to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle.

  12. Spermatogonial multiplication in the Chinese hamster. I. Cell cycle properties and synchronization of differentiating spermatogonia.

    PubMed

    Lok, D; de Rooij, D G

    1983-01-01

    The cell cycle properties of the six successive generations of differentiating spermatogonia in the Chinese hamster were analysed by the fraction of labelled mitoses technique (FLM). Except for the A1 spermatogonia most of which have a longer cell cycle time (Tc), Tc was found to be c. 60 hr for all types of differentiating spermatogonia. As in the mouse and the rat this represents c. 14% of the duration of the cycle of the seminiferous epithelium. With ongoing differentiation, ts of the differentiating spermatogonia increases from 14 to 25 hr, while tG2 shortens from 22 to 10 hr, ts + tG2 remaining at around 35 hr throughout. Autoradiography of whole mounted seminiferous tubules at 1 hr after injection of [3H]thymidine, and experiments with Ara-C revealed that the differentiating spermatogonia traverse S in sharply defined tubular segments. Thus adjacent clones of differentiating spermatogonia start and finish their S phase at virtually the same moment. This synchronization is not yet fully established among the first generation, as clones of A1 spermatogonia in the S phase were found intermingled with A1 cells in other phases of the cell cycle. Since there is little variation in tS and tG2 in the A1 spermatogonia, it was concluded that adjacent clones of A2 spermatogonia do not always arise at the same moment. Yet A2 spermatogonia do start S synchronously, and the FLM study confirms the expected variability in their tG1. A hypothesis is proposed that each generation of differentiating spermatogonia receives a stimulus to divide from outside the spermatogonial compartment. This would ensure the synchronous behaviour of adjacent clones and the strict relationship of the pattern of proliferation to the stages of the cycle of the seminiferous epithelium.

  13. Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice.

    PubMed

    Hasegawa, M; Zhang, Y; Niibe, H; Terry, N H; Meistrich, M L

    1998-03-01

    The effect of the p53 gene on the survival of mouse testicular cells was evaluated by analysis of degenerating and terminal transferase-mediated end labeling (TUNEL)-positive cells and the subsequent production of further differentiated progeny. In p53 null mice, in contrast to wild-type mice, radiation induced negligible levels of degenerating or TUNEL-positive differentiating spermatogonia within 24 h. This was correlated with higher production of differentiated progeny of the differentiating spermatogonia in p53 null mice. Contrary to the differentiating spermatogonia, the stem spermatogonia of p53 null mice produced fewer differentiated progeny after irradiation than did the stem cells of wild-type mice. We conclude that, because the degeneration and TUNEL positivity of the differentiating spermatogonia in mice of different genotypes were correlated with each other and were dependent on p53, this process is indeed apoptosis. In the differentiating spermatogonia, p53-dependent apoptosis accounted for the bulk of the loss of their progeny after irradiation. Furthermore, whereas the differentiating spermatogonia died by apoptosis that was dependent on p53, the stem spermatogonia, which are more radioresistant, did not.

  14. Sohlh2 knockout mice are male-sterile because of degeneration of differentiating type A spermatogonia.

    PubMed

    Hao, Jing; Yamamoto, Miwako; Richardson, Timothy E; Chapman, Karen M; Denard, Bray S; Hammer, Robert E; Zhao, Guang Quan; Hamra, F Kent

    2008-06-01

    The spermatogenesis and oogenesis-specific transcription factor Sohlh2 is normally expressed only in premeiotic germ cells. In this study, Sohlh2 and several other germ cell transcripts were found to be induced in mouse embryonic stem cells when cultured on a feeder cell line that overexpresses bone morphogenetic protein 4. To study the function of Sohlh2 in germ cells, we generated mice harboring null alleles of Sohlh2. Male Sohlh2-deficient mice were infertile because of a block in spermatogenesis. Although normal prior to birth, Sohlh2-null mice had reduced numbers of intermediate and type B spermatogonia by postnatal day 7. By day 10, development to the preleptotene spermatocyte stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia, and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type A2 spermatogonia and accumulated in M-phase prior to death. A similar phenotype was observed in Sohlh2-null mice on postnatal days 14, 21, 35, 49, 68, and 151. In adult Sohlh2-mutant mice, the ratio of undifferentiated type A spermatogonia (DAZL+/PLZF+) to differentiating type A spermatogonia (DAZL+/PLZF-) was twice normal levels. In culture, undifferentiated type A spermatogonia isolated from Sohlh2-null mice proliferated normally but linked the mutant phenotype to aberrant cell surface expression of the receptor-tyrosine kinase cKit. Thus, Sohlh2 is required for progression of differentiating type A spermatogonia into type B spermatogonia. One conclusion originating from these studies would be that testicular factors normally regulate the viability of differentiating spermatogonia by signaling through Sohlh2. This regulation would provide a crucial checkpoint to optimize the numbers of spermatocytes entering meiosis during each cycle of spermatogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

  15. A G-quadruplex DNA structure resolvase, RHAU, is essential for spermatogonia differentiation

    PubMed Central

    Gao, X; Ma, W; Nie, J; Zhang, C; Zhang, J; Yao, G; Han, J; Xu, J; Hu, B; Du, Y; Shi, Q; Yang, Z; Huang, X; Zhang, Y

    2015-01-01

    G-quadruplex (G4) DNA and G4 DNA resolvase are involved in a variety of biological processes. To understand the biological function of G4 DNA structures and their resolvases in spermatogenesis, we investigated the distribution of G4 structures in mouse testis and identified their alterations during spermatogenesis. Meanwhile, we studied the function of RNA helicase associated with AU-rich element (RHAU), a G4 DNA resolvase, in spermatogenesis with a germ-cell-specific knockout mouse model. The results showed that the ablation of RHAU in germ cells caused the increase of G4 structures and thus resulted in the decrease of spermatogonial differentiation. c-kit, a spermatogonia differentiation-related gene, contains two G4 DNA motifs on its promoter. We found its expression was significantly downregulated in RHAU conditional knockout testis. A further analysis demonstrated that RHAU directly bound to the G4 structures to activate c-kit expression. We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter. PMID:25611385

  16. In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay

    SciTech Connect

    Uchida, Aya; Kishi, Kasane; Aiyama, Yoshimi; Miura, Kento; Takase, Hinako M.; Suzuki, Hitomi; Kanai-Azuma, Masami; Iwamori, Tokuko; Kurohmaru, Masamichi; Tsunekawa, Naoki; Kanai, Yoshiakira

    2016-08-05

    In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted A{sub single} and marginal A{sub paired}–A{sub aligned} GFRα1-positive spermatogonia and was surrounded by A{sub aligned} GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. - Highlights: • A novel bead transplantation assay was developed to examine the in vivo effects of growth factors on spermatogonia. • A rapid aggregation of GFRα1-positive spermatogonia was induced by the transplanted GDNF-soaked beads. • Tightly-compacted A{sub single} and marginal A{sub paired}–A{sub aligned} spermatogonia were formed in each GFRα1-positive aggregate.

  17. Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia.

    PubMed

    Gallagher, Shannon J; Kofman, Amber E; Huszar, Jessica M; Dannenberg, Jan-Hermen; DePinho, Ronald A; Braun, Robert E; Payne, Christopher J

    2013-01-01

    Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating

  18. Undifferentiated primate spermatogonia and their endocrine control.

    PubMed

    Plant, Tony M

    2010-08-01

    The biology of spermatogonial stem cells is currently an area of intensive research and contemporary studies in primates are emerging. Quantitative regulation of sperm output by the primate testis seems to be exerted primarily on the transition from undifferentiated to differentiating spermatogonia. This review examines recent advances in our understanding of the mechanisms governing spermatogonial renewal and early differentiation in male primates, with a focus on the monkey. Emerging revisions to the classic view of dark and pale type A spermatogonia as reserve and renewing spermatogonial stem cells, respectively, are critically evaluated and essential features of endocrine control of undifferentiated spermatogonia throughout postnatal primate development are discussed. Obstacles in gaining a more complete understanding of primate spermatogonia are also identified.

  19. Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling.

    PubMed

    Pui, Han Pin; Saga, Yumiko

    2017-03-21

    Spermatogenesis is a continuous and highly coordinated process of spermatozoa production. In mice, this process is believed to initiate shortly after birth with the emergence of nascent spermatogonia in the testes. However, because the nascent spermatogonia originated from the gonocytes are morphologically indistinguishable from their predecessors and there is no clear definition for the gonocytes-to-spermatogonia transition (GST), it remains unclear when and how spermatogenesis is initiated in the mouse testes. To address these questions, we characterized the emergence of nascent spermatogonia in ICR mice. We found that GST is initiated in a subset of gonocytes as early as E18.5. These nascent spermatogonia express markers typical of undifferentiated spermatogonia residing in testes of adult mice. In addition to markers expression, we identified FOXO1 nuclear-to-cytoplasmic translocation as a novel feature of GST distinguishing nascent spermatogonia from the gonocytes. Using those criteria, we demonstrated that GST requires FGF signaling. When FGF signaling was inhibited pharmacologically, gonocytes retained nuclear FOXO1 expression, did not express spermatogonial markers and failed to proliferate. We found that FGF signaling acts upstream of GDNF and RA signalings for the activation of the MEK/ERK and PI3K/Akt pathways in germ cells during GST. Taken together, we defined the precise timing of GST and revealed FGF signaling as a master regulator of GST in the perinatal mouse testes.

  20. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes.

    PubMed

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    Spermatogenesis, a study of germ cell development, is a long, orderly, and well-defined process occurring in seminiferous tubules of the testis. It is a temporal event whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa over a period of several weeks. Spermatogenesis is characterized by three specific functional phases: proliferation, meiosis, and differentiation, and it involves spermatogonia, spermatocytes, and spermatids. Germ cells at steps of development form various cellular associations or stages, with 6, 12, and 14 specific stages being identified in human, mouse, and rat, respectively. The stages evolve over time in a given area of the seminiferous tubule forming a cycle of the seminiferous epithelium that has a well-defined duration for a given species. In this part, we discuss the proliferation and meiotic phase whereby spermatogonia undergo several mitotic divisions to form spermatocytes that undergo two meiotic divisions to form haploid spermatids. In the rat, spermatogonia can be subdivided into several classes: stem cells (A(s)), proliferating cells (A(pr), A(al)), and differentiating cells (A(1)-A(4), In, B). They are dependent on a specific microenvironment (niche) contributed by Sertoli, myoid, and Leydig cells for proper development. Spermatogonia possess several surface markers whereby they can be identified from each other. During meiosis, spermatocytes undergo chromosomal pairing, synapsis, and genetic exchange as well as transforming into haploid cells following meiosis. The meiotic cells form specific structural entities such as the synaptonemal complex and sex body. Many genes involved in spermatogonial renewal and the meiotic process have been identified and shown to be essential for this event.

  1. Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

    PubMed

    Ferder, Ianina C; Wang, Ning

    2015-06-30

    Like stem cells in other tissues, spermatogonia, including spermatogonial stem cells (SSCs) at the foundation of differentiation hierarchy, undergo age-related decline in function. The promyelocytic leukemia zinc finger (Plzf) protein plays an essential role in spermatogonia maintenance by preventing their differentiation. To evaluate whether there is an age-related change in Plzf expression, we found that aged mouse testes exhibited a robust "Plzf overexpression" phenotype, in that they showed not only a higher frequency of Plzf-expressing cells but also an increased level of Plzf expression in these cells. Moreover, some Plzf-expressing cells in aged testes even aberrantly appeared in the differentiating spermatogonia compartment, which is usually low or negative for Plzf expression. Importantly, ectopic Plzf expression in F9 cells suppressed retinoic acid (RA)-induced Stra8 activation, a gene required for meiosis initiation. These data, together with our observation of a lack of meiosis-initiating spermatocytes associated with high Plzf-expressing spermatogonia in the aged testes, particularly in the degenerative seminiferous tubules, suggest that age-related increase in Plzf expression represents a novel molecular signature of spermatogonia aging by functionally arresting their differentiation.

  2. Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia.

    PubMed

    Russell, Liane B; Hunsicker, Patricia R; Russell, William L

    2007-03-01

    Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem-cell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75mgMNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment - chemical or radiation - that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite

  3. Vitrification preserves proliferation capacity in human spermatogonia.

    PubMed

    Poels, Jonathan; Van Langendonckt, Anne; Many, Marie-Christine; Wese, François-Xavier; Wyns, Christine

    2013-03-01

    Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model? Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting. Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated. Ten ITT pieces issuing from 10 patients aged 2-12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared. Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3β-HSD). The entire piece of grafted tissue was assessed in each case. Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting. The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis. The findings of the present study have potential implications for

  4. Upregulation and nuclear translocation of testicular ghrelin protects differentiating spermatogonia from ionizing radiation injury

    PubMed Central

    Li, W; Zeng, Y; Zhao, J; Zhu, C-J; Hou, W-G; Zhang, S

    2014-01-01

    Proper control of apoptotic signaling is important for maintenance of testicular homeostasis after ionizing radiation (IR). Herein, we challenged the hypothesis that ghrelin, a pleiotropic modulator, is potentially involved in IR-induced germ cell injury. Lower body exposure to 2 Gy of IR induced a notable increase of ghrelin expression in the nuclear of differentiating spermatogonia at defined stages, with an impairment in the Leydig cells (LCs)-expressing ghrelin. Unexpectedly, inhibition of the ghrelin pathway by intraperitoneal injection of a specific GHS-R1α antagonist enhanced spermatogonia elimination by apoptosis during the early recovery following IR, and thereafter resulted in impaired male fertility, suggesting that the anti-apoptotic effects of evoked ghrelin, although transient along testicular IR injury, have a profound influence on the post-injury recovery. In addition, inhibition of ghrelin signaling resulted in a significant increase in the intratesticular testosterone (T) level at the end of 21 days after IR, which should stimulate the spermatogenic recovery from surviving spermatogonia to a certain extent during the late stage. We further demonstrated that the upregulation and nuclear trafficking of ghrelin, elaborately regulated by IR-elicited antioxidant system in spermatogonia, may act through a p53-dependent mechanism. The elicitation of ghrelin expression by IR stress, the regulation of ghrelin expression by IR-induced oxidative stress and the interaction between p53 and ghrelin signaling during IR injury were confirmed in cultured spermatogonia. Hence, our results represent the first evidence in support of a radioprotective role of ghrelin in the differentiating spermatogonia. The acutely, delicate regulation of local-produced ghrelin appears to be a fine-tune mechanism modulating the balance between testicular homeostasis and early IR injury. PMID:24853426

  5. Upregulation and nuclear translocation of testicular ghrelin protects differentiating spermatogonia from ionizing radiation injury.

    PubMed

    Li, W; Zeng, Y; Zhao, J; Zhu, C-J; Hou, W-G; Zhang, S

    2014-05-22

    Proper control of apoptotic signaling is important for maintenance of testicular homeostasis after ionizing radiation (IR). Herein, we challenged the hypothesis that ghrelin, a pleiotropic modulator, is potentially involved in IR-induced germ cell injury. Lower body exposure to 2 Gy of IR induced a notable increase of ghrelin expression in the nuclear of differentiating spermatogonia at defined stages, with an impairment in the Leydig cells (LCs)-expressing ghrelin. Unexpectedly, inhibition of the ghrelin pathway by intraperitoneal injection of a specific GHS-R1α antagonist enhanced spermatogonia elimination by apoptosis during the early recovery following IR, and thereafter resulted in impaired male fertility, suggesting that the anti-apoptotic effects of evoked ghrelin, although transient along testicular IR injury, have a profound influence on the post-injury recovery. In addition, inhibition of ghrelin signaling resulted in a significant increase in the intratesticular testosterone (T) level at the end of 21 days after IR, which should stimulate the spermatogenic recovery from surviving spermatogonia to a certain extent during the late stage. We further demonstrated that the upregulation and nuclear trafficking of ghrelin, elaborately regulated by IR-elicited antioxidant system in spermatogonia, may act through a p53-dependent mechanism. The elicitation of ghrelin expression by IR stress, the regulation of ghrelin expression by IR-induced oxidative stress and the interaction between p53 and ghrelin signaling during IR injury were confirmed in cultured spermatogonia. Hence, our results represent the first evidence in support of a radioprotective role of ghrelin in the differentiating spermatogonia. The acutely, delicate regulation of local-produced ghrelin appears to be a fine-tune mechanism modulating the balance between testicular homeostasis and early IR injury.

  6. Peroxisomes are present in murine spermatogonia and disappear during the course of spermatogenesis.

    PubMed

    Lüers, Georg H; Thiele, Samuel; Schad, Arno; Völkl, Alfred; Yokota, Sadaki; Seitz, Jürgen

    2006-06-01

    Peroxisomes are organelles that are almost ubiquitous in eukaryotic cells. They have, however, never been described in germ cells within the testis. Since some peroxisomal diseases like Adrenoleukodystrophy are associated with reduced fertility, we have re-investigated the peroxisomal compartment of the germinal epithelium of mice using in situ hybridization, immunohistochemistry, Western blotting and immunoelectron microscopy. Within the seminiferous tubules, peroxisomes are present in Sertoli cells and in germ cells. We could show that small-sized peroxisomes of typical ultrastructure are concentrated in spermatogonia and disappear during the course of spermatogenesis. Peroxisomes of spermatogonia differ in their relative protein composition from previously described peroxisomes of interstitial cells of Leydig. Since germ cells differentiate in mouse testis in a synchronized fashion, the disappearence of peroxisomes could be a suitable model system to investigate the degradation of an organelle as part of a physiological differentiation process in higher eukaryotes.

  7. E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro.

    PubMed

    Zhang, Yan; Su, Huimin; Luo, Fenhua; Wu, Sachula; Liu, Linhong; Liu, Taodi; Yu, Boyang; Wu, Yingji

    2011-09-01

    Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.

  8. The hot-spot p53R172H mutant promotes formation of giant spermatogonia triggered by DNA damage.

    PubMed

    Xue, Y; Raharja, A; Sim, W; Wong, E S M; Rahmat, S A B; Lane, D P

    2017-04-06

    Overexpression of mutant p53 is a common finding in most cancers but testicular tumours accumulate wild-type p53 (wtp53). In contrast to the accepted concept that p53 homozygous mutant mice do not accumulate mutant p53 in normal cells, our study on a mutant p53 mouse model of Li-Fraumeni syndrome harbouring the hot-spot p53R172H mutation described an elevated level of mutant p53 in non-cancerous mouse tissues. Here we use detailed immunohistochemical analysis to document the expression of p53R172H in mouse testis. In developing and adult testes, p53R172H was expressed in gonocytes, type A, Int, B spermatogonia as well as in pre-Sertoli cells and Leydig cells but was undetectable in spermatocytes and spermatids. A similar staining pattern was demonstrated for wtp53. However, the intensity of wtp53 staining was generally weaker than that of p53R172H, which indicates that the expression of p53R172H can be a surrogate marker of p53 gene transcription. Comparing the responses of wtp53 and p53R172H to irradiation, we found persistent DNA double-strand breaks in p53R172H testes and the formation of giant spermatogonia (GSG) following persistent DNA damage in p53R172H and p53-null mice. Strikingly, we found that p53R172H promotes spontaneous formation of GSG in non-stressed p53R172H ageing mice. Two types of GSG: Viable and Degenerative GSG were defined. We elucidate the factors involved in the formation of GSG: the loss of p53 function is a requirement for the formation of GSG whereas DNA damage acts as a promoting trigger. The formation of GSG does not translate to higher efficacy of testicular tumorigenesis arising from mutant p53 cells, which might be due to the presence of delayed-onset of p53-independent apoptosis.

  9. Proteomic analysis of proteins involved in spermiogenesis in mouse.

    PubMed

    Guo, Xuejiang; Shen, Jian; Xia, Zhengrong; Zhang, Rui; Zhang, Ping; Zhao, Chun; Xing, Jun; Chen, Ling; Chen, Wen; Lin, Min; Huo, Ran; Su, Bing; Zhou, Zuomin; Sha, Jiahao

    2010-03-05

    Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

  10. Depletion of the spermatogonia from the seminiferous epithelium of the rhesus monkey after X irradiation

    SciTech Connect

    van Alphen, M.M.; van de Kant, H.J.; de Rooij, D.G.

    1988-03-01

    In unirradiated testes large differences were found in the total number of spermatogonia among different monkeys, but the number of spermatogonia in the right and the left testes of the same monkey appeared to be rather similar. During the first 11 days after irradiation with 0.5 to 4.0 Gy of X rays the number of Apale spermatogonia (Ap) decreased to about 13% of the control level, while the number of Adark spermatogonia (Ad) did not change significantly. A significant decrease in the number of Ad spermatogonia was seen at Day 14 together with a significant increase in the number of Ap spermatogonia. It was concluded that the resting Ad spermatogonia are activated into proliferating Ap spermatogonia. After Day 16 the number of both Ap and Ad spermatogonia decreased to low levels. Apparently the new Ap spermatogonia were formed by lethally irradiated Ad spermatogonia and degenerated while attempting to divide. The activation of the Ad spermatogonia was found to take place throughout the cycle of the seminiferous epithelium. Serum FSH, LH, and testosterone levels were measured before and after irradiation. Serum FSH levels already had increased during the first week after irradiation to 160% of the control level. Serum LH levels increased between 18 and 25 days after irradiation. Serum testosterone levels did not change at all. The results found in the rhesus monkey are in line with those found in humans, but due to the presence of Ad spermatogonia they differ from those obtained in non-primates.

  11. The transition from stem cell to progenitor spermatogonia and male fertility requires the SHP2 protein tyrosine phosphatase.

    PubMed

    Puri, Pawan; Phillips, Bart T; Suzuki, Hitomi; Orwig, Kyle E; Rajkovic, Aleksandar; Lapinski, Philip E; King, Philip D; Feng, Gen-Sheng; Walker, William H

    2014-03-01

    SHP2 is a widely expressed protein tyrosine phosphatase required for signal transduction from multiple cell surface receptors. Gain and loss of function SHP2 mutations in humans are known to cause Noonan and LEOPARD syndromes, respectively, that are characterized by numerous pathological conditions including male infertility. Using conditional gene targeting in the mouse, we found that SHP2 is required for maintaining spermatogonial stem cells (SSCs) and the production of germ cells required for male fertility. After deleting SHP2, spermatogenesis was halted at the initial step during which transit-amplifying undifferentiated spermatogonia are produced from SSCs. In the absence of SHP2, proliferation of SSCs and undifferentiated spermatogonia was inhibited, thus germ cells cannot be replenished and SSCs cannot undergo renewal. However, germ cells beyond the undifferentiated spermatogonia stage of development at the time of SHP2 knockout were able to complete their maturation to become sperm. In cultures of SSCs and their progeny, inhibition of SHP2 activity reduced growth factor-mediated intracellular signaling that regulates SSC proliferation and cell fate. Inhibition of SHP2 also decreased the number of SSCs present in culture and caused SSCs to detach from supporting cells. Injection of mice with an SHP2 inhibitor blocked the production of germ cells from SSCs. Together, our studies show that SHP2 is essential for SSCs to maintain fertility and indicates that the pathogenesis of infertility in humans with SHP2 mutations is due to compromised SSC functions that block spermatogenesis. © AlphaMed Press.

  12. Hormonal suppression restores fertility in irradiated mice from both endogenous and donor-derived stem spermatogonia.

    PubMed

    Wang, Gensheng; Shao, Shan H; Weng, Connie C Y; Wei, Caimiao; Meistrich, Marvin L

    2010-09-01

    Irradiation interrupts spermatogenesis and causes prolonged sterility in male mammals. Hormonal suppression treatment with gonadotropin-releasing hormone (GnRH) analogues has restored spermatogenesis in irradiated rats, but similar attempts were unsuccessful in irradiated mice, monkeys, and humans. In this study, we tested a stronger hormonal suppression regimen (the GnRH antagonist, acyline, and plus flutamide) for efficacy both in restoring endogenous spermatogenesis and in enhancing colonization of transplanted stem spermatogonia in mouse testes irradiated with a total doses between 10.5 and 13.5 Gy. A 4-week hormonal suppression treatment, given immediately after irradiation, increased endogenous spermatogenic recovery 1.5-fold, and 11-week hormonal suppression produced twofold increases compared with sham-treated irradiated controls. Furthermore, 10-week hormonal suppression restored fertility from endogenous surviving spermatogonial stem cells in 90% of 10.5-Gy irradiated mice, whereas only 10% were fertile without hormonal suppression. Four- and 11-week hormonal suppression also enhanced spermatogenic development from transplanted stem spermatogonia in irradiated recipient mice, by 3.1- and 4.8-fold, respectively, compared with those not given hormonal treatment. Moreover, the 10-week hormonal suppression regimen, but not a sham treatment, restored fertility of some 13.5-Gy irradiated recipient mice from donor-derived spermatogonial stem cells. This is the first report of hormonal suppression inducing recovery of endogenous spermatogenesis and fertility in a mouse model treated with anticancer agents. The combination of spermatogonial transplantation with hormonal suppression should be investigated as a treatment to restore fertility in young men after cytotoxic cancer therapy.

  13. Sleeping Beauty mouse models identify candidate genes involved in gliomagenesis.

    PubMed

    Vyazunova, Irina; Maklakova, Vilena I; Berman, Samuel; De, Ishani; Steffen, Megan D; Hong, Won; Lincoln, Hayley; Morrissy, A Sorana; Taylor, Michael D; Akagi, Keiko; Brennan, Cameron W; Rodriguez, Fausto J; Collier, Lara S

    2014-01-01

    Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.

  14. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    SciTech Connect

    Yoneda, Akihiro; Watanabe, Tomomasa

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  15. Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis

    PubMed Central

    Vyazunova, Irina; Maklakova, Vilena I.; Berman, Samuel; De, Ishani; Steffen, Megan D.; Hong, Won; Lincoln, Hayley; Morrissy, A. Sorana; Taylor, Michael D.; Akagi, Keiko; Brennan, Cameron W.; Rodriguez, Fausto J.; Collier, Lara S.

    2014-01-01

    Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma. PMID:25423036

  16. N-acetyl endorphin in rat spermatogonia and primary spermatocytes.

    PubMed Central

    Cheng, M C; Clements, J A; Smith, A I; Lolait, S J; Funder, J W

    1985-01-01

    In previous reports modest levels of beta-endorphin have been found by radioimmunoassay in rat testis, and localized by immunofluorescence to the interstitial cells. We have confirmed these previous reports and extended them by showing that the majority of testicular endorphins are acetylated forms, N-acetyl gamma-endorphin, N-acetyl alpha-endorphin, and N-acetyl beta-endorphin1-27. In addition, N-acetylated endorphins are not found in interstitial cells, but are confined to spermatogonia and primary spermatocytes. Images PMID:3156881

  17. Isolation of undifferentiated and early differentiating Type A spermatogonia from Pou5f1-GFP reporter mice

    PubMed Central

    Garcia, Thomas; Hofmann, Marie-Claude

    2012-01-01

    Limited understanding of the mechanisms underlying self-renewal and differentiation of spermatogonial stem cells hampers our ability to develop new therapeutic and contraceptive approaches. Mouse models of spermatogonial stem cell development are key to developing new insights into the biology of both the normal and diseased testis. Advances in transgenic reporter mice have enabled the isolation, molecular characterization, and functional analysis of mouse Type A spermatogonia subpopulations from the normal testis, including populations enriched for spermatogonial stem cells. Application of these reporters both to the normal testis and to gene-deficient and over-expression models will promote a better understanding of the earliest steps of spermatogenesis, and the role of spermatogonial stem cells in germ cell tumor. PMID:22144234

  18. Isolation of undifferentiated and early differentiating type A spermatogonia from Pou5f1-GFP reporter mice.

    PubMed

    Garcia, Thomas; Hofmann, Marie-Claude

    2012-01-01

    Limited understanding of the mechanisms underlying self-renewal and differentiation of spermatogonial stem cells hampers our ability to develop new therapeutic and contraceptive approaches. Mouse models of spermatogonial stem cell development are key to developing new insights into the biology of both the normal and diseased testis. Advances in transgenic reporter mice have enabled the isolation, molecular characterization, and functional analysis of mouse Type A spermatogonia subpopulations from the normal testis, including populations enriched for spermatogonial stem cells. Application of these reporters both to the normal testis and to gene-deficient and over-expression models will promote a better understanding of the earliest steps of spermatogenesis, and the role of spermatogonial stem cells in germ cell tumor.

  19. Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia.

    PubMed

    Goel, Sandeep; Reddy, Niranjan; Mandal, Suman; Fujihara, Mayako; Kim, Sung-Min; Imai, Hiroshi

    2010-10-15

    Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.

  20. Involvement of Transient Receptor Potential Vanilloid (TRPV) 4 in mouse sperm thermotaxis

    PubMed Central

    HAMANO, Koh-ichi; KAWANISHI, Tae; MIZUNO, Atsuko; SUZUKI, Makoto; TAKAGI, Yuji

    2016-01-01

    Transient Receptor Potential Vanilloid (TRPV) 4 is one of the temperature-sensitive ion channels involved in temperature receptors, and it is known to be activated from 35 to 40ºC. Here we analyzed sperm motility function of Trpv4 knockout (KO) mouse in temperature-gradient conditions to elucidate the thermotaxis of mouse sperm and the involvement of TRPV4 in thermotaxis. The sperm were introduced at the vertical column end of a T-shaped chamber filled with medium in a plastic dish, and we measured the number of sperm that arrived at both ends of the wide column where we had established a temperature gradient of approx. 2ºC, and we evaluated the sperm’s thermotaxis. Large numbers of wild-type (WT) mouse sperm migrated into the high level of the temperature gradient that was set in the wide column, and thermotaxis was confirmed. The ratio of migrated sperm at the high temperature level of the T-shaped chamber was decreased in the KO sperm and Ruthenium red (a TRPV antagonist) treated sperm compared with the WT sperm. The thermotaxis of the mouse sperm was confirmed, and the involvement of TRPV4 in this thermotaxis was suggested. PMID:27180924

  1. Involvement of Transient Receptor Potential Vanilloid (TRPV) 4 in mouse sperm thermotaxis.

    PubMed

    Hamano, Koh-Ichi; Kawanishi, Tae; Mizuno, Atsuko; Suzuki, Makoto; Takagi, Yuji

    2016-08-25

    Transient Receptor Potential Vanilloid (TRPV) 4 is one of the temperature-sensitive ion channels involved in temperature receptors, and it is known to be activated from 35 to 40ºC. Here we analyzed sperm motility function of Trpv4 knockout (KO) mouse in temperature-gradient conditions to elucidate the thermotaxis of mouse sperm and the involvement of TRPV4 in thermotaxis. The sperm were introduced at the vertical column end of a T-shaped chamber filled with medium in a plastic dish, and we measured the number of sperm that arrived at both ends of the wide column where we had established a temperature gradient of approx. 2ºC, and we evaluated the sperm's thermotaxis. Large numbers of wild-type (WT) mouse sperm migrated into the high level of the temperature gradient that was set in the wide column, and thermotaxis was confirmed. The ratio of migrated sperm at the high temperature level of the T-shaped chamber was decreased in the KO sperm and Ruthenium red (a TRPV antagonist) treated sperm compared with the WT sperm. The thermotaxis of the mouse sperm was confirmed, and the involvement of TRPV4 in this thermotaxis was suggested.

  2. Evidences for the agmatine involvement in antidepressant like effect of bupropion in mouse forced swim test.

    PubMed

    Kotagale, Nandkishor R; Tripathi, Sunil J; Aglawe, Manish M; Chopde, Chandrabhan T; Umekar, Milind J; Taksande, Brijesh G

    2013-06-01

    Although bupropion has been widely used in the treatment of depression, the precise mechanism of its therapeutic actions is not fully understood. The present study investigated the role of agmatine in an antidepressant like effect of bupropion in mouse forced swim test. The antidepressant like effect of bupropion was potentiated by pretreatment with agmatine (10-20mg/kg, ip) and by the drugs known to increase endogenous agmatine levels in brain viz., l-arginine (40 μg/mouse, icv), an agmatine biosynthetic precursor, ornithine decarboxylase inhibitor, dl-α-difluoromethyl ornithine hydrochloride, DFMO (12.5 μg/mouse, icv), diamine oxidase inhibitor, aminoguanidine (6.5 μg/mouse, icv) and agmatinase inhibitor, arcaine (50 μg/mouse, icv) as well as imidazoline I1 receptor agonists, moxonidine (0.25mg/kg, ip) and clonidine (0.015 mg/kg, ip) and imidazoline I2 receptor agonist, 2-(2-benzofuranyl)-2-imidazoline hydrochloride, 2-BFI (5mg/kg, ip). Conversely, prior administration of I1 receptor antagonist, efaroxan (1mg/kg, ip) and I2 receptor antagonist, idazoxan (0.25mg/kg, ip) blocked the antidepressant like effect of bupropion and its synergistic combination with agmatine. These results demonstrate involvement of agmatine in the antidepressant like effect of bupropion and suggest agmatine and imidazoline receptors as a potential therapeutic target for the treatment of depressive disorders.

  3. Increased urinary lysophosphatidic acid in mouse with subtotal nephrectomy: potential involvement in chronic kidney disease.

    PubMed

    Mirzoyan, Koryun; Baïotto, Anna; Dupuy, Aude; Marsal, Dimitri; Denis, Colette; Vinel, Claire; Sicard, Pierre; Bertrand-Michel, Justine; Bascands, Jean-Loup; Schanstra, Joost P; Klein, Julie; Saulnier-Blache, Jean-Sébastien

    2016-12-01

    Increased incidence of chronic kidney disease (CKD) with consecutive progression to end-stage renal disease represents a significant burden to healthcare systems. Renal tubulointerstitial fibrosis (TIF) is a classical hallmark of CKD and is well correlated with the loss of renal function. The bioactive lysophospholipid lysophosphatidic acid (LPA), acting through specific G-protein-coupled receptors, was previously shown to be involved in TIF development in a mouse model of unilateral ureteral obstruction. Here, we study the role of LPA in a mouse subjected to subtotal nephrectomy (SNx), a more chronic and progressive model of CKD. Five months after surgical nephron reduction, SNx mice showed massive albuminuria, extensive TIF, and glomerular hypertrophy when compared to sham-operated animals. Urinary and plasma levels of LPA were analyzed using liquid chromatography tandem mass spectrometry. LPA was significantly increased in SNx urine, not in plasma, and was significantly correlated with albuminuria and TIF. Moreover, SNx mice showed significant downregulation in the renal expression of lipid phosphate phosphohydrolases (LPP1, 2, and 3) that might be involved in reduced LPA bioavailability through dephosphorylation. We concluded that SNx increases urinary LPA through a mechanism that could involve co-excretion of plasma LPA with albumin associated with a reduction of its catabolism in the kidney. Because of the previously demonstrated profibrotic activity of LPA, the association of urinary LPA with TIF suggests the potential involvement of LPA in the development of advanced CKD in the SNx mouse model. Targeting LPA metabolism might represent an interesting approach in CKD treatment.

  4. Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration.

    PubMed

    Martínez-López, María José; Alcántara, Soledad; Mascaró, Cristina; Pérez-Brangulí, Francesc; Ruiz-Lozano, Pilar; Maes, Tamara; Soriano, Eduardo; Buesa, Carlos

    2005-04-01

    The development of the nervous system (NS) requires the coordinated migration of multiple waves of neurons and subsequent processes of neurite maturation, both involving selective guidance mechanisms. In Caenorhabditis elegans, unc-53 codes for a new multidomain protein involved in the directional migration of a subset of cells. We describe here the first functional characterization of the mouse homologue, mouse Neuron navigator 1 (mNAV1), whose expression is largely restricted to the NS during development. EGFP-mNAV1 associates with microtubules (MTs) plus ends present in the growth cone through a new microtubule-binding (MTB) domain. Moreover, its overexpression in transfected cells leads to MT bundling. The abolition of mNAV1 causes loss of directionality in the leading processes of pontine-migrating cells, providing evidence for a role of mNAV1 in mediating Netrin-1-induced directional migration.

  5. Isolation of highly purified type A spermatogonia from prepubertal rat testis.

    PubMed

    Morena, A R; Boitani, C; Pesce, M; De Felici, M; Stefanini, M

    1996-01-01

    We have developed a new method that allows isolation of highly purified type A spermatogonia from prepubertal rats. The procedure is based on the maximal release of spermatogonia from the seminiferous epithelium obtained by the complete enzymatic digestion of the tubular basal lamina, followed by removal of contaminating somatic cells through adhesion to plastic dishes coated with the lectin Datura stramonium agglutinin and fractionation on a discontinuous Percoll gradient. The cell suspension obtained contains up to 85% type A spermatogonia. Besides morphological criteria, the identification of germ cells and somatic cells has been performed by means of immunocytochemical markers, such as c-kit receptor, which is present only in germ cells, and vimentin, which is present only in somatic cells. All type A spermatogonia isolated were c-kit positive, thus suggesting that c-kit receptor is present in both undifferentiated and differentiating type A spermatogonia. Preliminary culture experiments demonstrate that spermatogonia survival in vitro was significantly improved by the addition of 10% fetal calf serum or horse serum to the culture medium; however, optimal culture conditions remain to be established. In vitro studies on isolated spermatogonia may provide a significant contribution toward elucidation of the mechanisms regulating spermatogonial proliferation and differentiation.

  6. Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice.

    PubMed

    Oatley, Melissa J; Kaucher, Amy V; Racicot, Karen E; Oatley, Jon M

    2011-08-01

    Continual spermatogenesis at a quantitatively normal level is required to sustain male fertility. The foundation of this process relies on maintenance of an undifferentiated spermatogonial population consisting of spermatogonial stem cells (SSCs) that self-renew as well as transient amplifying progenitors produced by differentiation. In mammals, type A(single) spermatogonia form the SSC population, but molecular markers distinguishing these from differentiating progenitors are undefined and knowledge of mechanisms regulating their functions is limited. We show that in the mouse male germline the transcriptional repressor ID4 is expressed by a subpopulation of undifferentiated spermatogonia and selectively marks A(single) spermatogonia. In addition, we found that ID4 expression is up-regulated in isolated SSC-enriched fractions by stimulation from GDNF, a key growth factor driving self-renewal. In mice lacking ID4 expression, quantitatively normal spermatogenesis was found to be impaired due to progressive loss of the undifferentiated spermatogonial population during adulthood. Moreover, reduction of ID4 expression by small interfering RNA treatment abolished the ability of wild-type SSCs to expand in vitro during long-term culture without affecting their survival. Collectively, these results indicate that ID4 is a distinguishing marker of SSCs in the mammalian germline and plays an important role in the regulation of self-renewal.

  7. Axial elongation in mouse embryos involves mediolateral cell intercalation behavior in the paraxial mesoderm

    NASA Astrophysics Data System (ADS)

    Yen, WeiWei; Burdsal, Carol; Periasamy, Ammasi; Sutherland, Ann E.

    2006-02-01

    The cell mechanical and signaling pathways involved in gastrulation have been studied extensively in invertebrates and amphibians, such as Xenopus, and more recently in non-mammalian vertebrates such as zebrafish and chick. However, because culturing mouse embryos extra-utero is very difficult, this fundamental process has been least characterized in the mouse. As the primary mammalian model for genetics, biochemistry, and the study of human disease and birth defects, it is important to investigate how gastrulation proceeds in murine embryos. We have developed a method of using 4D multiphoton excitation microscopy and extra-utero culture to visualize and characterize the morphogenetic movements in mouse embryos dissected at 8.5 days of gestation. Cells are labeled by expression of an X chromosome-linked enhanced green fluorescent protein (EGFP) transgene. This method has provided a unique approach, where, for the first time, patterns of cell behavior in the notochord and surrounding paraxial mesoderm can be visualized and traced quantitatively. Our observations of mouse embryos reveal both distinct differences as well as striking similarities in patterned cell motility relative to other vertebrate models such as Xenopus, where axial extension is driven primarily by mediolateral oriented cell behaviors in the notochord and paraxial somitic mesoderm. Unlike Xenopus, the width of the mouse notochord remains the same between 4-somite stage and 8-somite stage embryos. This implies the mouse notochord plays a lesser role in driving axial extension compared to Xenopus, although intercalation may occur where the anterior region of the node becomes notochordal plate. In contrast, the width of mouse paraxial mesoderm narrows significantly during this period and cells within the paraxial mesoderm are both elongated and aligned perpendicular to the midline. In addition, these cells are observed to intercalate, consistent with a role for paraxial mesoderm in driving convergence

  8. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

    PubMed Central

    2013-01-01

    Background We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). Results We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Conclusions Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines. PMID:24025402

  9. AURKB and MAPK involvement in the regulation of the early stages of mouse zygote development.

    PubMed

    Xu, Lin; Liu, Tong; Han, Feng; Zong, Zhihong; Wang, Guoli; Yu, Bingzhi; Zhang, Jie

    2012-01-01

    Aurora kinases have become a hot topic for research as they have been found to play an important role in various stages of mitotic cell division and to participate in malignant conversions of tumors. The participation of Aurora kinases in the regulation of oocyte meiosis has been recently reported, but their participation in mammalian early embryonic development remained unclear. The object of our study was to establish the spatio-temporal expression pattern of Aurora kinase B (AURKB) in mouse zygotes during the first cleavage, to reveal its functions in the early development of mouse zygotes, and to define the involvement of AURKB in mitogen-activated protein kinase (MAPK) signaling. Our results showed that in mouse zygotes AURKB expression increased in G1 phase and peaked in M phase. AURKB protein distribution was found to be in association with nuclei and distributed throughout the cytoplasm in a cell cycle-dependent manner. Functional disruption of AURKB resulted in abnormal division phenotypes or mitotic impairments. U0126, a specific mitogen-activated protein kinase kinase (MEK) inhibitor, caused significantly altered morphologies of early embryos together with a decrease in protein expression and kinase activity of AURKB. Our results indicated that the activity of AURKB was required for regulating multiple stages of mitotic progression in the early development of mouse zygotes and was correlated with the activation of the MAPK pathway.

  10. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse.

    PubMed

    Daxinger, Lucia; Harten, Sarah K; Oey, Harald; Epp, Trevor; Isbel, Luke; Huang, Edward; Whitelaw, Nadia; Apedaile, Anwyn; Sorolla, Anabel; Yong, Joan; Bharti, Vandhana; Sutton, Joanne; Ashe, Alyson; Pang, Zhenyi; Wallace, Nathan; Gerhardt, Daniel J; Blewitt, Marnie E; Jeddeloh, Jeffrey A; Whitelaw, Emma

    2013-01-01

    We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.

  11. No evidence of deer mouse involvement in plague (Yersinia pestis) epizootics in prairie dogs.

    PubMed

    Salkeld, Daniel J; Stapp, Paul

    2008-06-01

    Plague, the disease caused by the bacterium Yersinia pestis, can have devastating impacts on black-tailed prairie dog (Cynomys ludovicianus) colonies. One suggested mechanism behind sporadic prairie dog die-offs involves an alternative mammal host, such as the deer mouse (Peromyscus maniculatus), which often inhabits prairie dog colonies. We examined the flea populations of deer mice to investigate the potential of flea-borne transmission of plague between deer mice and prairie dogs in northern Colorado, where plague is active in prairie dog colonies. Deer mice were predominantly infested with the flea Aetheca wagneri, and were rarely infested with prairie dog fleas, Oropsylla hirsuta. Likelihood of flea infestation increased with average monthly temperature, and flea loads were higher in reproductive animals. These results suggest that the deer mouse is an unlikely maintenance host of plague in this region.

  12. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration.

    PubMed

    Chu, Jessie; Hong, Nancy A; Masuda, Claudio A; Jenkins, Brian V; Nelms, Keats A; Goodnow, Christopher C; Glynne, Richard J; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A P; Kay, Steve A

    2009-02-17

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders.

  13. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration

    PubMed Central

    Chu, Jessie; Hong, Nancy A.; Masuda, Claudio A.; Jenkins, Brian V.; Nelms, Keats A.; Goodnow, Christopher C.; Glynne, Richard J.; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A. P.; Kay, Steve A.

    2009-01-01

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders. PMID:19196968

  14. Involvement of Fas and FasL in Ectromelia virus-induced apoptosis in mouse brain.

    PubMed

    Krzyzowska, Małgorzata; Cymerys, Joanna; Winnicka, Anna; Niemiałtowski, Marek

    2006-02-01

    In this study we showed that the virulent Moscow strain of Ectromelia virus (ECTV-MOS) infection leads to induction of apoptosis in the BALB/c mouse central nervous system. ECTV-MOS-infected cells and inflammation sites were found in brain parenchyma between 5 and 15 days after footpad infection with ECTV-MOS. Infected cells consisted of microglia and monocytes, astrocytes and oligodendrocytes and these type of cells underwent apoptosis within 5-15 days post infection (d.p.i.). The highest number of apoptotic cells was found at 5 and 10 d.p.i. and represented mainly microglia (61.4% and 38.6% of apoptotic cells, respectively) and astrocytes (21% and 8.9%, respectively). The number of apoptotic oligodendrocytes was 5.4% and 4.5%, respectively. Fluorometric assays demonstrated involvement of caspase-1, -3 and -8 but not caspase-9 in apoptosis in ECTV-MOS-infected mouse brains. Expression of Fas/FasL was significantly increased on ECTV-MOS-infected cells between 5 and 15 d.p.i., whereas Fas was up-regulated also on the surrounding, non-infected cells. Taking together we may conclude that ECTV-MOS infection of microglia and astrocytes leads to local inflammation resulting in Fas/FasL up-regulation and apoptosis, which limits mouse central nervous system infection with ECTV-MOS.

  15. Extraordinary Sequence Divergence at Tsga8, an X-linked Gene Involved in Mouse Spermiogenesis

    PubMed Central

    Good, Jeffrey M.; Vanderpool, Dan; Smith, Kimberly L.; Nachman, Michael W.

    2011-01-01

    The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion–deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5′ and 3′ ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice. PMID:21186189

  16. Single-cell gene expression analysis reveals diversity among human spermatogonia.

    PubMed

    Neuhaus, N; Yoon, J; Terwort, N; Kliesch, S; Seggewiss, J; Huge, A; Voss, R; Schlatt, S; Grindberg, R V; Schöler, H R

    2017-02-10

    Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. The heterogeneity of human

  17. AZFc deletions do not affect the function of human spermatogonia in vitro.

    PubMed

    Nickkholgh, B; Korver, C M; van Daalen, S K M; van Pelt, A M M; Repping, S

    2015-07-01

    Azoospermic factor c (AZFc) deletions are the underlying cause in 10% of azoo- or severe oligozoospermia. Through extensive molecular analysis the precise genetic content of the AZFc region and the origin of its deletion have been determined. However, little is known about the effect of AZFc deletions on the functionality of germ cells at various developmental steps. The presence of normal, fertilization-competent sperm in the ejaculate and/or testis of the majority of men with AZFc deletions suggests that the process of differentiation from spermatogonial stem cells (SSCs) to mature spermatozoa can take place in the absence of the AZFc region. To determine the functionality of AZFc-deleted spermatogonia, we compared in vitro propagated spermatogonia from six men with complete AZFc deletions with spermatogonia from three normozoospermic controls. We found that spermatogonia of AZFc-deleted men behave similar to controls during culture. Short-term (18 days) and long-term (48 days) culture of AZFc-deleted spermatogonia showed the same characteristics as non-deleted spermatogonia. This similarity was revealed by the same number of passages, the same germ cell clusters formation and similar level of genes expression of spermatogonial markers including ubiquitin carboxyl-terminal esterase L1 (UCHL1), zinc finger and BTB domain containing 16 (ZBTB16) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), as well as germ cell differentiation markers including signal transducer and activator of transcription 3 (STAT3), spermatogenesis and oogenesis specific basic helix-loophelix 2 (SOHLH2), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and synaptonemal complex protein 3 (SYCP3). The only exception was melanoma antigen family A4 (MAGEA4) which showed significantly lower expression in AZFc-deleted samples than controls in short-term culture while in long-term culture it was hardly detected in both AZFc-deleted and control

  18. Differential Gene Expression Profiling of Enriched Human Spermatogonia after Short- and Long-Term Culture

    PubMed Central

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Renninger, Markus; Skutella, Thomas

    2014-01-01

    This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen−/laminin+ matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the “spermatogonial” gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future. PMID:24738045

  19. Effect of continuous gamma-radiation on the stem and differentiating spermatogonia of the adult rat.

    PubMed

    Erickson, B H

    1978-10-01

    Sprague-Dawley rats were irradiated continuously (60Co, gamma) at a dose-rate of either 1 (0.0007 rad/min), 3 or 6 rad/23-h day for monthly intervals of 1 to 6. At one month after irradiation, counts of differentiating spermatogonia (A1 and A4) were reduced to a level that remained essentially unchanged during the succeeding 5 months of irradiation. The magnitude of the irradiation effect was greatest at stage 1 of the spermatogenic cycle where numbers of differentiating spermatogonia were reduced to 50% of control by 1 rad/day and to 30 and 20% of control by 3 and 6 rad/day, respectively. Number of stem spermatogonia was not significantly affected by 1 rad/day (P greater than 0.10). At 3 and 6 rad, however, number of stem cells declined from 80% of control at 1 month to 60 and 40% of control at 6 months, respectively. At a dose-rate of 1 rad/day there was neither a reduction in number of A1 spermatogonia per clone nor significant evidence of necrosis among either stem or differentiating spermatogonia; therefore, a reduction of stem-cell mitotic activity appears to be the principal effect of continuous low-level irradiation on spermatogenesis in the rat.

  20. Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis

    PubMed Central

    Guo, Caixia; Fischhaber, Paula L.; Luk-Paszyc, Margaret J.; Masuda, Yuji; Zhou, Jing; Kamiya, Kenji; Kisker, Caroline; Friedberg, Errol C.

    2003-01-01

    Polκ and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Polκ. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Polζ) and with two other Y-family polymerases, Polι and Polη. Mouse Polκ, Rev7, Polι and Polη each bind to the same ∼100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Polκ for binding to the Rev1 C-terminus. Notwith standing the physical interaction between Rev1 and Polκ, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein–protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined. PMID:14657033

  1. Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes.

    PubMed

    Piano, Ilaria; Novelli, Elena; Della Santina, Luca; Strettoi, Enrica; Cervetto, Luigi; Gargini, Claudia

    2016-01-01

    The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

  2. Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes

    PubMed Central

    Piano, Ilaria; Novelli, Elena; Della Santina, Luca; Strettoi, Enrica; Cervetto, Luigi; Gargini, Claudia

    2016-01-01

    The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis. PMID:26924963

  3. Proteomic analysis of male 4C germ cell proteins involved in mouse meiosis.

    PubMed

    Guo, Xuejiang; Zhang, Ping; Qi, Yujuan; Chen, Wen; Chen, Xiangxiang; Zhou, Zuomin; Sha, Jiahao

    2011-01-01

    Male meiosis is a specialized type of cell division that gives rise to sperm. Errors in this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Until now, there has been a lack of a large-scale identification of proteins involved in male meiosis in mammals. In this study, we report the high-confidence identification of 3625 proteins in mouse male germ cells with 4C DNA content undergoing meiosis I. Of these, 397 were found to be testis specific. Bioinformatics analysis of the proteome led to the identification of 28 proteins known to be essential for male meiosis in mice. We also found 172 proteins that had yeast orthologs known to be essential for meiosis. Chromosome distribution analysis of the proteome showed underrepresentation of the identified proteins on the X chromosome, which may be due to meiotic sex chromosome inactivation. Characterization of the proteome of 4C germ cells from mouse testis provides an inventory of proteins, which is useful for understanding meiosis and the mechanisms of male infertility.

  4. Positive genetic hazard predictions from short-term tests have proved false for results in mammalian spermatogonia with all environmental chemicals so far tested

    SciTech Connect

    Russell, W.L.

    1985-01-01

    Eleven chemicals for which there has been considerable human exposure were studied by the mouse specific-locus method because of their positive mutagenic action in other test systems. All were positive in the Drosophila sex-linked recessive lethal test, and in mammalian somatic cells proved mutagenic. In mouse stem-cell spermatogonia none of the chemicals, even at maximum tolerated dose, has given a specific-locus mutation frequency higher than the control, and the mutation frequency for all eleven combined (12 mutations in 298,502 offspring) was actually less than the historical control. Absence of mutation induction cannot be attributed to: (a) failure of the chemicals to reach the testis (10 of them are known to reach the testis in active form), (b) small sample size (the samples are large), (c) insensitivity of the test (the test is not insensitive: a positive control gave a mutation frequency 132 times higher than the historical control). It is concluded that mammalian stem-cell spermatogonia have an effective repair capability. 9 refs., 1 tab.

  5. Imatinib has deleterious effects on differentiating spermatogonia while sparing spermatogonial stem cell self renewal.

    PubMed

    Heim, Crystal; Minniear, Kayla; Dann, Christina Tenenhaus

    2011-05-01

    Imatinib mesylate is among a growing number of effective cancer drugs that provide molecularly targeted therapy; however, imatinib causes reproductive defects in rodents. The availability of an in vitro system for screening the effect of drugs on spermatogenesis would be beneficial. The imatinib targets, KIT and platelet derived growth factor receptor beta (PDGFRB), were shown here to be expressed in "germline stem" (GS) cell cultures that contain spermatogonia, including spermatogonial stem cells (SSCs). GS cell cultures were utilized to determine whether imatinib affects SSC self renewal or differentiation. GS cells grown in imatinib retained self renewal based on multiple assays, including transplantation. However, growth in imatinib led to decreased numbers of differentiated spermatogonia and reduced culture growth consistent with the known requirement for KIT in survival and proliferation of spermatogonia. These results build upon the in vivo studies and support the possibility of utilizing GS cell cultures for preclinical drug tests.

  6. Dedifferentiating spermatogonia outcompete somatic stem cells for niche occupancy in the Drosophila testis.

    PubMed

    Sheng, X Rebecca; Brawley, Crista M; Matunis, Erika L

    2009-08-07

    Differentiating cells can dedifferentiate to replace stem cells in aged or damaged tissues, but the underlying mechanisms are unknown. In the Drosophila testis, a cluster of stromal cells called the hub creates a niche by locally activating Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling in adjacent germline and somatic stem cells. Here, we establish a system to study spermatogonial dedifferentiation. Ectopically expressing the differentiation factor bag-of-marbles (Bam) removes germline stem cells from the niche. However, withdrawing ectopic Bam causes interconnected spermatogonia to fragment, move into the niche, exchange positions with resident somatic stem cells, and establish contact with the hub. Concomitantly, actin-based protrusions appear on subsets of spermatogonia, suggesting acquired motility. Furthermore, global downregulation of Jak-STAT signaling inhibits dedifferentiation, indicating that normal levels of pathway activation are required to promote movement of spermatogonia into the niche during dedifferentiation, where they outcompete somatic stem cells for niche occupancy.

  7. Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa.

    PubMed

    Vicens, Alberto; Lüke, Lena; Roldan, Eduardo R S

    2014-01-01

    Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

  8. Igf Binding Proteins Protect Undifferentiated Spermatogonia in the Zebrafish Testis Against Excessive Differentiation.

    PubMed

    Safian, Diego; Morais, Roberto D V S; Bogerd, Jan; Schulz, Rüdiger W

    2016-11-01

    IGF binding proteins (IGFBPs) modulate the availability of IGFs for their cognate receptors. In zebrafish testes, IGF3 promotes the proliferation and differentiation of type A undifferentiated (Aund) spermatogonia, and igf3 expression is strongly elevated by FSH but also responds to T3. Here we report the effects of FSH and T3 on igfbp transcript levels in adult zebrafish testis. We then examined T3 and FSH effects on zebrafish spermatogenesis and explored the relevance of IGFBPs in modulating these T3 or FSH effects, using a primary tissue culture system for adult zebrafish testis. T3 up-regulated igfbp1a and igfbp3 expression, whereas FSH reduced igfbp1a transcript levels. To quantify effects on spermatogenesis, we determined the mitotic index and relative section areas occupied by Aund, type A differentiating, or type B spermatogonia. In general, T3 and FSH stimulated spermatogonial proliferation and increased the areas occupied by spermatogonia, suggesting that both self-renewal and differentiating divisions were stimulated. Preventing IGF/IGFBP interaction by NBI-31772 further increased T3- or FSH-induced spermatogonial proliferation. However, under these conditions the more differentiated type A differentiating and B spermatogonia occupied larger surface areas at the expense of the area held by Aund spermatogonia. Clearly decreased nanos2 transcript levels are in agreement with this finding, and reduced amh expression may have facilitated spermatogonial differentiation. We conclude that elevating IGF3 bioactivity by blocking IGFBPs shifted T3- or FSH-induced signaling from stimulating spermatogonial self-renewal as well as differentiation toward predominantly stimulating spermatogonial differentiation, which leads to a depletion of type Aund spermatogonia.

  9. Involvement of Notch1 in the development of mouse mammary tumors.

    PubMed

    Diévart, A; Beaulieu, N; Jolicoeur, P

    1999-10-28

    The MMTV/neu transgenic (Tg) mice spontaneously develop mammary tumors stochastically after a long latent period, suggesting that the c-neu/erbB2 oncogene is not sufficient for tumor formation. To identify putative collaborator(s) of the c-neu/erbB2, we used the provirus insertional mutagenesis approach with mammary tumors arising in MMTV/neu Tg mice infected with the mouse mammary tumor virus (MMTV). The Notch1 gene was identified as a novel target for MMTV provirus insertional activation. In Notch1-rearranged tumors, the Notch1 gene was interrupted by the MMTV provirus insertion upstream of the exons coding for the TM domain. These insertions led to overexpression of novel 5' truncated approximately 7 kb RNA coding for 280 kDa mutant protein harboring only the Notch1 ectodomain, N(EC)mut. These may be involved in tumor formation. Another consequence of these insertions was the expression of truncated 3' Notch1 transcripts (3.5 - 4.5 kb) and proteins (86 - 110 kDa) deleted of most of the extracellular sequences (Notch1intra). We found that 3' truncated Notch1intra can transform HC11 mouse mammary epithelial cells in vitro. Deletion analysis revealed that the ankyrin-repeats and the domain 1 (aa 1751 - 1821) are required, while a signal peptide, the two conserved cysteines (C1652 and C1685) and the OPA and PEST sequences are dispensable for transformation. These results indicate that the N-terminally truncated Notch1intra protein behaves as an oncogene in this system.

  10. Involvement of Stat3 in mouse brain development and sexual dimorphism: a proteomics approach.

    PubMed

    Di Domenico, Fabio; Casalena, Gabriella; Sultana, Rukhsana; Cai, Jian; Pierce, William M; Perluigi, Marzia; Cini, Chiara; Baracca, Alessandra; Solaini, Giancarlo; Lenaz, Giorgio; Jia, Jia; Dziennis, Suzan; Murphy, Stephanie J; Alkayed, Nabil J; Butterfield, D Allan

    2010-11-29

    Although the role of STAT3 in cell physiology and tissue development has been largely investigated, its involvement in the development and maintenance of nervous tissue and in the mechanisms of neuroprotection is not yet known. The potentially wide range of STAT3 activities raises the question of tissue- and gender-specificity as putative mechanisms of regulation. To explore the function of STAT3 in the brain and the hypothesis of a gender-linked modulation of STAT3, we analyzed a neuron-specific STAT3 knockout mouse model investigating the influence of STAT3 activity in brain protein expression pattern in both males and females in the absence of neurological insult. We performed a proteomic study aimed to reveal the molecular pathways directly or indirectly controlled by STAT3 underscoring its role in brain development and maintenance. We identified several proteins, belonging to different neuronal pathways such as energy metabolism or synaptic transmission, controlled by STAT3 that confirm its crucial role in brain development and maintenance. Moreover, we investigated the different processes that could contribute to the sexual dimorphic behavior observed in the incidence of neurological and mental disease. Interestingly both STAT3 KO and gender factors influence the expression of several mitochondrial proteins conferring to mitochondrial activity high importance in the regulation of brain physiology and conceivable relevance as therapeutic target.

  11. Involvement of activin signaling in abnormalities of mouse vagina exposed neonatally to diethylstilbestrol.

    PubMed

    Nakajima, Tadaaki; Iguchi, Taisen; Sato, Tomomi

    2011-06-01

    Perinatal exposure to a synthetic estrogen, diethylstilbestrol (DES), causes cervicovaginal adenosis and permanent hyperplastic cornified vaginal epithelium with keratinization in mice. To investigate the mechanisms of the induction of vaginal abnormalities by DES, we have focused on activin A signaling. We have found that the βA-subunit mRNA is mainly expressed in the neonatal vaginal stroma, whereas activin A receptor type IB is localized in the neonatal vaginal epithelium. SMAD2, the intracellular signaling protein, is phosphorylated in the neonatal vagina. Cell proliferation in the vaginal epithelium grown in vitro is reduced by DES treatment or by activin signaling suppression through inhibin treatment. Thus, activin A (a homodimer of the βA-subunit) in the stroma stimulates epithelial cell proliferation in the neonatal vagina. DES treatment decreases the expression of the βA-subunit and activin receptor IIB but increases the expression of the βB-subunit and inhibin receptor. Neonatal DES treatment inhibits the phosphorylation of SMAD2 in the vaginal epithelium, indicating the inhibition of activin A signaling in the vaginal epithelium by neonatal DES treatment. Treatment with DES or inhibin, a native antagonist of activin, induces adenosis-like structures and keratinization in the vagina grown in vitro. These data suggest that the suppression of activin A signaling by DES is involved in the induction of cervicovaginal adenosis and keratinization in the neonatal mouse vaginal epithelium.

  12. INVOLVEMENT OF STAT3 IN MOUSE BRAIN DEVELOPMENT AND SEXUAL DIMORPHISM: A PROTEOMICS APPROACH

    PubMed Central

    Di Domenico, Fabio; Casalena, Gabriella; Sultana, Rukhsana; Cai, Jian; Pierce, William M.; Perluigi, Marzia; Cini, Chiara; Baracca, Alessandra; Solaini, Giancarlo; Lenaz, Giorgio; Jia, Jia; Dziennis, Suzan; Murphy, Stephanie J.; Alkayed, Nabil J.; Butterfield, D. Allan

    2010-01-01

    Although the role of STAT3 in cell physiology and tissue development has been largely investigated, its involvement in the development and maintenance of nervous tissue and in the mechanisms of neuroprotection is not yet known. The potentially wide range of STAT3 activities raise the question of tissue- and gender-specificity as putative mechanisms of regulation. To explore the function of STAT3 in the brain and the hypothesis of a gender-linked modulation of STAT3, we analyzed a neuron-specific STAT3 knockout mouse model investigating the influence of STAT3 activity in brain protein expression pattern in both males and females in the absence of neurological insult. We performed a proteomic study aimed to reveal the molecular pathways directly or indirectly controlled by STAT3 underscoring its role in brain development and maintenance. We identified several proteins, belonging to different neuronal pathways such as energy metabolism or synaptic transmission, controlled by STAT3 that confirm its crucial role in brain development and maintenance. Moreover, we investigated the different processes that could contribute to the sexual dimorphic behavior observed in the incidence of neurological and mental disease. Interestingly both STAT3 KO and gender factors influence the expression of several mitochondrial proteins conferring to mitochondrial activity high importance in the regulation of brain physiology and conceivable relevance as therapeutic target. PMID:20875800

  13. Phosphodiesterase isozymes involved in regulating acid secretion in the isolated mouse stomach.

    PubMed

    Okuda, S; Honda, M; Ito, Y; Aihara, E; Kato, S; Mitsufuji, S; Yoshikawa, T; Takeuchi, K

    2009-12-01

    The effect of subtype-selective phosphodiesterase (PDE) inhibitors on acid secretion was examined in mouse stomachs to investigate which PDE isozymes are involved in the local regulation of this secretion. Male DDY mice were used after 18 h fasting. An isolated stomach was incubated in an organ bath containing buffered solution gassed with 95% O(2)/5% CO(2), while the lumen was perfused with unbuffered solution gassed with 100% O(2). Acid secretion was measured at pH 5.4 using a pH-stat method. Histamine or pituitary adenylate cyclase activating polypeptide (PACAP) was added to the serosal solution. PDE inhibitors were added to the serosal solution 30 min before histamine or PACAP. The secretion of acid in the isolated stomach was increased by histamine or PACAP, and these responses were totally inhibited by famotidine. IBMX alone increased basal acid secretion and significantly enhanced the acid responses to histamine and PACAP. Among the PDE inhibitors tested, only rolipram (PDE4 inhibitor) significantly increased basal acid secretion and potentiated the acid responses to histamine and PACAP. The latter peptide increased histamine release into the medium, and this response was also enhanced by rolipram. Furthermore, rolipram significantly increased cAMP production induced in the isolated stomach by histamine and PACAP. These results suggest that PDE4 is involved in the local regulation of gastric acid secretion via the degradation of cAMP and that the PDE4 inhibitor rolipram increases the secretion of acid by potentiating acid production in parietal cells and enhancing histamine release from enterochromaffin-like cells.

  14. High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells.

    PubMed

    Pech, Matthew F; Garbuzov, Alina; Hasegawa, Kazuteru; Sukhwani, Meena; Zhang, Ruixuan J; Benayoun, Bérénice A; Brockman, Stephanie A; Lin, Shengda; Brunet, Anne; Orwig, Kyle E; Artandi, Steven E

    2015-12-01

    Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans. © 2015 Pech et al.; Published by Cold Spring Harbor Laboratory Press.

  15. Prion Infection of Mouse Brain Reveals Multiple New Upregulated Genes Involved in Neuroinflammation or Signal Transduction

    PubMed Central

    Striebel, James F.; Race, Brent; Phillips, Katie; Chesebro, Bruce

    2014-01-01

    ABSTRACT Gliosis is often a preclinical pathological finding in neurodegenerative diseases, including prion diseases, but the mechanisms facilitating gliosis and neuronal damage in these diseases are not understood. To expand our knowledge of the neuroinflammatory response in prion diseases, we assessed the expression of key genes and proteins involved in the inflammatory response and signal transduction in mouse brain at various times after scrapie infection. In brains of scrapie-infected mice at pre- and postclinical stages, we identified 15 previously unreported differentially expressed genes related to inflammation or activation of the STAT signal transduction pathway. Levels for the majority of differentially expressed genes increased with time postinfection. In quantitative immunoblotting experiments of STAT proteins, STAT1α, phosphorylated-STAT1α (pSTAT1α), and pSTAT3 were increased between 94 and 131 days postinfection (p.i.) in brains of mice infected with strain 22L. Furthermore, a select group of STAT-associated genes was increased preclinically during scrapie infection, suggesting early activation of the STAT signal transduction pathway. Comparison of inflammatory markers between mice infected with scrapie strains 22L and RML indicated that the inflammatory responses and gene expression profiles in the brains were strikingly similar, even though these scrapie strains infect different brain regions. The endogenous interleukin-1 receptor antagonist (IL-1Ra), an inflammatory marker, was newly identified as increasing preclinically in our model and therefore might influence scrapie pathogenesis in vivo. However, in IL-1Ra-deficient or overexpressor transgenic mice inoculated with scrapie, neither loss nor overexpression of IL-1Ra demonstrated any observable effect on gliosis, protease-resistant prion protein (PrPres) formation, disease tempo, pathology, or expression of the inflammatory genes analyzed. IMPORTANCE Prion infection leads to Pr

  16. L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding.

    PubMed

    Hubbe, M; Kowitz, A; Schirrmacher, V; Schachner, M; Altevogt, P

    1993-11-01

    L1 is a cell surface glycoprotein of the immunoglobulin superfamily which was initially shown to mediate adhesion between neural cells. Recently we have reported that L1 is expressed by bone marrow cells and the majority of mature lymphocytes (Kowitz et al., Eur. J. Immunol. 1992. 22: 1199-1205). To analyze the function of L1 on leukocytes we studied its regulation following cell activation. In vitro activation of B lymphocytes with lipopolysaccharide or T lymphocytes with phorbol 12-myristate 13-acetate/Ca2+ ionophore, concanavalin A or anti-CD3 monoclonal antibody as well as in vivo activation of V beta 8+ T cells with staphylococcal enterotoxin B (SEB) revealed a down-regulation of L1 within 48 h. A rapid loss of L1 expression was seen when mouse neutrophils were activated with PMA alone. This rapid loss paralleled the shedding of L-selectin. We also studied a possible role of L1 in the binding of leukocytes to endothelial cells. ESb-MP lymphoma cells with a high expression of L1 (L1hi) could bind to bend3 endothelioma cells without prior activation with inflammatory cytokines. The interaction was inhibited by anti-L1 antibodies. In contrast, ESb-MP cells with low L1 expression (L1lo) were only marginally bound. Latex beads coated with affinity-isolated L1 antigen were also able to bind to the endothelioma cells in a specific fashion. The binding of ESb-MP lymphoma cells required Ca2+ and Mg2+ ions and was sensitive to cold temperature. Since the endothelioma cells did not express L1 the binding mechanism studied here is distinct from the established L1-L1 homotypic interaction. It is possible that the novel L1-mediated adhesion pathway involves an unidentified ligand and could play a role in leukocyte migration.

  17. Involvement of catecholaminergic neurons in motor innervation of striated muscle in the mouse esophagus.

    PubMed

    van der Keylen, Piet; Garreis, Fabian; Steigleder, Ruth; Sommer, Daniel; Neuhuber, Winfried L; Wörl, Jürgen

    2016-05-01

    Enteric co-innervation is a peculiar innervation pattern of striated esophageal musculature. Both anatomical and functional data on enteric co-innervation related to various transmitters have been collected in different species, although its function remains enigmatic. However, it is unclear whether catecholaminergic components are involved in such a co-innervation. Thus, we examined to identify catecholaminergic neuronal elements and clarify their relationship to other innervation components in the esophagus, using immunohistochemistry with antibodies against tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT) and protein gene product 9.5 (PGP 9.5), α-bungarotoxin (α-BT) and PCR with primers for amplification of cDNA encoding TH and dopamine-β-hydroxylase (DBH). TH-positive nerve fibers were abundant throughout the myenteric plexus and localized on about 14% of α-BT-labelled motor endplates differing from VAChT-positive vagal nerve terminals. TH-positive perikarya represented a subpopulation of only about 2.8% of all PGP 9.5-positive myenteric neurons. Analysis of mRNA showed both TH and DBH transcripts in the mouse esophagus. As ChAT-positive neurons in the compact formation of the nucleus ambiguus were negative for TH, the TH-positive nerve varicosities on motor endplates are presumably of enteric origin, although a sympathetic origin cannot be excluded. In the medulla oblongata, the cholinergic ambiguus neurons were densely supplied with TH-positive varicosities. Thus, catecholamines may modulate vagal motor innervation of esophageal-striated muscles not only at the peripheral level via enteric co-innervation but also at the central level via projections to the nucleus ambiguus. As Parkinson's disease, with a loss of central dopaminergic neurons, also affects the enteric nervous system and dysphagia is prevalent in patients with this disease, investigation of intrinsic catecholamines in the esophagus may

  18. Involvement of posttranscriptional regulation of Clock in the emergence of circadian clock oscillation during mouse development

    PubMed Central

    Umemura, Yasuhiro; Koike, Nobuya; Ohashi, Munehiro; Tsuchiya, Yoshiki; Meng, Qing Jun; Minami, Yoichi; Hara, Masayuki; Hisatomi, Moe; Yagita, Kazuhiro

    2017-01-01

    Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10–12 hearts (63, no core circadian genes) than in E17–19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development. PMID:28827343

  19. Involvement of posttranscriptional regulation of Clock in the emergence of circadian clock oscillation during mouse development.

    PubMed

    Umemura, Yasuhiro; Koike, Nobuya; Ohashi, Munehiro; Tsuchiya, Yoshiki; Meng, Qing Jun; Minami, Yoichi; Hara, Masayuki; Hisatomi, Moe; Yagita, Kazuhiro

    2017-09-05

    Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10-12 hearts (63, no core circadian genes) than in E17-19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development.

  20. Acute Inflammation Loci Are Involved in Wound Healing in the Mouse Ear Punch Model.

    PubMed

    Canhamero, Tatiane; Garcia, Ludmila Valino; De Franco, Marcelo

    2014-09-01

    Significance: Molecular biology techniques are being used to aid in determining the mechanisms responsible for tissue repair without scar formation. Wound healing is genetically determined, but there have been few studies that examine the genes responsible for tissue regeneration in mammals. Research using genetic mapping is extremely important for understanding the molecular mechanisms involved in the different phases of tissue regeneration. This process is complex, but an early inflammatory phase appears to influence lesion closure, and the present study demonstrates that acute inflammation loci influence tissue regeneration in mice in a positive manner. Recent Advances: Mapping studies of quantitative trait loci (QTL) have been undertaken in recent years to examine candidate genes that participate in the regeneration phenotype. Our laboratory has identified inflammation modifier QTL for wound healing. Mouse lines selected for the maximum (AIRmax) or minimum (AIRmin) acute inflammatory reactivity (AIR) have been used to study not only the tissue repair but also the impact of the genetic control of inflammation on susceptibility to autoimmune, neoplasic, and infectious diseases. Murphy Roths Large and AIRmax mice are exclusive in their complete epimorphic regeneration, although middle-aged inbred mice may also be capable of healing. Critical Issues: Inflammatory reactions have traditionally been described in the literature as negative factors in the process of skin injury closure. Inflammation is exacerbated due to the early release of mediators or the intense release of factors that cause cell proliferation after injury. The initial release of these factors as well as the clean-up of the lesion microenvironment are both crucial for following events. In addition, the activation and repression of some genes related to the regeneration phenotype may modulate lesion closure, demonstrating the significance of genetic studies to better understand the mechanisms

  1. Acute Inflammation Loci Are Involved in Wound Healing in the Mouse Ear Punch Model

    PubMed Central

    Canhamero, Tatiane; Garcia, Ludmila Valino; De Franco, Marcelo

    2014-01-01

    Significance: Molecular biology techniques are being used to aid in determining the mechanisms responsible for tissue repair without scar formation. Wound healing is genetically determined, but there have been few studies that examine the genes responsible for tissue regeneration in mammals. Research using genetic mapping is extremely important for understanding the molecular mechanisms involved in the different phases of tissue regeneration. This process is complex, but an early inflammatory phase appears to influence lesion closure, and the present study demonstrates that acute inflammation loci influence tissue regeneration in mice in a positive manner. Recent Advances: Mapping studies of quantitative trait loci (QTL) have been undertaken in recent years to examine candidate genes that participate in the regeneration phenotype. Our laboratory has identified inflammation modifier QTL for wound healing. Mouse lines selected for the maximum (AIRmax) or minimum (AIRmin) acute inflammatory reactivity (AIR) have been used to study not only the tissue repair but also the impact of the genetic control of inflammation on susceptibility to autoimmune, neoplasic, and infectious diseases. Murphy Roths Large and AIRmax mice are exclusive in their complete epimorphic regeneration, although middle-aged inbred mice may also be capable of healing. Critical Issues: Inflammatory reactions have traditionally been described in the literature as negative factors in the process of skin injury closure. Inflammation is exacerbated due to the early release of mediators or the intense release of factors that cause cell proliferation after injury. The initial release of these factors as well as the clean-up of the lesion microenvironment are both crucial for following events. In addition, the activation and repression of some genes related to the regeneration phenotype may modulate lesion closure, demonstrating the significance of genetic studies to better understand the mechanisms

  2. Digital Gene Expression Tag Profiling Analysis of the Gene Expression Patterns Regulating the Early Stage of Mouse Spermatogenesis

    PubMed Central

    Meng, Lijun; Liu, Meiling; Zhao, Lina; Hu, Fen; Ding, Cunbao; Wang, Yang; He, Baoling; Pan, Yuxin; Fang, Wei; Chen, Jing; Hu, Songnian; Jia, Mengchun

    2013-01-01

    Detailed characterization of the gene expression patterns in spermatogonia and primary spermatocytes is critical to understand the processes which occur prior to meiosis during normal spermatogenesis. The genome-wide expression profiles of mouse type B spermatogonia and primary spermatocytes were investigated using the Solexa/Illumina digital gene expression (DGE) system, a tag based high-throughput transcriptome sequencing method, and the developmental processes which occur during early spermatogenesis were systematically analyzed. Gene expression patterns vary significantly between mouse type B spermatogonia and primary spermatocytes. The functional analysis revealed that genes related to junction assembly, regulation of the actin cytoskeleton and pluripotency were most significantly differently expressed. Pathway analysis indicated that the Wnt non-canonical signaling pathway played a central role and interacted with the actin filament organization pathway during the development of spermatogonia. This study provides a foundation for further analysis of the gene expression patterns and signaling pathways which regulate the molecular mechanisms of early spermatogenesis. PMID:23554914

  3. The KIT is a putative marker for differentiating spermatogonia in stallions.

    PubMed

    Jung, Heejun; Song, Hyuk; Yoon, Minjung

    2015-01-01

    Putative markers have been discovered and are used to identify and separate certain lineage of spermatogonia. The KIT is a marker for differentiating spermatogonial stem cells in several species including mice and goats. The objectives of this study were (1) to investigate reproductive stage-dependent KIT expression patterns in stallions and (2) to identify spermatogonia subpopulations expressing KIT in stallion testes. To achieve these objectives, testicular samples were obtained during routine field castration of stallions. The reproductive stage of the stallions was classified as pre-pubertal (<1 year, n=3), pubertal (1-1.5 year, n=4), post-pubertal (2-3 year, n=6), or adult (4-8 year, n=6). For immunohistochemistry, KIT was used at a dilution of 1:200. In the pre-pubertal and pubertal stage, most germ cells were immunolabeled with KIT. In the post-pubertal and adult stages, immunolabeling of KIT was evident in the germ cells attached to the basement membrane of the seminiferous tubules with exception of some spermatogonia. Co-immunolabeling with KIT and deleted in azoospermia like (DAZL) showed different co-staining patterns, including KIT only, both KIT and DAZL), or DAZL positive germ cell populations alone. The KIT was not immunolabeled in Sertoli or Leydig cells at any reproductive stages. The result of Western blot analysis verified the cross-activity of the KIT antibody with horse testes tissue. In conclusion, KIT appears to be expressed in differentiating spermatogonia, and may be used to identify and isolate differentiating germ cells from stallions.

  4. An epigenetic switch is crucial for spermatogonia to exit the undifferentiated state toward a Kit-positive identity.

    PubMed

    Shirakawa, Takayuki; Yaman-Deveci, Ruken; Tomizawa, Shin-Ichi; Kamizato, Yoshito; Nakajima, Kuniko; Sone, Hidetoshi; Sato, Yasuyuki; Sharif, Jafar; Yamashita, Akio; Takada-Horisawa, Yuki; Yoshida, Shosei; Ura, Kiyoe; Muto, Masahiro; Koseki, Haruhiko; Suda, Toshio; Ohbo, Kazuyuki

    2013-09-01

    Epigenetic modifications influence gene expression and chromatin remodeling. In embryonic pluripotent stem cells, these epigenetic modifications have been extensively characterized; by contrast, the epigenetic events of tissue-specific stem cells are poorly understood. Here, we define a new epigenetic shift that is crucial for differentiation of murine spermatogonia toward meiosis. We have exploited a property of incomplete cytokinesis, which causes male germ cells to form aligned chains of characteristic lengths, as they divide and differentiate. These chains revealed the stage of spermatogenesis, so the epigenetic differences of various stages could be characterized. Single, paired and medium chain-length spermatogonia not expressing Kit (a marker of differentiating spermatogonia) showed no expression of Dnmt3a2 and Dnmt3b (two de novo DNA methyltransferases); they also lacked the transcriptionally repressive histone modification H3K9me2. By contrast, spermatogonia consisting of ~8-16 chained cells with Kit expression dramatically upregulated Dnmt3a2/3b expression and also displayed increased H3K9me2 modification. To explore the function of these epigenetic changes in spermatogonia in vivo, the DNA methylation machinery was destabilized by ectopic Dnmt3b expression or Np95 ablation. Forced Dnmt3b expression induced expression of Kit; whereas ablation of Np95, which is essential for maintaining DNA methylation, interfered with differentiation and viability only after spermatogonia become Kit positive. These data suggest that the epigenetic status of spermatogonia shifts dramatically during the Kit-negative to Kit-positive transition. This shift might serve as a switch that determines whether spermatogonia self-renew or differentiate.

  5. Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

    PubMed

    Raverdeau, Mathilde; Gely-Pernot, Aurore; Féret, Betty; Dennefeld, Christine; Benoit, Gérard; Davidson, Irwin; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

    2012-10-09

    Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

  6. Direct Transdifferentiation of Stem/Progenitor Spermatogonia Into Reproductive and Nonreproductive Tissues of All Germ Layers

    PubMed Central

    Simon, Liz; Ekman, Gail C.; Kostereva, Natalia; Zhang, Zhen; Hess, Rex A.; Hofmann, Marie-Claude; Cooke, Paul S.

    2010-01-01

    Pluripotent stem cells have great clinical potential for tissue regeneration/repair in humans. The use of embryonic stem (ES) cells is ethically controversial, leading to searches for other sources of pluripotent stem cells. Testicular spermatogonial stem cells (SSCs) produce the spermatogenic lineage. Under in vitro conditions, SSCs have the ability to give rise to pluripotent ES-like cells. We hypothesized that stem/progenitor spermatogonia could directly transdifferentiate into different tissue types if they were recombined with inductive mesenchymes from fetal/neonatal organs using a tissue separation/recombination methodology and grown in vivo. Green fluorescent protein transgenic mice were used to track cell lineages. Our results indicate that stem/progenitor spermatogonia recombined with the appropriate mesenchyme can directly transdifferentiate in vivo into tissues of all germ layers, including prostatic, uterine, and skin epithelium. In addition, transdifferentiated tissue expressed molecular, histological, and functional markers of the appropriate epithelium. The ability of stem/progenitor spermatogonia to directly generate various epithelia emphasizes their clinical potential, and if adult human SSCs have similar properties, this may have applications in human regenerative medicine. PMID:19544441

  7. Survival and renewal of murine stem Spermatogonia following /sup 60/Co radiation

    SciTech Connect

    Erickson, B.H.

    1981-04-01

    Spermatogonia, with the exception of the few that are either isolated or paired, exist in clones of varying size, and even though the spermatogonium has been the object of much study, our understanding of the radioresponse of the components of this triumvirate is incomplete. Isolated, paired, and clonal spermatogonia are distinguished most reliably when viewed in segments of seminiferous tubules mounted in toto. Tubular segments were isolated from the testes of 80- to 90-day-old BALB/c mice following irradiation doses of 25 to 800 rad and intervals of 1 to 14 days. Neither isolated nor paired spermatogonia were apparently affected by doses up to 200 rad. In contrast the population of A/sub 1/-spermatogonial clones was markedly reduced by a dose of 25 rad. The D/sub 0/ and n for the clone were 217 rad and 0.5, respectively. In the apparent absence of a significant elevation in necrosis at doses of 20 rad and less, the loss of A/sub 1/ clones was attributed to mitotic inhibition. The comparative unresponsiveness of the isolated spermatogonium to irradiation, its low mitotic rate when contrasted with the clone's high radiosensitivity and mitotic activity, and its apparent ability to excise an abnormal cell and otherwise fragment to give rise to additional clones led to the tentative conclusion that the clone, and not the isolated spermatogonium, is the stem cell.

  8. A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory.

    PubMed

    Ostroveanu, Anghelus; Van der Zee, Eddy A; Dolga, Amalia M; Luiten, Paul G M; Eisel, Ulrich L M; Nijholt, Ingrid M

    2007-05-11

    A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to neuronal membranes and postsynaptic densities and plays an important role in synaptic function. Several studies suggested that AKAP79/150 anchored PKA contributes to mechanisms associated with synaptic plasticity and memory processes, but the precise role of AKAPs in these processes is still unknown. In this study we established the mouse brain distribution of AKAP150 using two well-characterized AKAP150 antibodies. Using Western blotting and immunohistochemistry we showed that AKAP150 is widely distributed throughout the mouse brain. The highest AKAP150 expression levels were observed in striatum, cerebral cortex and several other forebrain regions (e.g. olfactory tubercle), relatively high expression was found in hippocampus and olfactory bulb and low/no expression in cerebellum, hypothalamus, thalamus and brain stem. Although there were some minor differences in mouse AKAP150 brain distribution compared to the distribution in rat brain, our data suggested that rodents have a characteristic AKAP150 brain distribution pattern. In general we observed that AKAP150 is strongly expressed in mouse brain regions involved in learning and memory. These data support its suggested role in synaptic plasticity and memory processes.

  9. Temporal and spatial mouse brain expression of cereblon, an ionic channel regulator involved in human intelligence.

    PubMed

    Higgins, Joseph J; Tal, Adit L; Sun, Xiaowei; Hauck, Stefanie C R; Hao, Jin; Kosofosky, Barry E; Rajadhyaksha, Anjali M

    2010-03-01

    A mild form of autosomal recessive, nonsyndromal intellectual disability (ARNSID) in humans is caused by a homozygous nonsense mutation in the cereblon gene (mutCRBN). Rodent crbn protein binds to the intracellular C-terminus of the large conductance Ca(2+)-activated K(+)channel (BK(Ca)). An mRNA variant (human SITE 2 INSERT or mouse strex) of the BK(Ca) gene (KCNMA1) that is normally expressed during embryonic development is aberrantly expressed in mutCRBN human lymphoblastoid cell lines (LCLs) as compared to wild-type (wt) LCLs. The present study analyzes the temporal and spatial distribution of crbn and kcnma1 mRNAs in the mouse brain by the quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR). The spatial expression pattern of endogenous and exogenous crbn proteins is characterized by immunostaining. The results show that neocortical (CTX) crbn and kcnma1 mRNA expression increases from embryonic stages to adulthood. The strex mRNA variant is >3.5-fold higher in embryos and decreases rapidly postnatally. Mouse crbn mRNA is abundant in the cerebellum (CRBM), with less expression in the CTX, hippocampus (HC), and striatum (Str) in adult mice. The intracytoplasmic distribution of endogenous crbn protein in the mouse CRBM, CTX, HC, and Str is similar to the immunostaining pattern described previously for the BK(Ca) channel. Exogenous hemagglutinin (HA) epitope-tagged human wt- and mutCRBN proteins using cDNA transfection in HEK293T cell lines showed the same intracellular expression distribution as endogenous mouse crbn protein. The results suggest that mutCRBN may cause ARNSID by disrupting the developmental regulation of BK(Ca) in brain regions that are critical for memory and learning.

  10. Conotruncal anomalies in the trisomy 16 mouse: an immunohistochemical analysis with emphasis on the involvement of the neural crest.

    PubMed

    Waller, B R; McQuinn, T; Phelps, A L; Markwald, R R; Lo, C W; Thompson, R P; Wessels, A

    2000-11-01

    The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse.

  11. The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

    PubMed

    Ramaswamy, Suresh; Walker, William H; Aliberti, Paula; Sethi, Rahil; Marshall, Gary R; Smith, Alyxzandria; Nourashrafeddin, Seyedmehdi; Belgorosky, Alicia; Chandran, Uma R; Hedger, Mark P; Plant, Tony M

    2017-10-01

    were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These

  12. FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix.

    PubMed

    Ito, R; Abé, S I

    1999-03-01

    We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.

  13. Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

    PubMed

    Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

    2005-08-01

    This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

  14. Cardiac fibrosis in mouse expressing DsRed tetramers involves chronic autophagy and proteasome degradation insufficiency

    PubMed Central

    Chen, Tsung-Hsien; Chen, Mei-Ru; Chen, Tzu-Yin; Wu, Tzu-Chin; Liu, Shan-Wen; Hsu, Ching-Han; Liou, Gan-Guang; Kao, Yu-Ying; Dong, Guo-Chung; Chu, Pao-Hsien; Liao, Jiunn-Wang; Lin, Kurt Ming-Chao

    2016-01-01

    Proteinopathy in the heart which often manifests excessive misfolded/aggregated proteins in cardiac myocytes can result in severe fibrosis and heart failure. Here we developed a mouse model, which transgenically express tetrameric DsRed, a red fluorescent protein (RFP), in an attempt to mimic the pathological mechanisms ofcardiac fibrosis. Whilst DsRed is expressed and forms aggregation in most mouse organs, certain pathological defects are specifically recapitulated in cardiac muscle cells including mitochondria damages, aggresome-like residual bodies, excessive ubiquitinated proteins, and the induction of autophagy. The proteinopathy and cellular injuries caused by DsRed aggregates may be due to impaired or overburdened ubiquitin-proteasome system and autophagy-lysosome systems. We further identified that DsRed can be ubiquitinated and associated with MuRF1, a muscle-specific E3 ligase. Concomitantly, an activation of NF-κB signaling and a strong TIMP1 induction were noted, suggesting that RFP-induced fibrosis was augmented by a skewed balance between TIMP1 and MMPs. Taken together, our study highlights the molecular consequences of uncontrolled protein aggregation leading to congestive heart failure, and provides novel insights into fibrosis formation that can be exploited for improved therapy. PMID:27494843

  15. Gene repressive mechanisms in the mouse brain involved in memory formation.

    PubMed

    Yu, Nam-Kyung; Kaang, Bong-Kiun

    2016-04-01

    Gene regulation in the brain is essential for long-term plasticity and memory formation. Despite this established notion, the quantitative translational map in the brain during memory formation has not been reported. To systematically probe the changes in protein synthesis during memory formation, our recent study exploited ribosome profiling using the mouse hippocampal tissues at multiple time points after a learning event. Analysis of the resulting database revealed novel types of gene regulation after learning. First, the translation of a group of genes was rapidly suppressed without change in mRNA levels. At later time points, the expression of another group of genes was downregulated through reduction in mRNA levels. This reduction was predicted to be downstream of inhibition of ESR1 (Estrogen Receptor 1) signaling. Overexpressing Nrsn1, one of the genes whose translation was suppressed, or activating ESR1 by injecting an agonist interfered with memory formation, suggesting the functional importance of these findings. Moreover, the translation of genes encoding the translational machineries was found to be suppressed, among other genes in the mouse hippocampus. Together, this unbiased approach has revealed previously unidentified characteristics of gene regulation in the brain and highlighted the importance of repressive controls. [BMB Reports 2016; 49(4): 199-200].

  16. Failure of spermatogenesis to recover despite the presence of a spermatogonia in the irradiated LBNF1 rat.

    PubMed

    Kangasniemi, M; Huhtaniemi, I; Meistrich, M L

    1996-06-01

    The dose and time response of LBNF1, rat testis to gamma irradiation was studied with use of single doses from 2.5 Gy to 6.0 Gy. Germ cells were initially depleted as a result of killing the radiosensitive differentiating spermatogonia. Some recovery of spermatogenesis was observed at 4 and 6 wk after irradiation as indicated by the repopulation of tubules with germ cells derived from surviving stem spermatogonia. Although spermatogenesis showed additional recovery and was maintained throughout the 60-wk follow-up period after 2.5 Gy, at doses from 3.5 Gy to 6.0 Gy, repopulation indices declined after 6 wk to less than 2%. The numbers of Sertoli cells per nonrepopulating tubule were constant, independent of radiation dose or time. In addition, the nonrepopulating tubules contained an average of one A spermatogonium per 100 Sertoli cells. The size and shape of these cells corresponded to undifferentiated A spermatogonia in nonirradiated control tests. Despite high labeling (40%) and mitotic (20%) indices, the numbers of A spermatogonia changed very little with time, and no differentiated cells were produced in these tubules. The failure of spermatogenesis to recover was not due to hormone deficiency: serum gonadotropin levels increased after irradiation, and serum testosterone remained at control levels. The irradiated LBNF1 rat model may be useful for studying the regulation of differentiation of A spermatogonia.

  17. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells.

    PubMed

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio; Campagnolo, Luisa

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.

  18. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    SciTech Connect

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio; Campagnolo, Luisa

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75{sup NTR}), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75{sup NTR} and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75{sup NTR} and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75{sup NTR}/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75{sup NTR} or TrkA. Interestingly, immunoreactivity to anti-p75{sup NTR} antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75{sup NTR}, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75{sup NTR} is turned on.

  19. Transcriptome analysis of genes and gene networks involved in aggressive behavior in mouse and zebrafish.

    PubMed

    Malki, Karim; Du Rietz, Ebba; Crusio, Wim E; Pain, Oliver; Paya-Cano, Jose; Karadaghi, Rezhaw L; Sluyter, Frans; de Boer, Sietse F; Sandnabba, Kenneth; Schalkwyk, Leonard C; Asherson, Philip; Tosto, Maria Grazia

    2016-09-01

    Despite moderate heritability estimates, the molecular architecture of aggressive behavior remains poorly characterized. This study compared gene expression profiles from a genetic mouse model of aggression with zebrafish, an animal model traditionally used to study aggression. A meta-analytic, cross-species approach was used to identify genomic variants associated with aggressive behavior. The Rankprod algorithm was used to evaluated mRNA differences from prefrontal cortex tissues of three sets of mouse lines (N = 18) selectively bred for low and high aggressive behavior (SAL/LAL, TA/TNA, and NC900/NC100). The same approach was used to evaluate mRNA differences in zebrafish (N = 12) exposed to aggressive or non-aggressive social encounters. Results were compared to uncover genes consistently implicated in aggression across both studies. Seventy-six genes were differentially expressed (PFP < 0.05) in aggressive compared to non-aggressive mice. Seventy genes were differentially expressed in zebrafish exposed to a fight encounter compared to isolated zebrafish. Seven genes (Fos, Dusp1, Hdac4, Ier2, Bdnf, Btg2, and Nr4a1) were differentially expressed across both species 5 of which belonging to a gene-network centred on the c-Fos gene hub. Network analysis revealed an association with the MAPK signaling cascade. In human studies HDAC4 haploinsufficiency is a key genetic mechanism associated with brachydactyly mental retardation syndrome (BDMR), which is associated with aggressive behaviors. Moreover, the HDAC4 receptor is a drug target for valproic acid, which is being employed as an effective pharmacological treatment for aggressive behavior in geriatric, psychiatric, and brain-injury patients. © 2016 Wiley Periodicals, Inc.

  20. Circadian abnormalities in mouse models of Smith-Magenis syndrome: evidence for involvement of RAI1.

    PubMed

    Lacaria, Melanie; Gu, Wenli; Lupski, James R

    2013-07-01

    Smith-Magenis syndrome (SMS; OMIM 182290) is a genomic disorder characterized by multiple congenital anomalies, intellectual disability, behavioral abnormalities, and disordered sleep resulting from an ~3.7 Mb deletion copy number variant (CNV) on chromosome 17p11.2 or from point mutations in the gene RAI1. The reciprocal duplication of this region results in another genomic disorder, Potocki-Lupski syndrome (PTLS; OMIM 610883), characterized by autism, intellectual disability, and congenital anomalies. We previously used chromosome-engineering and gene targeting to generate mouse models for PTLS (Dp(11)17/+), and SMS due to either deletion CNV or gene knock-out (Df(11)17-2/+ and Rai1(+/-) , respectively) and we observed phenotypes in these mouse models consistent with their associated human syndromes. To investigate the contribution of individual genes to the circadian phenotypes observed in SMS, we now report the analysis of free-running period lengths in Rai1(+/-) and Df(11)17-2/+ mice, as well as in mice deficient for another known circadian gene mapping within the commonly deleted/duplicated region, Dexras1, and we compare these results to those previously observed in Dp(11)17/+ mice. Reduced free-running period lengths were seen in Df(11)17-2/+, Rai1(+/-) , and Dexras1(-/-) , but not Dexras1(+/-) mice, suggesting that Rai1 may be the primary gene underlying the circadian defects in SMS. However, we cannot rule out the possibility that cis effects between multiple haploinsufficient genes in the SMS critical interval (e.g., RAI1 and DEXRAS1) either exacerbate the circadian phenotypes observed in SMS patients with deletions or increase their penetrance in certain environments. This study also confirms a previous report of abnormal circadian function in Dexras1(-/-) mice.

  1. Vitamin B12-induced spermatogenesis recovery in cimetidine-treated rats: effect on the spermatogonia number and sperm concentration.

    PubMed

    Beltrame, Flávia L; Sasso-Cerri, Estela

    2017-01-01

    The H2-receptor antagonist cimetidine is an antiulcer drug also used for the treatment of cancer due to its antiangiogenic effect. However, this drug has caused structural changes in the seminiferous tubules. Vitamin B12 has been used as a therapeutic agent for the treatment of male infertility. The supplementation of rats with vitamin B12 during cimetidine treatment has recovered the damaged seminiferous tubules, but how this vitamin restores the seminiferous epithelium has not been clarified. In this study, we evaluated whether vitamin B12 improves the number of spermatogonia, spermatocytes, and sperm concentration in cimetidine-treated rats. Adult male rats were treated for 50 days as follows: cimetidine group received 100 mg kg-1 b.w. of cimetidine, cimetidine-B12 group received cimetidine and 3 μg of vitamin B12-hydroxocobalamin, B12 group received only 3 μg of vitamin, and control group received saline. Sperm concentration was calculated and historesin-embedded testes sections were used for the quantitative analyses of spermatogonia (A; In/B) and spermatocytes. TUNEL method and PCNA immunofluorescence were performed. Cimetidine caused a significant reduction in sperm concentration. TUNEL-positive spermatogonia and spermatocytes were correlated to a significant reduction in the number of these cells. In cimetidine-B12 group, sperm concentration was higher than cimetidine group and a significant increase in the number of spermatogonia (stages II-VI) was correlated to a high incidence of PCNA-immunolabeled spermatogonia and spermatocytes. The results show that the supplementation of rats with vitamin B12 during cimetidine treatment increases sperm concentration and exerts a potential effect in the recovery of spermatogonia and spermatocytes.

  2. OCT2, SSX and SAGE1 reveal the phenotypic heterogeneity of spermatocytic seminoma reflecting distinct subpopulations of spermatogonia

    PubMed Central

    Lim, Jasmine; Goriely, Anne; Turner, Gareth DH; Ewen, Katherine A; Jacobsen, Grete Krag; Graem, Niels; Wilkie, Andrew OM; Rajpert-De Meyts, Ewa

    2011-01-01

    Spermatocytic seminoma (SS) is a rare testicular neoplasm that occurs predominantly in older men. In this study, we aimed to shed light on the histogenesis of SS by investigating the developmental expression of protein markers that identify distinct subpopulations of human spermatogonia in the normal adult testis. We analysed the expression pattern of OCT2, SSX2-4, and SAGE1 in 36 SS cases and four intratubular SS (ISS) as well as a series of normal testis samples throughout development. We describe for the first time two different types of SS characterized by OCT2 or SSX2-4 immunoexpression. These findings are consistent with the mutually exclusive antigenic profile of these markers during different stages of testicular development and in the normal adult testis. OCT2 was expressed predominantly in Adark spermatogonia, SSX2-4 was present in Apale and B spermatogonia and leptotene spermatocytes, whilst SAGE1 was exclusively present in a subset of post-pubertal germ cells, most likely B spermatogonia. The presence of OCT2 and SSX2-4 in distinct subsets of germ cells implies that these markers represent germ cells at different maturation stages. Analysis of SAGE1 and SSX2-4 in ISS showed spatial differences suggesting ongoing maturation of germ cells during progression of SS tumourigenesis. We conclude that the expression pattern of OCT2, SSX2-4, and SAGE1 supports the origin of SS from spermatogonia and provides new evidence for heterogeneity of this tumour, potentially linked either to the cellular origin of SS or to partial differentiation during tumour progression, including a hitherto unknown OCT2-positive variant of the tumour likely derived from Adark spermatogonia. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:21706474

  3. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

    PubMed

    Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

    2016-09-01

    Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

  4. Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture.

    PubMed

    Zheng, Y; Thomas, A; Schmidt, C M; Dann, C T

    2014-11-01

    Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia? Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures. The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures. Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures. Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4). UTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were

  5. Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture

    PubMed Central

    Zheng, Y.; Thomas, A.; Schmidt, C.M.; Dann, C.T.

    2014-01-01

    STUDY QUESTION Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia? SUMMARY ANSWER Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1–2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures. WHAT IS KNOWN ALREADY The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures. STUDY DESIGN, SIZE, DURATION Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures. PARTICIPANTS/MATERIALS, SETTING, METHODS Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1–2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4). MAIN RESULTS AND THE ROLE OF CHANCE UTF1, FGFR3, DAZL and ZBTB16 qRT–PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in

  6. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

    PubMed Central

    Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.

    2016-01-01

    STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no

  7. An inversion involving the mouse Shh locus results in brachydactyly through dysregulation of Shh expression

    PubMed Central

    Niedermaier, Michael; Schwabe, Georg C.; Fees, Stephan; Helmrich, Anne; Brieske, Norbert; Seemann, Petra; Hecht, Jochen; Seitz, Volkhard; Stricker, Sigmar; Leschik, Gundula; Schrock, Evelin; Selby, Paul B.; Mundlos, Stefan

    2005-01-01

    Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5–E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development. PMID:15841179

  8. Involvement of ceramide in ethanol-induced apoptotic neurodegeneration in the neonatal mouse brain.

    PubMed

    Saito, Mariko; Chakraborty, Goutam; Hegde, Medha; Ohsie, Jason; Paik, Sun-Mee; Vadasz, Csaba; Saito, Mitsuo

    2010-10-01

    Acute administration of ethanol to 7-day-old mice is known to cause robust apoptotic neurodegeneration in the brain. Our previous studies have shown that such ethanol-induced neurodegeneration is accompanied by increases in lipids, including ceramide, triglyceride (TG), cholesterol ester (ChE), and N-acylphosphatidylethanolamine (NAPE) in the brain. In this study, the effects of ethanol on lipid profiles as well as caspase 3 activation were examined in the cortex, hippocampus, cerebellum, and inferior colliculus of the postnatal day 7 mouse brain. We found that the cortex, hippocampus, and inferior colliculus, which showed substantial caspase 3 activation by ethanol, manifested significant elevations in ceramide, TG, and NAPE. In contrast, the cerebellum, with the least caspase 3 activation, failed to show significant changes in ceramide and TG, and exhibits much smaller increases in NAPE than other brain regions. Ethanol-induced increases in ChE were observed in all brain regions tested. Inhibitors of serine palmitoyltransferase effectively blocked ethanol-induced caspase 3 activation as well as elevations in ceramide, ChE, and NAPE. Immunohistochemical studies indicated that the expression of serine palmitoyltransferase was mainly localized in neurons and was enhanced in activated caspase 3-positive neurons generated by ethanol. These results indicate that de novo ceramide synthesis has a vital role in ethanol-induced apoptotic neurodegeneration in the developing brain.

  9. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models

    PubMed Central

    Liu, Huiping; Patel, Manishkumar R.; Prescher, Jennifer A.; Patsialou, Antonia; Qian, Dalong; Lin, Jiahui; Wen, Susanna; Chang, Ya-Fang; Bachmann, Michael H.; Shimono, Yohei; Dalerba, Piero; Adorno, Maddalena; Lobo, Neethan; Bueno, Janet; Dirbas, Frederick M.; Goswami, Sumanta; Somlo, George; Condeelis, John; Contag, Christopher H.; Gambhir, Sanjiv Sam; Clarke, Michael F.

    2010-01-01

    To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44+ cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy. PMID:20921380

  10. Autocrine growth factors are involved in branching morphogenesis of mouse lung epithelium.

    PubMed

    Okada, Kimiko; Noda, Masatsugu; Nogawa, Hiroyuki

    2013-01-01

    The current model for branching morphogenesis of mouse lung proposes that the epithelium bifurcates as cells pursue separate sources of fibroblast growth factor (FGF) 10, secreted from mesenchymal tissue through interactions with epithelial tissue. If so, it may be assumed that the lung epithelium will grow into a uniform, expanding ball (without branching) when uniformly exposed to a constant concentration of FGF10. To test this hypothesis, we cultured Matrigel-embedded lung epithelium explants in FGF10-supplemented medium while shaking the culture dishes. Shaking cultures with FGF10 resulted in inferior epithelial branching compared to control cultures at rest. However, this effect was unexpectedly accompanied by poor growth rather than by ball-like expansion. When using FGF1, epithelial cultures grew and branched similarly well under either culture condition. Thus, we hypothesized that FGF10 signaling must be mediated by autocrine FGFs, such as FGF1, which might easily diffuse through the culture medium in the shaking culture. Reverse transcription-polymerase chain reaction analyses showed that FGF9 as well as FGF1 were expressed in the epithelium in vivo and in FGF10-stimulated epithelium in vitro, and FGF9 induced epithelial branching at a much lower concentration than FGF10. These results suggest that FGF1 and FGF9 may mediate FGF10 signaling and induce branching in the lung epithelium via autocrine signaling.

  11. Antihistoplasma effect of activated mouse splenic macrophages involves production of reactive nitrogen intermediates.

    PubMed Central

    Lane, T E; Wu-Hsieh, B A; Howard, D H

    1994-01-01

    The mechanism by which recombinant murine gamma interferon (rMuIFN-gamma) and bacterial lipopolysaccharide (LPS) activate mouse resident splenic macrophages to inhibit the intracellular growth of the fungus Histoplasma capsulatum was examined. Growth inhibition depended on L-arginine metabolism. The growth inhibitory state normally induced by rMuIFN-gamma and LPS in resident splenic macrophages did not occur when the macrophages were cultured in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of L-arginine metabolism. Resident splenic macrophages treated with rMuIFN-gamma and LPS produced nitrite (NO2-), an end product of L-arginine metabolism. When macrophages were cultured in the presence of NG-monomethyl-L-arginine together with rMuIFN-gamma and LPS, only baseline levels of NO2- were detected. Spleen cells from H. capsulatum-infected mice produced high levels of NO2- in culture. The production of NO2- correlated with in vitro inhibition of the intracellular growth of H. capsulatum. Anti-tumor necrosis factor alpha antibody did not block NO2- production by the immigrant splenic macrophages and did not abolish the antihistoplasma activity. PMID:8168960

  12. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes.

    PubMed

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-20

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA.

  13. Perfluorooctanoic acid affects endocytosis involving clathrin light chain A and microRNA-133b-3p in mouse testes.

    PubMed

    Lu, Yin; Wang, Jianshe; Guo, Xuejiang; Yan, Shengmin; Dai, Jiayin

    2017-03-01

    Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. Among its potential health hazards, testicular toxicity is of major concern. To explore the potential effect of miRNA on post-translational regulation after PFOA exposure, changes in miRNAs were detected via miRNA array. Seventeen miRNAs were differentially expressed (eight upregulated, nine downregulated) in male mouse testes after exposure to 5mg/kg/d of PFOA for 28d (>1.5-fold and P<0.05 compared with the control). Eight of these miRNAs were further selected for TaqMan qPCR analysis. Proteomic profile analysis indicated that many changed proteins after PFOA treatment, including intersectin 1 (ITSN1), serine protease inhibitor A3K (Serpina3k), and apolipoprotein a1 (APOA1), were involved in endocytosis and blood-testis barrier (BTB) processes. These changes were further verified by immunohistochemical and Western blot analyses. Endocytosis-related genes were selected for qPCR analysis, with many found to be significantly changed after PFOA treatment, including epidermal growth factor receptor pathway substrate 8 (Eps8), Eps15, cortactin, cofilin, espin, vinculin, and zyxin. We further predicted the potential interaction between changed miRNAs and proteins, which indicated that miRNAs might play a role in the post-translational regulation of gene expression after PFOA treatment in mouse testes. Among them, miR-133b-3p/clathrin light chain A (CLTA) was selected and verified in vitro by transfection and luciferase activity assay. Results showed that PFOA exposure affects endocytosis in mouse testes and that CLTA is a potential target of miR-133b-3p. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Convergent Evidence from Mouse and Human Studies Suggests the Involvement of Zinc Finger Protein 326 Gene in Antidepressant Treatment Response

    PubMed Central

    Liou, Ying-Jay; Chen, Chien-Hsiun; Cheng, Chih-Ya; Chen, Shiow-Yi; Chen, Tai-Jui; Yu, Younger W-Y; Nian, Fang-Shin; Tsai, Shih-Jen; Hong, Chen-Jee

    2012-01-01

    Objectives The forced swim test (FST) is a commonly used model to predict antidepressant efficacy. Uncovering the genetic basis of the model may unravel the mechanism of antidepressant treatment. Methods FVB/NJ (FVB) and C57BL/6J (B6) were first identified as the response and non-response strains to fluoxetine (a serotonin-specific reuptake inhibitor antidepressant) treatment in the mouse FST. Simple-interval (SIM) and composite-interval (CIM) mappings were applied to map the quantitative trait loci (QTLs) of the anti-immobility effect of fluoxetine in FST (FSTFLX) in 865 male B6×FVB-F2 mice. The brain mRNA expressions of the gene with the maximum QTL-linkage signal for FSTFLX after the FST were compared between B6 and FVB mice and also compared between fluoxetine and saline treatment. The association of the variants in the human homologue of the mouse FSTFLX-QTL gene with major depressive disorder (MDD) and antidepressant response were investigated in 1080 human subjects (MDD/control = 582/498). Results One linkage signal for FSTFLX-QTL was detected at an intronic SNP (rs6215396) of the mouse Zfp326 gene (maximal CIM-LOD = 9.36). The Zfp326 mRNA expression in the FVB thalamus was significantly down-regulated by fluoxetine in the FST, and the higher FVB-to-B6 Zfp326 mRNA expressions in the frontal cortex, striatum and hypothalamus diminished after fluoxetine treatment. Two coding-synonymous SNPs (rs2816881 and rs10922744) in the human homologue of Zfp326, ZNF326, were significantly associated with the 8-week antidepressant treatment response in the MDD patients (Bonferroni-corrected p = 0.004–0.028). Conclusions The findings suggest the involvement of the Zfp326 and ZNF326 genes in antidepressant treatment response. PMID:22666313

  15. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes

    PubMed Central

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-01

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA. PMID:26805874

  16. STRUCTURAL ORGANIZATION AND FUNCTION OF MOUSE PHOTORECEPTOR RIBBON SYNAPSES INVOLVE THE IMMUNOGLOBULIN ADHESION PROTEIN SYNCAM 1

    PubMed Central

    Ribic, Adema; Liu, Xinran; Crair, Michael C.; Biederer, Thomas

    2013-01-01

    Adhesive interactions in the retina instruct the developmental specification of inner retinal layers. However, potential roles of adhesion in the development and function of photoreceptor synapses remain incompletely understood. This contrasts with our understanding of synapse development in the central nervous system (CNS), which can be guided by select adhesion molecules such as the Synaptic Cell Adhesion Molecule 1 (SynCAM 1/CADM1/Nectin-like 2 protein). This immunoglobulin superfamily protein modulates the development and plasticity of classical excitatory synapses. We now show by immuno-electron microscopy and immunoblotting that SynCAM 1 is expressed on mouse rod photoreceptors and their terminals in the outer nuclear and plexiform layers (ONL and OPL) in a developmentally regulated manner. Expression of SynCAM 1 on rods is low in early postnatal stages (P3-P7), but increases after eye opening (P14). In support of functional roles in the photoreceptors, electroretinogram recordings demonstrate impaired responses to light stimulation in SynCAM 1 knockout (KO) mice. In addition, the structural integrity of synapses in the OPL requires SynCAM 1. Quantitative ultrastructural analysis of SynCAM 1 KO retina measured fewer fully assembled, triadic rod ribbon synapses. Further, rod synapse ribbons are shortened in KO mice and protein levels of Ribeye, a major structural component of ribbons, are reduced in SynCAM 1 KO retina. Together, our results implicate SynCAM 1 in the synaptic organization of the rod visual pathway and provide evidence for novel roles of synaptic adhesion in the structural and functional integrity of ribbon synapses. PMID:23982969

  17. Nrf2 signalling and autophagy are involved in diabetes mellitus-induced defects in the development of mouse placenta

    PubMed Central

    Han, Sha-sha; Jin, Ya; Li, He; Wu, Xia; Ma, Zheng-lai; cheng, Xin; Tang, Xiuwen

    2016-01-01

    It is widely accepted that diabetes mellitus impairs placental development, but the mechanism by which the disease operates to impair development remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in placental development in mice are mainly characterized by the changes of morphological structure of placenta. The alteration of differentiation-related gene expressions in trophoblast cells rather than cell proliferation/apoptosis is responsible for the phenotypes found in mouse placenta. Meanwhile, excess reactive oxygen species (ROS) production and activated nuclear factor erythroid2-related factor 2 (Nrf2) signalling were observed in the placenta of mice suffering from PGDM. Using BeWo cells, we also demonstrated that excess ROS was produced and Nrf2 signalling molecules were activated in settings characterized by a high concentration of glucose. More interestingly, differentiation-related gene expressions in trophoblast cells were altered when endogenous Nrf2 expression is manipulated by transfecting Nrf2-wt or Nrf2-shRNA. In addition, PGDM interferes with autophagy in both mouse placenta and BeWo cells, implying that autophagy is also involved, directly or indirectly, in PGDM-induced placental phenotypes. Therefore, we revealed that dysfunctional oxidative stress-activated Nrf2 signalling and autophagy are probably responsible for PGDM-induced defects in the placental development of mice. The mechanism was through the interference with differentiation-related gene expression in trophoblast cells. PMID:27383629

  18. Indoleamine 2,3-Dioxygenase 1 (Ido1) Is Involved in the Control of Mouse Caput Epididymis Immune Environment

    PubMed Central

    Damon-Soubeyrand, Christelle; Saez, Fabrice; Kocer, Ayhan; Janny, Laurent; Pons-Rejraji, Hanae; Munn, David H.; Mellor, Andrew L.; Gharbi, Najoua; Cadet, Rémi; Guiton, Rachel; Aitken, Robert J.; Drevet, Joël R.

    2013-01-01

    The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1−/− model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1−/− animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed. PMID:23840489

  19. Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model

    PubMed Central

    Sato, Kenji; Takaishi, Mikiro; Tokuoka, Shota; Sano, Shigetoshi

    2014-01-01

    TNF-α plays a crucial role in psoriasis; therefore, TNF inhibition has become a gold standard for the treatment of psoriasis. TNF-α is processed from a membrane-bound form by TNF-α converting enzyme (TACE) to soluble form, which exerts a number of biological activities. EGF receptor (EGFR) ligands, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor (TGF)-α are also TACE substrates and are psoriasis-associated growth factors. Vascular endothelial growth factor (VEGF), one of the downstream molecules of EGFR and TNF signaling, plays a key role in angiogenesis for developing psoriasis. In the present study, to assess the possible role of TACE in the pathogenesis of psoriasis, we investigated the involvement of TACE in TPA-induced psoriasis-like lesions in K5.Stat3C mice, which represent a mouse model of psoriasis. In this mouse model, TNF-α, amphiregulin, HB-EGF and TGF-α were significantly up-regulated in the skin lesions, similar to human psoriasis. Treatment of K5.Stat3C mice with TNF-α or EGFR inhibitors attenuated the skin lesions, suggesting the roles of TACE substrates in psoriasis. Furthermore, the skin lesions of K5.Stat3C mice showed down-regulation of tissue inhibitor of metalloproteinase-3, an endogenous inhibitor of TACE, and an increase in soluble TNF-α. A TACE inhibitor abrogated EGFR ligand-dependent keratinocyte proliferation and VEGF production in vitro, suggesting that TACE was involved in both epidermal hyperplasia and angiogenesis during psoriasis development. These results strongly suggest that TACE contributes to the development of psoriatic lesions through releasing two kinds of psoriasis mediators, TNF-α and EGFR ligands. Therefore, TACE could be a potential therapeutic target for the treatment of psoriasis. PMID:25384035

  20. Involvement of TNF-α converting enzyme in the development of psoriasis-like lesions in a mouse model.

    PubMed

    Sato, Kenji; Takaishi, Mikiro; Tokuoka, Shota; Sano, Shigetoshi

    2014-01-01

    TNF-α plays a crucial role in psoriasis; therefore, TNF inhibition has become a gold standard for the treatment of psoriasis. TNF-α is processed from a membrane-bound form by TNF-α converting enzyme (TACE) to soluble form, which exerts a number of biological activities. EGF receptor (EGFR) ligands, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor (TGF)-α are also TACE substrates and are psoriasis-associated growth factors. Vascular endothelial growth factor (VEGF), one of the downstream molecules of EGFR and TNF signaling, plays a key role in angiogenesis for developing psoriasis. In the present study, to assess the possible role of TACE in the pathogenesis of psoriasis, we investigated the involvement of TACE in TPA-induced psoriasis-like lesions in K5.Stat3C mice, which represent a mouse model of psoriasis. In this mouse model, TNF-α, amphiregulin, HB-EGF and TGF-α were significantly up-regulated in the skin lesions, similar to human psoriasis. Treatment of K5.Stat3C mice with TNF-α or EGFR inhibitors attenuated the skin lesions, suggesting the roles of TACE substrates in psoriasis. Furthermore, the skin lesions of K5.Stat3C mice showed down-regulation of tissue inhibitor of metalloproteinase-3, an endogenous inhibitor of TACE, and an increase in soluble TNF-α. A TACE inhibitor abrogated EGFR ligand-dependent keratinocyte proliferation and VEGF production in vitro, suggesting that TACE was involved in both epidermal hyperplasia and angiogenesis during psoriasis development. These results strongly suggest that TACE contributes to the development of psoriatic lesions through releasing two kinds of psoriasis mediators, TNF-α and EGFR ligands. Therefore, TACE could be a potential therapeutic target for the treatment of psoriasis.

  1. Involvement of MAPK ERK activation in upregulation of water channel protein aquaporin 1 in a mouse model of Bell's palsy.

    PubMed

    Fang, Fan; Liu, Cai-Yue; Zhang, Jie; Zhu, Lie; Qian, Yu-Xin; Yi, Jing; Xiang, Zheng-Hua; Wang, Hui; Jiang, Hua

    2015-05-01

    The aim of this study is to immunolocalize the aquaporin 1 water channel protein (AQP1) in Schwann cells of idiopathic facial nerve and explore its possible role during the development of facial palsy induced by herpes simplex virus type 1 (HSV-1). HSV-1 was inoculated into the surface of posterior auricle of mouse to establish a paralyzed animal model. In HSV-1-induced facial palsy mice, protein levels of AQP1 significantly increased on the 9th to 16th day after inoculation of HSV-1. The upregulation of AQP1 was closely related to the intratemporal facial nerve edema in facial nerve canal, which was also consistent with the symptom of facial palsy in mice. In a hypoxia model of Schwann cells in vitro, we found that U0126, an ERK antagonist, inhibited not only morphological changes of cultures Schwann cells but also upregulation of both AQP1 and phosphorylated ERK. Combined with increased phosphorylated ERK in HSV-1-induced facial palsy mice, we inferred that ERK MAPK pathway might also be involved in increased AQP1 in mouse model of Bell's palsy. Although the precise mechanism needs to be further explored, our findings suggest that AQP1 in Schwann cells of intratemporal facial nerve is involved in the evolution of facial palsy induced by HSV-1 and may play an important role in the pathogenesis of this disease. AQP1 might be a potential target, and the ERK antagonist U0126 could be a new drug for the treatment of HSV-1-induced Bell's palsy in an early stage.

  2. Noggin and Sonic hedgehog are involved in compensatory changes within the motoneuron-depleted mouse spinal cord.

    PubMed

    Gulino, Rosario; Gulisano, Massimo

    2013-09-15

    Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of the neural tube, but recent findings suggest that they could participate in regeneration and functional recovery after injury. Here, in order to verify if these mechanisms could occur in the spinal cord and involve synaptic plasticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline Acetyltransferase, Synapsin-I and Glutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal cord lesion model obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expression changes of the analyzed proteins. Moreover, motor performance was found correlated with Sonic hedgehog and Noggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modulating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsic potential for repair and that some proteins classically involved in development, such as Sonic hedgehog and Noggin could have important roles in regeneration and functional restoration, by mechanisms including synaptic plasticity.

  3. Hyperhomocysteinemia and bleomycin hydrolase modulate the expression of mouse brain proteins involved in neurodegeneration.

    PubMed

    Suszyńska-Zajczyk, Joanna; Luczak, Magdalena; Marczak, Lukasz; Jakubowski, Hieronim

    2014-01-01

    Homocysteine (Hcy) is a risk factor for Alzheimer's disease (AD). Bleomycin hydrolase (BLMH) participates in Hcy metabolism and is also linked to AD. The inactivation of the Blmh gene in mice causes accumulation of Hcy-thiolactone in the brain and increases susceptibility to Hcy-thiolactone-induced seizures. To gain insight into brain-related Blmh function, we used two-dimensional IEF/SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry to examine brain proteomes of Blmh-/- mice and their Blmh+/+ littermates fed with a hyperhomocysteinemic high-Met or a control diet. We found that: (1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Aop1), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (Tbcb, CapZa2) were differentially expressed in brains of Blmh-null mice; (2) hyperhomocysteinemia amplified effects of the Blmh-/- genotype on brain protein expression; (3) proteins involved in brain-specific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in Blmh-null brains only in hyperhomocysteinemic animals; (4) most proteins regulated by the Blmh-/- genotype were also regulated by high-Met diet, albeit in the opposite direction; and (5) the differentially expressed proteins play important roles in neural development, learning, plasticity, and aging and are linked to neurodegenerative diseases, including AD. Taken together, our findings suggest that Blmh interacts with diverse cellular processes from energy metabolism and anti-oxidative defenses to cell cycle, cytoskeleton dynamics, and synaptic plasticity essential for normal brain homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in AD.

  4. Paraoxonase 1 and dietary hyperhomocysteinemia modulate the expression of mouse proteins involved in liver homeostasis.

    PubMed

    Suszyńska-Zajczyk, Joanna; Jakubowski, Hieronim

    2014-01-01

    Homocysteine (Hcy), a product of methionine metabolism, is elevated by the consumption of a high-methionine diet that can cause fatty liver disease. Paraoxonase 1 (Pon1), a hydrolase expressed mainly in the liver and carried in the circulation on high-density lipoprotein, participates in Hcy metabolism. Low Pon1 activity is linked to fatty liver disease. We hypothesize that hyperhomocysteinemia and low Pon1 induce changes in gene expression that could impair liver homeostasis. To test this hypothesis, we analyzed the liver proteome of Pon1(-/-) and Pon1(+/+) mice fed a high methionine diet (1% methionine in the drinking water) for 8 weeks using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We identified seven liver proteins whose expression was significantly altered in Pon1(-/-) mice. In animals fed with a control diet, the expression of three liver proteins involved in lipoprotein metabolism (ApoE), iron metabolism (Ftl), and regulation of nitric oxide generation (Ddah1) was up-regulated by the Pon1(-/-) genotype. In mice fed with a high-methionine diet, expression of four liver proteins was up-regulated and of three proteins was down-regulated by the Pon1(-/-) genotype. The up-regulated proteins are involved in lipoprotein metabolism (ApoE), energy metabolism (Atp5h), oxidative stress response (Prdx2), and nitric oxide regulation (Ddah1). The down-regulated proteins are involved in energy metabolism (Gamt), iron metabolism (Ftl), and catechol metabolism (Comt). Expression of one protein (Ftl) was up-regulated both by the Pon1(-/-) genotype and a high-methionine diet. Our findings suggest that Pon1 interacts with diverse cellular processes - from lipoprotein metabolism, nitric oxide regulation, and energy metabolism to iron transport and antioxidant defenses - that are essential for normal liver homeostasis and modulation of these interactions by a high-methionine diet may contribute to fatty liver disease.

  5. Identification of two quantitative trait loci involved in antibody production on mouse chromosome 8.

    PubMed

    Puel, A; Mevel, J C; Bouthillier, Y; Decreusefond, C; Fridman, W H; Feingold, N; Mouton, D

    1998-03-01

    Several quantitative trait loci (QTLs) contributing to the extreme phenotypes of the selected high (H) and low (L) antibody-responder lines of mice were mapped on distinct chromosomes. Successive backcrosses were bred to reduce the length of the QTL-bearing segment detected on chromosome 8 and to produce congenic lines to test gene effect independently of the other QTLs. An increase in antibody responses was repeatedly found to be associated with inheritance of the H-line allele at two markers separated by 30 cM on that chromosome. In the successive backcrosses, background and unlinked involved genes of H-line origin were progressively eliminated; however, unexpected within-progeny variations persisted in the third and even fourth backcross. Nevertheless, the presence of two QTLs within the considered interval was definitely demonstrated in distinct progenies of the fourth backcross which separately inherited one of the two gene-marker H-line alleles. The previously identified chromosome 8 segment therefore contains at least two QTLs involved in antibody responsiveness.

  6. The involvement of the TRPA1 receptor in a mouse model of sympathetically maintained neuropathic pain.

    PubMed

    Pinheiro, Francielle de Vargas; Villarinho, Jardel Gomes; Silva, Cássia Regina; Oliveira, Sara Marchesan; Pinheiro, Kelly de Vargas; Petri, Delia; Rossato, Mateus Fortes; Guerra, Gustavo Petri; Trevisan, Gabriela; Antonello Rubin, Maribel; Geppetti, Pierangelo; Ferreira, Juliano; André, Eunice

    2015-01-15

    Sympathetic fibres maintain some forms of neuropathic pain, but the underlying mechanisms are poorly understood. Therefore, this study investigated the possible involvement of transient receptor potential ankyrin 1 (TRPA1) and the role of the sympathetic nervous system (involved in sympathetically maintained neuropathic pain) in a model of neuropathic pain induced by sciatic nerve chronic constriction injury (CCI) in mice. Systemic injection of the selective TRPA1 antagonist HC-030031 reversed the mechanical and cold allodynia that was induced by sciatic nerve chronic constriction injury (CCI). Nerve injury also sensitised mice to nociception, which was induced by the intraplantar injection of a low dose of the TRPA1 agonist allyl isothiocyanate without changing TRPA1 immunoreactivity in the injected paw. Furthermore, chemical sympathectomy produced by guanethidine largely prevented CCI-induced mechanical and cold allodynia. CCI also induced a norepinephrine-triggered nociception that was inhibited by an α-adrenoceptor antagonist, norepinephrine transporter block and monoamine oxidase inhibition. Finally, the peripheral injection of HC-030031 also largely reduced CCI-induced norepinephrine nociception and mechanical or cold allodynia. Taken together, the present findings reveal a critical role of TRPA1 in mechanical and cold hypersensitivity and norepinephrine hypersensitivity following nerve injury. Finally, our results suggest that TRPA1 antagonism may be useful to treat patients who present sympathetically maintained neuropathic pain. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens)

    NASA Astrophysics Data System (ADS)

    Higaki, Shogo; Shimada, Manami; Kawamoto, Kazuaki; Todo, Takaaki; Kawasaki, Toshihiro; Tooyama, Ikuo; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

    2017-02-01

    Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2‧-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.

  8. In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens)

    PubMed Central

    Higaki, Shogo; Shimada, Manami; Kawamoto, Kazuaki; Todo, Takaaki; Kawasaki, Toshihiro; Tooyama, Ikuo; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

    2017-01-01

    Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials. PMID:28211534

  9. Autophagy is involved in mouse kidney development and podocyte differentiation regulated by Notch signalling.

    PubMed

    Zhang, Chuyue; Li, Wen; Wen, Junkai; Yang, Zhuo

    2017-02-03

    Podocyte dysfunction results in glomerular diseases accounted for 90% of end-stage kidney disease. The evolutionarily conserved Notch signalling makes a crucial contribution in podocyte development and function. However, the underlying mechanism of Notch pathway modulating podocyte differentiation remains less obvious. Autophagy, reported to be related with Notch signalling pathways in different animal models, is regarded as a possible participant during podocyte differentiation. Here, we found the dynamic changes of Notch1 were coincided with autophagy: they both increased during kidney development and podocyte differentiation. Intriguingly, when Notch signalling was down-regulated by DAPT, autophagy was greatly diminished, and differentiation was also impaired. Further, to better understand the relationship between Notch signalling and autophagy in podocyte differentiation, rapamycin was added to enhance autophagy levels in DAPT-treated cells, and as a result, nephrin was recovered and DAPT-induced injury was ameliorated. Therefore, we put forward that autophagy is involved in kidney development and podocyte differentiation regulated by Notch signalling.

  10. Regulation of crucial lncRNAs in differentiation of chicken embryonic stem cells to spermatogonia stem cells.

    PubMed

    Li, D; Ji, Y; Wang, F; Wang, Y; Wang, M; Zhang, C; Zhang, W; Lu, Z; Sun, C; Ahmed, M F; He, N; Jin, K; Cheng, S; Wang, Y; He, Y; Song, J; Zhang, Y; Li, B

    2017-04-01

    Regulation of crucial lncRNAs involved in differentiation of chicken embryonic stem cells (ESCs) to spermatogonia stem cells (SSCs) was explored by sequencing the transcriptome of ESCs, primordial germ cells (PGCs) and SSCs with RNA-Seq; analytical bioinformatic methods were used to excavate candidate lncRNAs. We detected expression of candidate lncRNAs in ESCs, PGCs and SSCs and forecasted related target genes. Utilizing wego, david and string, function and protein-protein interactions of target genes were analyzed. Finally, based on string analysis, interaction diagrams and relevant signaling pathways were established. Our results indicate a total of 9657 lncRNAs in ESCs, PGCs and SSCs, with 3549 defined as significantly different. We screened 20 candidate lncRNAs, each demonstrating a greater than eight-fold difference in |logFC| value between groups (ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs) or specifically expressed in an individual cell type. qRT-PCR results indicated that expression tendencies of candidate lncRNAs were consistent with RNA-Seq. Fifteen cis and four trans target genes were forecasted. Based on wego and string analyses, we found lnc-SSC1, lnc-SSC5, lnc-SSC2 and lnc-ESC2 negatively regulated target genes SUFU, EPHA3, KLF3, ARL3 and TRIM8, whereas SHH, NOTCH, TGF-β, cAMP/cGMP and JAK/STAT signaling pathways were promoted, causing differentiation of ESCs into SSCs. Our findings represent a preliminary unveiling of lncRNA-associated regulatory mechanisms during differentiation of chicken ESCs into SSCs, filling a research void in male germ cell differentiation related to lncRNA. Our results also provide basic information for improving in vitro induction systems for differentiation of chicken ESCs into SSCs.

  11. Differential involvement of CCK-A and CCK-B receptors in the regulation of locomotor activity in the mouse.

    PubMed

    Vasar, E; Harro, J; Lang, A; Pôld, A; Soosaar, A

    1991-01-01

    The influence of the CCK-A antagonist devazepide and the CCK-B/gastrin antagonist L-365,260 on the locomotor activity of mice was studied. Devazepide and L-365,260 had opposite effects on spontaneous locomotor activity, and on caerulein- and apomorphine-induced hypomotility in the mouse. Devazepide in high doses (0.1-1 mg/kg IP) reduced spontaneous motor activity, whereas L-365,260 at a high dose (1 mg/kg IP) increased the activity of mice. Devazepide (0.1-10 micrograms/kg) moderately antagonized the sedative effect of apomorphine (0.1 mg/kg SC) and caerulein (25 micrograms/kg SC), whereas L-365,260 (1-10 micrograms/kg) significantly potentiated the actions of dopamine and CCK agonists. Concomitant administration of caerulein (15 micrograms/kg SC) and apomorphine (0.1 mg/kg SC) caused an almost complete loss of locomotor activity in the mouse. Devazepide and L-365,260 (0.1-10 micrograms/kg) were completely ineffective against caerulein-induced potentiation of apomorphine hypomotility. Devazepide in high doses (0.1-1 mg/kg), reducing the spontaneous motor activity of mice, counteracted the motor excitation induced by d-amphetamine (5 mg/kg IP). The CCK agonist caerulein (100 micrograms/kg SC) had a similar antiamphetamine effect. Devazepide (1-100 micrograms/kg) and L-365,260 (1 micrograms/kg) reversed completely the antiamphetamine effect of caerulein. The results of present study reflect apparently distinct role of CCK-A and CCK-B receptors in the regulation of motor activity. The opposite effect of devazepide and L-365,260 on caerulein- and apomorphine-induced hypolocomotion is probably related to the antagonistic role of CCK-A and CCK-B receptor subtypes in the regulation of mesencephalic dopaminergic neurons. The antiamphetamine effect of caerulein is possibly linked to the stimulation of CCK-A receptors in the mouse brain, whereas the blockade of both subtypes of the CCK-8 receptor is involved in the antiamphetamine effect of devazepide.

  12. Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells.

    PubMed

    Yamauchi, Mika; Yamaguchi, Toru; Kaji, Hiroshi; Sugimoto, Toshitsugu; Chihara, Kazuo

    2005-03-01

    We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether the CaR plays any role in regulating osteoblast differentiation. In this study, we found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS-R467 (1 and 3 microM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. Alkaline phosphatase activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen and osteopontin mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin, which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing alkaline phosphatase activity, osteocalcin expression, and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.

  13. Differential expression of genes involved in the degeneration and regeneration pathways in mouse models for muscular dystrophies.

    PubMed

    Onofre-Oliveira, P C G; Santos, A L F; Martins, P M; Ayub-Guerrieri, D; Vainzof, M

    2012-03-01

    The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd (mdx), SJL/J, Large (myd) and Lama2 (dy2J) /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-β1 and Pro-collagen 1α2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-β1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

  14. A monoclonal antibody that specifically reacts with human embryonal carcinomas, spermatogonia and oocytes is able to induce human EC cell death.

    PubMed

    Nakano, T; Umezawa, A; Abe, H; Suzuki, N; Yamada, T; Nozawa, S; Hata, J

    1995-02-01

    We developed a mouse monoclonal antibody, 6E2 (IgG3), against a human embryonal carcinoma (EC) cell line, NCR-G3, that possesses totipotent differentiation capabilities. Culturing human EC cells in the presence of 6E2 causes their death. It has been shown that 6E2 kills EC cells dose dependently. In immunohistochemical examination with normal human germ cells, 6E2 reacted specifically with spermatogonia and oocytes. Among human germ cell tumor tissues on aceton-fixed frozen sections, 6E2 reacted with embryonal carcinomas, seminomas and dysgerminomas, but it did not react with choriocarcinomas or with yolk sac tumors. Consistently, in flow cytometric analysis of cultured human germ cell tumor cell lines, 6E2 reacted exclusively with EC cells including NCR-G3 cells. It was revealed, by preserving its antigenicity after treatment with periodic acid and tunicamycin and by radiolabeling cells followed by immunoprecipitation, that the molecule defined by 6E2 is a cell surface protein having a molecular weight of approximately 80 kDa. These data illustrate that the molecule defined by 6E2 links human germ cell tumors, especially embryonal carcinoma, seminoma and dysgerminoma, to their normal counterparts and that it may play a role in survival and proliferation of human EC cells.

  15. Selection against Robertsonian fusions involving housekeeping genes in the house mouse: integrating data from gene expression arrays and chromosome evolution.

    PubMed

    Ruiz-Herrera, Aurora; Farré, Marta; Ponsà, Montserrat; Robinson, Terence J

    2010-11-01

    Monobrachial homology resulting from Robertsonian (Rb) fusions is thought to contribute to chromosomal speciation through underdominance. Given the karyotypic diversity characterizing wild house mouse populations [Mus musculus domesticus, (MMU)], variation that results almost exclusively from Rb fusions (diploid numbers range from 22 to 40) and possibly whole arm reciprocal translocations (WARTs), this organism represents an excellent model for testing hypotheses of chromosomal evolution. Previous studies of chromosome size and recombination rates have failed to explain the bias for certain chromosomes to be involved more frequently than others in these rearrangements. Here, we show that the pericentromeric region of one such chromosome, MMU19, which is infrequently encountered as a fusion partner in wild populations, is significantly enriched for housekeeping genes when compared to other chromosomes in the genome. These data suggest that there is selection against breakpoints in the pericentromeric region and provide new insights into factors that constrain chromosomal reorganizations in house mice. Given the anticipated increase in vertebrate whole genome sequences, the examination of gene content and expression profiles of the pericentromeric regions of other mammalian lineages characterized by Rb fusions (i.e., other rodents, bats, and bovids, among others) is both achievable and crucial to developing broadly applicable models of chromosome evolution.

  16. Mmu-miR-193 Is Involved in Embryo Implantation in Mouse Uterus by Regulating GRB7 Gene Expression

    PubMed Central

    Li, Rong; He, Junlin; Chen, Xuemei; Ding, YuBin; Wang, Yingxiong; Long, ChunLan; Shen, Lianju

    2014-01-01

    Embryo implantation is a complicated process involving a series of endometrial changes that depend on differential gene expression. MicroRNAs (miRNAs) are important for regulation of gene expression. Previous studies have shown that miRNAs may participate in the regulation of gene expression during embryo implantation. To explore the role of endometrial miRNAs in early murine pregnancy, we used microarrays to investigate whether miRNAs were differentially expressed in the mouse endometrium on pregnancy day 4 (D4) and day 6 (D6). The results demonstrated that 17 miRNAs were upregulated and 18 were downregulated (>2-fold) in D6 endometria compared to D4. We identified that mmu-miR-193 exhibited the highest upregulation on D6, and the upregulation of mmu-miR-193 before embryo implantation could reduce the embryo implantation rate. Further, we demonstrated that mmu-miR-193 influenced embryo implantation by regulating growth factor receptor-bound protein 7 expression. In summary, our study suggests that mmu-miR-193 plays an important role in embryo implantation. PMID:24336674

  17. MicroRNA-224 is involved in the regulation of mouse cumulus expansion by targeting Ptx3.

    PubMed

    Yao, Guidong; Liang, Meng; Liang, Ning; Yin, Mianmian; Lü, Mingrong; Lian, Jie; Wang, Yong; Sun, Fei

    2014-01-25

    MicroRNAs (miRNAs) are indicated to regulate ovarian development in a cell- or stage-specific manner. Our previous study showed that miR-224 is involved in TGF-β1-mediated follicular granulosa cell (GC) growth and estradiol (E2) production during the transition from the preantral to early antral stage by targeting Smad4. In this study, miR-224 was found to target pentraxin 3 (Ptx3), a gene critical for cumulus expansion during ovulation. In addition, PTX3 was up-regulated in mouse mural GCs and cumulus-oocyte complexes (COCs) by TGF-β1 treatment, which was partially mediated by miR-224. The effect of miR-224 during ovulation was further examined in vitro and in vivo by construction of an adenovirus-mediated expression vector for miR-224 (Ad-miR-224). In vitro studies demonstrated that miR-224 could perturb cumulus expansion in EGF-stimulated COCs by decreasing PTX3 secretion. In vivo studies also showed that injection of Ad-miR-224 into ovarian bursa decreased PTX3 expression and disrupted ovulation, which led to a decreased number of implantation sites and offspring being born. These results indicate that miR-224 may affect ovulation and subsequent embryo development by targeting Ptx3, suggesting potential roles for miRNAs in offering new treatments for ovulation disorder-associated infertility, or, conversely, designing new contraceptives.

  18. Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies

    PubMed Central

    Lee, Wei-Hua; Higuchi, Hitoshi; Ikeda, Sakae; Macke, Erica L; Takimoto, Tetsuya; Pattnaik, Bikash R; Liu, Che; Chu, Li-Fang; Siepka, Sandra M; Krentz, Kathleen J; Rubinstein, C Dustin; Kalejta, Robert F; Thomson, James A; Mullins, Robert F; Takahashi, Joseph S; Pinto, Lawrence H; Ikeda, Akihiro

    2016-01-01

    While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases. DOI: http://dx.doi.org/10.7554/eLife.19264.001 PMID:27863209

  19. Involvement of hypoxia-inducible factor-1 α (HIF-1α) in inhibition of benzene on mouse hematopoietic system.

    PubMed

    Meng, Xing; Zhang, Juan; Yin, Lihong; Pu, Yuepu

    2016-01-01

    Benzene is an occupational and environmental pollutant that damages the hematopoietic system through oxidant mechanisms. The aims of this study were to assess the role of oxidation in benzene-mediated damage by determination of the levels of reactive oxygen species (ROS) and to evaluate the role of hypoxia-inducible factor-1α (HIF-1α) in this process. C57BL/6 mice were exposed to benzene at varying concentrations of 60, 150, or 300 mg/kg/d for 15 d. Mice in the benzene groups displayed weight loss, and hematologic consequences including decreased red and white blood cell counts, reduced platelet count, diminished hemoglobin content, and lower number of hematopoietic stem cells in bone marrow (BM). There was an elevated proportional neutrophil count and decrease in relative thymus weight. In BM there was a significant increase in ROS levels at 150 mg/kg benzene. However, as a result of diminished cellular viability, ROS levels were not markedly different between the 300-mg/kg benzene dose and the control, as the number of hematopoietic stem cells was reduced. HIF-1α expression and protein levels were decreased in BM cells at all doses of benzene. In conclusion, data indicated that HIF-1α may be involved in benzene-induced inhibition of mouse hematopoiesis and that oxidative stress may play a role in the observed toxicity.

  20. Antidepressant-like effect of chromium chloride in the mouse forced swim test: involvement of glutamatergic and serotonergic receptors.

    PubMed

    Piotrowska, Anna; Młyniec, Katarzyna; Siwek, Agata; Dybała, Małgorzata; Opoka, Włodzimierz; Poleszak, Ewa; Nowak, Gabriel

    2008-01-01

    Chromium (Cr) (III), an essential microelement of living organisms, was reported to exhibit potential antidepressant properties in preclinical and clinical studies. The aim of the present study was to examine the effect of CrCl(3) ip administration in the forced swim test (FST) in mice and the involvement of glutamatergic and serotonergic receptors in the antidepressant-like activity of chromium. CrCl(3) in a dose of 12 mg/kg, but not in doses of 6 or 32 mg/kg, reduced the immobility time in the FST. The locomotor activity was reduced by CrCl(3) in a dose of 32 mg/kg. Moreover, the reduction of the immobility time induced by the active dose (12 mg/kg) of CrCl(3) was completely abolished by NBQX (10 mg/kg; an antagonist of the AMPA receptor) pretreatment and partially inhibited by ritanserin (4 mg/kg; an antagonist of 5-HT(2A/C) receptor), WAY 1006335 (0.1 mg/kg; an antagonist of 5-HT(1A) receptor) and N-methyl-D-aspartate (75 mg/kg; agonist of NMDA receptor) administration. The present study demonstrates the antidepressant-like activity of chromium in the mouse FST and indicates the major role of the AMPA receptor and participation of NMDA glutamatergic and 5-HT(1) and 5-HT(2A/C) serotonin receptors in this activity.

  1. Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein.

    PubMed

    Green, Teresa D; Park, Joungjoa; Yin, Qi; Fang, Shijing; Crews, Anne L; Jones, Samuel L; Adler, Kenneth B

    2012-09-01

    A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N-terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS-related peptide could affect migration of macrophages, using the mouse macrophage-like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP-1 (25-100 ng/ml) or C5a (5-20 ng/ml). Cells were preincubated (15 min) with MANS or a mis-sense control peptide (RNS), both at 50 μM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP-1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP-1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.

  2. Stem cell activity of type A spermatogonia is seasonally regulated in rainbow trout.

    PubMed

    Sato, Mana; Hayashi, Makoto; Yoshizaki, Goro

    2017-06-01

    Spermatogonial stem cells (SSCs) support continuous production of sperm throughout the male's life. However, the biological characteristics of SSCs are poorly understood in animals exhibiting seasonal reproduction, even though most wild animals are seasonal breeders. During the spermiation season in rainbow trout, the lumen of the testes contains only spermatozoa and scattered type A spermatogonia (ASG) along the walls of the testicular lobules. These few remaining ASG, designated "residual ASG," are the only germ cells capable of supporting the next spermatogenesis, suggesting that the residual ASG are true SSCs. However, whether residual ASG can behave as SSCs in any teleost species is unknown. In this study, we attempted to clarify the biological characteristics of SSCs associated with seasonal reproduction in rainbow trout using spermatogonial transplantation. We found that the stem cell activity was clearly regulated seasonally during the annual reproductive cycle. Although the residual ASG exhibited moderate transplantability and colony-forming ability at the beginning of the spermiation season, these parameters decreased dramatically later and remained low until the next spermatogenesis was initiated. Furthermore, no clear correlations were observed between these qualitative changes and previously described morphologic characteristics of ASG or plasma sex steroid levels. Our results suggest that the biological properties of SSC populations in rainbow trout are seasonally regulated by a novel mechanism. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Taurine plays an important role in the protection of spermatogonia from oxidative stress.

    PubMed

    Higuchi, Masato; Celino, Fritzie T; Shimizu-Yamaguchi, Sonoko; Miura, Chiemi; Miura, Takeshi

    2012-12-01

    It has been demonstrated that taurine has various physiological functions in the body. We demonstrated that taurine is abundant in the serum, liver, muscle and testis of the Japanese eel (Anguilla japonica). In the eel testis, taurine is found mainly in spermatogonia and is weakly expressed also in the Sertoli cells. We have further found in the eel testis that taurine is actively accumulated via the sodium/chloride-dependent taurine transporter (TauT; SLC6A6), which is expressed in germ cells. In our current study, the effects of taurine on the anti-oxidant response were examined. Taurine was found to promote the total superoxide dismutase (SOD) activity in the testis. Moreover, our results indicate that taurine does not affect the mRNA levels of copper-zinc (Cu/Zn) SOD or manganese SOD, but promotes the translation of Cu/Zn SOD. Overall, our present data suggest that taurine may modulate Cu/Zn SOD at the translational level and thereby may play an important role in the protection of germ cells from oxidative stress.

  4. Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss).

    PubMed

    Falahatkar, Bahram; Poursaeid, Samaneh; Kitada, Ryota; Yoshizaki, Goro

    2017-03-14

    A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.

  5. Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis.

    PubMed

    de Waal, Paul P; Leal, Marcelo C; García-López, Angel; Liarte, Sergio; de Jonge, Hugo; Hinfray, Nathalie; Brion, François; Schulz, Rüdiger W; Bogerd, Jan

    2009-08-01

    Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17beta-oestradiol (E(2)) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E(2) ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.

  6. Involvement of FLIP in 2-methoxyestradiol-induced tumor regression in transgenic adenocarcinoma of mouse prostate model.

    PubMed

    Ganapathy, Manonmani; Ghosh, Rita; Jianping, Xie; Zhang, Xiaoping; Bedolla, Roble; Schoolfield, John; Yeh, I-Tien; Troyer, Dean A; Olumi, Aria F; Kumar, Addanki P

    2009-03-01

    The purpose of this study is to investigate whether Fas-associated death domain interleukin-1 converting enzyme like inhibitory protein (FLIP) inhibition is a therapeutic target associated with 2-methoxyestradiol (2-ME2)-mediated tumor regression. Expression and levels of FLIP were analyzed using (a) real-time PCR and immunoblot analysis in androgen-independent PC-3 cells treated with the newly formulated 2-ME2 and (b) immunohistochemistry in different Gleason pattern human prostate tumors. Transient transfections and chromatin immunoprecipitation (ChIP) assays were used to identify the transcription factors that regulate FLIP. Involvement of FLIP in 2-ME2-induced tumor regression was evaluated in transgenic adenocarcinoma mouse prostate (TRAMP) mice. High Gleason pattern (5+5) human prostate tumors exhibit significant increase in FLIP compared with low Gleason pattern 3+3 (P=or<0.04). 2-ME2 reduced the levels and promoter activity of FLIP (P=0.001) in PC-3 cells. Transient expression assays show sequences between -503/+242 being sufficient for 2-ME2-induced inhibition of FLIP promoter activity. Cotransfection experiments show that overexpression of Sp1 activated, whereas Sp3 inhibited, Sp1 transactivation of FLIP promoter activity (P=0.0001). 2-ME2 treatment reduced binding of Sp1 to the FLIP promoter as evidenced by ChIP. Further, levels of FLIP associated with Fas or FADD decreased, whereas cleavage of caspase-8, levels of Bid, and apoptosis increased in response to 2-ME2 treatment in PC-3 cells. Administration of 2-ME2 regressed established prostate tumors in TRAMP mice that were associated with reduced expression of FLIP and Sp1. Targeting Sp1-mediated FLIP signaling pathway may provide a novel approach for prostate cancer management.

  7. Involvement of FLIP in 2-methoxyestradiol induced tumor regression in transgenic adenocarcinoma of mouse prostate (TRAMP) model

    PubMed Central

    Ganapathy, Manonmani; Ghosh, Rita; Jianping, Xie; Zhang, Xiaoping; Bedolla, Roble; Schoolfield, John; Yeh, I-Tien; Troyer, Dean A; Olumi, Aria F; Kumar, Addanki P

    2009-01-01

    Purpose The purpose of this study is to investigate whether Fas-associated death domain interleukin-1 converting enzyme like inhibitory protein (FLIP) inhibition is a therapeutic target associated 2-methoxyestradiol (2-ME2) mediated tumor regression. Experimental Design Expression and levels of FLIP was analyzed using (i) Real-Time PCR and immunoblot analysis in androgen independent PC-3 cells treated with the newly formulated 2-methoxyestradiol (2-ME2) and (ii) immunohistochemistry in different Gleason pattern human prostate tumors. Transient transfections and Chromatin Immunoprecipitation (ChIP) assays were used to identify the transcription factors that regulate FLIP. Involvement of FLIP in 2-ME2 -induced tumor regression was evaluated in transgenic adenocarcinoma mouse prostate (TRAMP) mice. Results High Gleason pattern (5+5) human prostate tumors exhibit significant increase in FLIP compared to low Gleason pattern 3+3 (p=<0.04). 2-ME2 reduced the levels and promoter activity of FLIP (p=0.001) in PC-3 cells. Transient expression assays show sequences between −503/+242 being sufficient for 2-ME2 induced inhibition of FLIP promoter activity. Co-transfection experiments show that overexpression of Sp1 activated, while Sp3 inhibited Sp1 transactivation of FLIP promoter activity (p=0.0001). 2-ME2 treatment reduced binding of Sp1 to the FLIP promoter as evidenced by ChIP. Further, levels of FLIP associated with Fas or FADD decreased, while cleavage of caspase-8, levels of Bid and apoptosis increased in response to 2-ME2 treatment in PC-3 cells. Administration of 2-ME2 regressed established prostate tumors in TRAMP mice that was associated with reduced expression of FLIP and Sp1. Conclusion Targeting Sp1 mediated FLIP signaling pathway may provide a novel approach for prostate cancer management. PMID:19223508

  8. c-Jun Amino-Terminal Kinase is Involved in Valproic Acid-Mediated Neuronal Differentiation of Mouse Embryonic NSCs and Neurite Outgrowth of NSC-Derived Neurons.

    PubMed

    Lu, Lu; Zhou, Hengxing; Pan, Bin; Li, Xueying; Fu, Zheng; Liu, Jun; Shi, Zhongju; Chu, Tianci; Wei, Zhijian; Ning, Guangzhi; Feng, Shiqing

    2017-04-01

    Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, can induce neuronal differentiation, promote neurite extension and exert a neuroprotective effect in central nervous system (CNS) injuries; however, comparatively little is known regarding its action on mouse embryonic neural stem cells (NSCs) and the underlying molecular mechanism. Recent studies suggested that c-Jun N-terminal kinase (JNK) is required for neurite outgrowth and neuronal differentiation during neuronal development. In the present study, we cultured mouse embryonic NSCs and treated the cells with 1 mM VPA for up to 7 days. The results indicate that VPA promotes the neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons; moreover, VPA induces the phosphorylation of c-Jun by JNK. In contrast, the specific JNK inhibitor SP600125 decreased the VPA-stimulated increase in neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons. Taken together, these results suggest that VPA promotes neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSC-derived neurons. Moreover, JNK activation is involved in the effects of VPA stimulation.

  9. Myosin light chain phosphatase activation is involved in the hydrogen sulfide-induced relaxation in mouse gastric fundus.

    PubMed

    Dhaese, Ingeborg; Lefebvre, Romain A

    2009-03-15

    The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP.

  10. Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloid-β peptide in mouse brain capillary endothelial cells.

    PubMed

    Ito, Shingo; Ohtsuki, Sumio; Murata, Sho; Katsukura, Yuki; Suzuki, Hiroya; Funaki, Miho; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-01-01

    Cerebral clearance of amyloid-β peptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

  11. Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts.

    PubMed

    Pedersen, Stine F; Beisner, Kristine H; Hougaard, Charlotte; Willumsen, Berthe M; Lambert, Ian H; Hoffmann, Else K

    2002-06-15

    The role of Rho GTPases in the regulatory volume decrease (RVD) process following osmotic cell swelling is controversial and has so far only been investigated for the swelling-activated Cl- efflux. We investigated the involvement of RhoA in the RVD process in NIH3T3 mouse fibroblasts, using wild-type cells and three clones expressing constitutively active RhoA (RhoAV14). RhoAV14 expression resulted in an up to fourfold increase in the rate of RVD, measured by large-angle light scattering. The increase in RVD rate correlated with RhoAV14 expression. RVD in wild-type cells was unaffected by the Rho kinase inhibitor Y-27632 and the phosphatidyl-inositol 3 kinase (PI3K) inhibitor wortmannin. The maximal rates of swelling-activated K+ (86 Rb+ as tracer) and taurine ([3H]taurine as tracer) efflux after a 30 % reduction in extracellular osmolarity were increased about twofold in cells with maximal RhoAV14 expression compared to wild-type cells, but were unaffected by Y-27632. The volume set points for activation of release of both osmolytes appeared to be reduced by RhoAV14 expression. The maximal taurine efflux rate constant was potentiated by the tyrosine phosphatase inhibitor Na(3)VO(4), and inhibited by the tyrosine kinase inhibitor genistein. The magnitude of the swelling-activated Cl- current (I(Cl,swell) ) was higher in RhoAV14 than in wild-type cells after a 7.5 % reduction in extracellular osmolarity, but, in contrast to 86Rb+ and [3H]taurine efflux, similar in both strains after a 30 % reduction in extracellular osmolarity. I(Cl,swell) was inhibited by Y-27632 and strongly potentiated by the myosin light chain kinase inhibitors ML-7 and AV25. It is suggested that RhoA, although not the volume sensor per se, is an important upstream modulator shared by multiple swelling-activated channels on which RhoA exerts its effects via divergent signalling pathways.

  12. Conditional Allele Mouse Planner (CAMP): software to facilitate the planning and design of breeding strategies involving mice with conditional alleles.

    PubMed

    Hoffert, Jason D; Pisitkun, Trairak; Miller, R Lance

    2012-06-01

    Transgenic and conditional knockout mouse models play an important role in biomedical research and their use has grown exponentially in the last 5-10 years. Generating conditional knockouts often requires breeding multiple alleles onto the background of a single mouse or group of mice. Breeding these mice depends on parental genotype, litter size, transmission frequency, and the number of breeding rounds. Therefore, a well planned breeding strategy is critical for keeping costs to a minimum. However, designing a viable breeding strategy can be challenging. With so many different variables this would be an ideal task for a computer program. To facilitate this process, we created a Java-based program called Conditional Allele Mouse Planner (CAMP). CAMP is designed to provide an estimate of the number of breeders, amount of time, and costs associated with generating mice of a particular genotype. We provide a description of CAMP, how to use it, and offer it freely as an application.

  13. Tumor growth affects the metabonomic phenotypes of multiple mouse non-involved organs in an A549 lung cancer xenograft model

    PubMed Central

    Xu, Shan; Tian, Yuan; Hu, Yili; Zhang, Nijia; Hu, Sheng; Song, Dandan; Wu, Zhengshun; Wang, Yulan; Cui, Yanfang; Tang, Huiru

    2016-01-01

    The effects of tumorigenesis and tumor growth on the non-involved organs remain poorly understood although many research efforts have already been made for understanding the metabolic phenotypes of various tumors. To better the situation, we systematically analyzed the metabolic phenotypes of multiple non-involved mouse organ tissues (heart, liver, spleen, lung and kidney) in an A549 lung cancer xenograft model at two different tumor-growth stages using the NMR-based metabonomics approaches. We found that tumor growth caused significant metabonomic changes in multiple non-involved organ tissues involving numerous metabolic pathways, including glycolysis, TCA cycle and metabolisms of amino acids, fatty acids, choline and nucleic acids. Amongst these, the common effects are enhanced glycolysis and nucleoside/nucleotide metabolisms. These findings provided essential biochemistry information about the effects of tumor growth on the non-involved organs. PMID:27329570

  14. Ultrastructure of spermatogonia and spermatocyte lobules in Taenia solium strobilae (Cestoda, Cyclophyllidea, Taeniidae) from golden hamsters.

    PubMed

    Willms, Kaethe; Caro, Jose Antonio; Robert, Lilia

    2003-08-01

    Golden hamsters ( Mesocricetus auratus) were infected with Taenia solium metacestodes dissected from infected pig meat. Adult worms were collected from hamster intestines of animals killed 5-60 days post-infection (dpi), incubated in RPMI 1640 medium with or without colchicine, fixed and processed for transmission electron microscopy (TEM). Sections for light microscopy from 40 different blocks with scolex, immature and mature proglottids were photographed. Thin sections were cut from 25 selected blocks, examined and photographed with TEM. Metaphase mitosis figures were observed in the subtegument of the germinative tissue and interpreted as germ cell precursors. In immature proglottids (20 dpi), discrete cell clusters of three to four cells surrounded by a thin cytoplasmic envelope were identified along the inner border of the lateral excretory ducts. These were also observed in more mature proglottids (40-60 dpi) as clusters of eight cells enclosed in a cytoplasmic envelope, with nuclei of spermatogonia exhibiting the synaptolems of primary meiotic cells. In mature proglottids from 45 dpi, a large number of spermatocyte lobules were found, exhibiting different stages of spermatogenesis from primary spermatocytes to mature filiform spermatids with a single axoneme, annular nucleus and spiral cortical microtubules, similar to spermatozoa described for type III spermiogenesis of species of the family Taeniidae. All mature spermatocyte lobules were enclosed in a highly organized cellular envelope and surrounded by a basal lamina. The envelopes contained a number of distinct organelles, seen in cross-section as discrete lattices of microtubules located between two layers of plasma membrane, as well as thickened furled cytoplasm with numerous strands of rough endoplasmic reticulum and pockets of microtubules.

  15. Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos.

    PubMed

    Jin, Yong-Xun; Zheng, Zhong; Yu, Xian-Feng; Zhang, Jia-Bao; Namgoong, Suk; Cui, Xiang-Shun; Hyun, Sang-Hwan; Kim, Nam-Hyung

    2016-02-01

    The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

  16. Mono-(2-ethylhexyl)-phthalate (MEHP) affects ERK-dependent GDNF signalling in mouse stem-progenitor spermatogonia.

    PubMed

    Lucas, Benjamin E G; Fields, Christopher; Joshi, Neeraj; Hofmann, Marie-Claude

    2012-09-04

    Many commercial and household products such as lubricants, cosmetics, plastics, and paint contain phthalates, in particular bis-(2-ethyhexyl)-phthalate (DEHP). As a consequence, phthalates have been found in a number of locations and foods (streambeds, household dust, bottled water and dairy products). Epidemiological and animal studies analysing phthalate exposure in males provide evidence of degradation in sperm quality, associated to an increase in the incidence of genital birth defects and testicular cancers. In the testis, spermatogenesis is maintained throughout life by a small number of spermatogonial stem cells (SSCs) that self-renew or differentiate to produce adequate numbers of spermatozoa. Disruption or alteration of SSC self-renewal induce decreased sperm count and sperm quality, or may potentially lead to testicular cancer. GDNF, or glial cell-line-derived neurotrophic factor, is a growth factor that is essential for the self-renewal of SSCs and continuous spermatogenesis. In the present study, the SSC-derived cell line C18-4 was used as a model for preliminary assessment of the effects of mono-(2-ethylhexyl)-phthalate (MEHP, main metabolite of DEHP) on spermatogonial stem cells. Our data demonstrate that MEHP disrupts one of the known GDNF signalling pathways in these cells. MEHP induced a decrease of C18-4 cell viability in a time- and dose-dependent manner, as well as a disruption of ERK1/2 activation but not of SRC signalling. As a result, we observed a decrease of expression of the transcription factor FOS, which is downstream of the GDNF/ERK1/2 axis in these cells. Taken together, our data suggest that MEHP exposure affects SSC proliferation through inhibition of specific signalling molecules. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Adaptation of the targeted capture Methyl-Seq platform for the mouse genome identifies novel tissue-specific DNA methylation patterns of genes involved in neurodevelopment

    PubMed Central

    Hing, Benjamin; Ramos, Enrique; Braun, Patricia; McKane, Melissa; Jancic, Dubravka; Tamashiro, Kellie L K; Lee, Richard S; Michaelson, Jacob J; Druley, Todd E; Potash, James B

    2015-01-01

    Methyl-Seq was recently developed as a targeted approach to assess DNA methylation (DNAm) at a genome-wide level in human. We adapted it for mouse and sought to examine DNAm differences across liver and 2 brain regions: cortex and hippocampus. A custom hybridization array was designed to isolate 99 Mb of CpG islands, shores, shelves, and regulatory elements in the mouse genome. This was followed by bisulfite conversion and sequencing on the Illumina HiSeq2000. The majority of differentially methylated cytosines (DMCs) were present at greater than expected frequency in introns, intergenic regions, near CpG islands, and transcriptional enhancers. Liver-specific enhancers were observed to be methylated in cortex, while cortex specific enhancers were methylated in the liver. Interestingly, commonly shared enhancers were differentially methylated between the liver and cortex. Gene ontology and pathway analysis showed that genes that were hypomethylated in the cortex and hippocampus were enriched for neuronal components and neuronal function. In contrast, genes that were hypomethylated in the liver were enriched for cellular components important for liver function. Bisulfite-pyrosequencing validation of 75 DMCs from 19 different loci showed a correlation of r = 0.87 with Methyl-Seq data. We also identified genes involved in neurodevelopment that were not previously reported to be differentially methylated across brain regions. This platform constitutes a valuable tool for future genome-wide studies involving mouse models of disease. PMID:25985232

  18. Sp7/Osterix is involved in the up-regulation of the mouse pro-α1(V) collagen gene (Col5a1) in osteoblastic cells.

    PubMed

    Wu, Yun-Feng; Matsuo, Noritaka; Sumiyoshi, Hideaki; Yoshioka, Hidekatsu

    2010-10-01

    Sp7/Osterix, a transcription factor whose expression is restricted in osteoblasts, belongs to the Sp family of transcription factor that bind to G/C-rich sequences. Previous studies have identified a Sp1binding site in the proximal promoter region of the mouse Col5a1 gene, but it did not activate or repress this gene in a mouse fibroblast cell line and a human rhabdomyosarcoma cell line. The purpose of the present study was to clarify the involvement of Sp7/Osterix in the mouse Col5a1 gene. A functional analysis revealed that mutation of the Sp1 binding site specifically decreased the promoter activity in osteoblastic cells. An overexpression of Sp7/Osterix significantly increased the promoter activity and the endogenous mRNA levels of the Col5a1 gene in osteoblastic cells. Conversely, siRNA-mediated knockdown of Sp7/Osterix decreased the promoter activity and the endogenous mRNA levels of the Col5a1 gene. These effects on promoter activity were canceled when the mutant construct of Sp1 binding site was introduced. Consistent with these data, the experiments using an osteoblast differentiation model showed increased promoter activity and endogenous mRNA levels, along with increased Sp7/Osterix during differentiation. Therefore, type V collagen appears to be involved in bone formation.

  19. Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels.

    PubMed

    Brennan, Sarah C; Finney, Brenda A; Lazarou, Maria; Rosser, Anne E; Scherf, Caroline; Adriaensen, Dirk; Kemp, Paul J; Riccardi, Daniela

    2013-01-01

    Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+) channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match

  20. Spermatogonia, Germline Cells, and Testicular Organization in the Characiform Prochilodus lineatus Studied Using Histological, Stereological, and Morphometric Approaches.

    PubMed

    De Melo Dias, Gisele Cristiane; Cassel, Mônica; Oliveira De Jesus, Lázaro Wender; Batlouni, Sergio Ricardo; Borella, Maria Inés

    2017-03-01

    Prochilodus lineatus is an important representative of the order Characiformes and a species that offers great advantages to fish farming. Therefore, detailed knowledge of its reproductive biology can be applied to various fields of production and biotechnology. In this study, we have identified testicular germ cells during spermatogenesis and have evaluated the volumetric proportion of the testes occupied by structures of the tubular and intertubular compartments. In addition, the individual volume of type A spermatogonia was measured and used to estimate the mean number of these cells per testis. Gonads of adult P. lineatus males were extracted and fixed. Light and transmission electron microscopy were applied to fragments of three testicular regions. Histological, stereological, and morphometric analyses were performed. The stereological data suggest that components of the tubular and intertubular compartments of the P. lineatus testes present a uniform distribution in all three regions and therefore reflect regions with similar distributions of cell types. In addition, P. lineatus testes showed ∼0.6% of type A spermatogonia, as well as a predominance of cysts of primary spermatocytes and spermatids during the reproductive phase evaluated. The results from this study provide a better understanding of the morphology and structure of the testis and of the characterization of the type A spermatogonia in P. lineatus. The nuclear diameter of germ cells also decreases significantly during spermatogenesis. The data presented herein are the first of its kind for the order Characiformes and may be useful for future biotechnology studies on fish reproduction. Anat Rec, 300:589-599, 2017. © 2016 Wiley Periodicals, Inc.

  1. In-depth behavioral characterization of the corticosterone mouse model and the critical involvement of housing conditions.

    PubMed

    Demuyser, Thomas; Deneyer, Lauren; Bentea, Eduard; Albertini, Giulia; Van Liefferinge, Joeri; Merckx, Ellen; De Prins, An; De Bundel, Dimitri; Massie, Ann; Smolders, Ilse

    2016-03-15

    Depression and anxiety are disabling and highly prevalent psychiatric disorders. To better understand the neurobiological basis of mood and anxiety disorders, relevant animal models are needed. The corticosterone mouse model is frequently used to study depression. Chronic stress and accompanying glucocorticoid elevation causes pathological changes in the central nervous system, which are related to psychiatric symptoms. Exogenous administration of corticosterone is therefore often used to induce depressive-like behavior in mice and in some cases also features of anxiety-like behavior are shown. However, a thorough characterization of this model has never been conducted and housing conditions of the used subjects often differ between the implemented protocols. We chronically administered a subcutaneous corticosterone bolus injection to single- and group-housed mice, and we subsequently evaluated the face validity of this model by performing a battery of behavioral tests (forced swim test, mouse-tail suspension test, saccharin intake test, novelty-suppressed feeding test, elevated plus maze, light/dark paradigm and open field test). Our results show that corticosterone treatment has a substantial overall effect on depressive-like behavior. Increases in anxiety-like behavior on the other hand are mainly seen in single housed animals, independent of treatment. The current study therefore does not only show a detailed behavioral characterization of the corticosterone mouse model, but furthermore also elucidates the critical influence of housing conditions on the behavioral outcome in this model.

  2. TRAIL is involved in the tumoricidal activity of mouse natural killer cells stimulated by Newcastle disease virus in vitro.

    PubMed

    Song, De-Zhi; Liang, Ying; Xiao, Qing; Yin, Jun; Gong, Jin-Ling; Lai, Zhen-Ping; Zhang, Zeng-Feng; Gao, Ling-Xi; Fan, Xiao-Hui

    2013-10-01

    Newcastle disease virus (NDV) is a potential antitumor agent, and its antitumor effect has been evaluated in preclinical tests. However, the mechanisms of NDV-based antitumor therapy are still not completely clear. In the present study we found that NDV-stimulation enhanced the killing ability of mouse spleen natural killer (NK) cells towards mouse hepatoma cell lines, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) plays an important role in this tumoricidal activity. NDV stimulation induced up-regulation of TRAIL both at the mRNA and protein levels in NK cells. Blocking TRAIL by antibody (Ab) almost completely eliminated the killing effect of NK cells on hepatoma cell lines. Furthermore, neutralizing interferon (IFN)-γ by Ab could inhibit TRAIL expression and tumoricidal activity of NDV-stimulated NK cells. These results indicated a substantial role of TRAIL as an effector molecule in NDV-induced NK cells mediated tumoricidal activity. The NDV stimulation triggered TRAIL expression in mouse spleen NK cells could be mediated by IFN-γ induction. Copyright © 2013 Wiley Periodicals, Inc.

  3. The SMC5/6 complex is involved in crucial processes during human spermatogenesis.

    PubMed

    Verver, Dideke E; Langedijk, Nathalia S M; Jordan, Philip W; Repping, Sjoerd; Hamer, Geert

    2014-07-01

    Genome integrity is crucial for safe reproduction. Therefore, chromatin structure and dynamics should be tightly regulated during germ cell development. Chromatin structure and function are in large part determined by the structural maintenance of chromosomes (SMC) protein complexes, of which SMC5/6 recently has been shown to be involved in both spermatogonial differentiation and meiosis during mouse spermatogenesis. We therefore investigated the role of this complex in human spermatogenesis. We found SMC6 to be expressed in the human testis and present in a subset of type Adark and type Apale spermatogonia, all spermatocytes, and round spermatids. During human meiosis, SMC5/6 is located at the synaptonemal complex (SC), the XY body, and at the centromeres during meiotic metaphases. However, in contrast to mouse spermatogenesis, SMC6 is not located at pericentromeric heterochromatin in human spermatogenic cells, indicating subtle but perhaps important differences in not only SMC5/6 function but maybe also in maintenance of genomic integrity at the repetitive pericentromeric regions. Nonetheless, our data clearly indicate that the SMC5/6 complex, as shown in mice, is involved in numerous crucial processes during human spermatogenesis, such as in spermatogonial development, on the SC between synapsed chromosomes, and in DNA double-strand break repair on unsynapsed chromosomes during pachynema.

  4. Possible involvement of nitric oxide (NO) signaling pathway in the antidepressant-like effect of MK-801(dizocilpine), a NMDA receptor antagonist in mouse forced swim test.

    PubMed

    Dhir, Ashish; Kulkarni, S K

    2008-03-01

    L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) is an important signaling pathway involved in depression. With this information, the present study aimed to study the involvement of this signaling pathway in the antidepressant-like action of MK-801 (dizocilpine; N-methyl-d-aspartate receptor antagonist) in the mouse forced-swim test. Total immobility period was recorded in mouse forced swim test for 6 min. MK-801 (5-25 microg/kg., ip) produced a U-shaped curve in reducing the immobility period. The antidepressant-like effect of MK-801 (10 microg/kg, ip) was prevented by pretreatment with L-arginine (750 mg/kg, ip) [substrate for nitric oxide synthase (NOS)]. Pretreatment of mice with 7-nitroindazole (7-NI) (25 mg/kg, ip) [a specific neuronal nitric oxide synthase inhibitor] produced potentiation of the action of subeffective dose of MK-801 (5 microg/kg, ip). In addition, treatment of mice with methylene blue (10 mg/kg, ip) [direct inhibitor of both nitric oxide synthase and soluble guanylate cyclase] potentiated the effect of MK-801 (5 microg/kg, ip) in the forced-swim test. Further, the reduction in the immobility period elicited by MK-801 (10 microg/kg, ip) was also inhibited by pretreatment with sildenafil (5 mg/kg, ip) [phosphodiesterase 5 inhibitor]. The various modulators used in the study and their combination did not produce any changes in locomotor activity per se and in combination with MK-801. MK-801 however, at higher doses (25 microg/kg, ip) produced hyperlocomotion. The results demonstrated the involvement of nitric oxide signaling pathway in the antidepressant-like effect of MK-801 in mouse forced-swim test.

  5. Revealing Transcriptome Landscape of Mouse Spermatogonial Cells by Tiling Microarray

    PubMed Central

    Lee, Tin-Lap.; Rennert, Owen M.; Chan, Wai-Yee.

    2014-01-01

    Summary Spermatogenesis is a highly regulated developmental process by which spermatogonia develop into mature spermatozoa. This process involves many testis- or male germ cell-specific events through tightly regulated gene expression programs. In the past decade the advent of microarray technologies has allowed functional genomic studies of male germ cell development, resulting in the identification of genes governing various processes. A major limitation with conventional gene expression microarray is that there is a bias from gene probe design. The gene probes for expression microarrays are usually represented by a small number probes located at the 3’ end of a transcirpt. Tiling microarrays eliminate such issue by interrogating the genome in an unbiased fashion through probes tiled for the entire genome. These arrays provide a higher genomic resolution and allow identification of novel transcripts. To reveal the complexity of the genomic landscape of developing male germ cells, we applied tiling microarray to evaluate the transcriptome in spermatogonial cells. Over 50% of the mouse and rat genome are expressed during testicular development. More than 47% of transcripts are uncharacterized. The results suggested the transcription machinery in spermaotogonial cells are more complex than previously envisioned. PMID:22144238

  6. Involvement of dopamine D1 receptors and alpha1-adrenoceptors in the antidepressant-like effect of chlorpheniramine in the mouse tail suspension test.

    PubMed

    Hirano, Shoko; Miyata, Shigeo; Onodera, Kenji; Kamei, Junzo

    2007-05-07

    It has been reported that chlorpheniramine, a classical antihistamine, has antidepressant-like effects in animal models of depression. In this study, we examined the involvement of dopaminergic (dopamine D(1) and dopamine D(2) receptors), noradrenergic (alpha(1)- and beta-adrenoceptors) and serotonergic (5-HT(1A) and 5-HT(2) receptors) receptors in the antidepressant-like effect of chlorpheniramine in the mouse tail suspension test. We also investigated the involvement of these monoamine receptors in the antidepressant-like effect of imipramine for comparison with the mechanisms of the effect of chlorpheniramine. Both imipramine and chlorpheniramine significantly reduced the duration of immobility in the tail suspension test without affecting spontaneous locomotor activity in mice. The anti-immobility effect of imipramine (30 mg/kg, i.p.) was significantly antagonized by the selective dopamine D(1) receptor antagonist SCH23390 but not by the other receptor antagonists. In contrast, the anti-immobility effect of chlorpheniramine was significantly inhibited by SCH23390 and the selective alpha(1)-adrenoceptor antagonist prazosin, but not by the other receptor antagonists. In conclusion, these results suggest that chlorpheniramine exerts an antidepressant-like effect in the mouse tail suspension test that is mediated by at least the activation of dopamine D(1) receptors and alpha(1)-adrenoceptors. In addition, the antidepressant-like effect of chlorpheniramine may be induced by several mechanisms that are different from those involved in the antidepressant-like effect of imipramine.

  7. Rapamycin-sensitive mTORC1 signaling is involved in physiological primordial follicle activation in mouse ovary.

    PubMed

    Tong, Yuanyuan; Li, Fei; Lu, Yi; Cao, Yanlan; Gao, Jimin; Liu, Jianghuai

    2013-12-01

    In mammals, resting female oocytes reside in primordial ovarian follicles. An individual primordial follicle may stay quiescent for a protracted period of time before initiating follicular growth, which is also termed “activation.” Female reproductive capacity is sustained by the gradual, streamlined activation of the entire population of primordial follicles, but this process also results in reproductive senescence in older animals. Based on the recent findings that genetically triggered, excessive mammalian target of rapamycin complex 1 (mTORC1) activation in mouse oocytes leads to accelerated primordial follicle activation, we examined the necessity of mTORC1 signaling in physiological primordial follicle activation. We found that induction of oocyte mTORC1 activity is associated with early follicular growth in neonatal mouse ovaries. Pharmacological inhibition of mTORC1 activity in vivo by rapamycin treatment leads to a marked, but partial, suppression of primordial follicle activation. The suppressive effect of rapamycin on primordial follicle activation was reproduced in cultured ovaries. While rapamycin did not apparently affect several plausible cellular targets in neonatal mouse ovaries, such as mTORC2, AKT, or cyclin-dependent kinase (CDK) inhibitor p27-KIP1, its inhibitory effect on Cyclin A2 gene expression implies that mTORC1 signaling in oocytes may engage a Cyclin A/CDK regulatory network that promotes primordial follicle activation. The current work strengthens the concept that mTORC1-dependent events in the oocytes of primordial follicles may represent potential targets for intervention in humans to slow the depletion of the ovarian reserve. © 2013 Wiley Periodicals, Inc.

  8. mTOR/P70S6K promotes spermatogonia proliferation and spermatogenesis in Sprague Dawley rats.

    PubMed

    Xu, Hao; Shen, Lianju; Chen, Xuemei; Ding, Yubin; He, Junlin; Zhu, Jing; Wang, Yingxiong; Liu, Xueqing

    2016-02-01

    Spermatogenesis is a critical process for maintaining male fertility. Sustained spermatogonial stem cell self-renewal and differentiation ensures constant spermatogenesis, and several signalling pathways regulate this process. An increasing number of studies have suggested that the mammalian target of rapamycin (mTOR) signalling pathway plays an important role in spermatogenesis; however, the mechanism remains unknown. Our study showed that mTOR was positively related with spermatogenesis by detecting mTOR expression and the expression of its target p70s6k, rps6 and 4e-bp1 at different developmental stages. Phosphorylated p70s6k, rps6 and 4ebp1 levels were independently and gradually down-regulated with age. Subsequently, we showed in vivo and in vitro that, upon mTOR inactivation by rapamycin, the number of sperm significantly decreased (P < 0.05) and spermatogonia proliferation was blocked. Phosphorylated p70s6k and rps6 levels were down-regulated, but the levels of phosphorylated 4e-bp1 did not change. Spermatogonia were treated with the specific PI3K inhibitor LY294002, and p70s6k, rps6 and 4ebp1 phosphorylation overtly decreased. Therefore, we suggest that mTOR plays an important role in spermatogenesis by regulating p70s6k activation and that 4e-bp1 is either directly or indirectly regulated by PI3K.

  9. Stage-specific DNA synthesis of rat spermatogenesis as an indicator of genotoxic effects of vinblastine, mitomycin C and ionizing radiation on rat spermatogonia and spermatocytes.

    PubMed

    Sjöblom, T; Parvinen, M; Lähdetie, J

    1995-10-01

    We have studied the effects of three known mutagens: vinblastine sulphate, mitomycin C and local irradiation of testes on the stage-specific DNA synthesis in the rat testis by using transillumination assisted microdissection of rat seminiferous tubules. It enables us to investigate the sensitivity of different types of spermatogonia and preleptotene spermatocytes to the genotoxic effects of these agents. According to our results, spermatogonia and preleptotene spermatocytes are quite resistant to the action of vinblastine at the treatment times and the doses used. After treatment with mitomycin C, type A2, A3 and A4 spermatogonia seem to be the first cell types affected, which shows itself as a reduction in the DNA synthesis at stages I, II-III, XIII-XIV of the epithelial cycle two and/or three days after the treatment. It also seems that they are mostly affected during the S-phase of their cell cycles. In addition, preleptotene spermatocytes are also sensitive to the action of mitomycin C when they are treated in the G1 phase of the cell cycle. The local irradiation of 3 Gy has severe effects on the spermatogonia of rat testis which can be seen already 18 h after the treatment and becomes more evident 42 and 66 h after the treatment as a reduction of DNA synthesis at stages XII-V. Type A spermatogonia (A1-A4) seem to be the most sensitive cell types to the action of irradiation. This study indicates that the novel method of stage-specific DNA synthesis in rat spermatogenesis allows detailed studies of sensitivities in differentiating spermatogonia to genotoxic agents.

  10. The guidance molecule Semaphorin3A is differentially involved in the arealization of the mouse and primate neocortex.

    PubMed

    Homman-Ludiye, Jihane; Bourne, James A

    2014-11-01

    The visual cortex is organized into discrete domains characterized by their specific function, connectivity, chemoarchitecture, and cytoarchitecture. Gradients of transcription factors across the anteroposterior and mediolateral axes of the neocortex have previously been demonstrated to specify the main sensory regions. However, they do not account for the establishment of multiple areas in the primate visual cortex, which occupies approximately 50% of the neocortical surface. We demonstrate that the guidance molecule Semaphorin3A (Sema3A) is initially secreted in the cortical plate of the embryonic marmoset monkey and acts as an intrinsic cue to control the migration of subpopulations of neuronal progenitors and projection neurons expressing the receptor Neuropilin 1 (Npn1). During the first 2 postnatal weeks, Sema3A expression becomes primarily associated with ventral visual cortical areas, leading to the specific migration of Npn1+ neurons in the late maturing visual areas. In the mouse, Sema3A distribution is not arealized, but Npn1 expression becomes restricted to the posterior neocortex at embryonic day 16.5. The selective reduction in the striate cortex we observe in Sema3A-/- animals potentially results from the differential distribution of Npn1+ cells. Therefore, the Sema3A/Npn1 pathway participates to the parcellation of the visual neocortex in both the mouse and the marmoset, however, through different regulatory processes.

  11. Naegleria fowleri glycoconjugates with residues of α-D-mannose are involved in adherence of trophozoites to mouse nasal mucosa.

    PubMed

    Carrasco-Yepez, Maricela; Campos-Rodriguez, Rafael; Godinez-Victoria, Marycarmen; Rodriguez-Monroy, Marco Aurelio; Jarillo-Luna, Adriana; Bonilla-Lemus, Patricia; De Oca, Arturo Contis-Montes; Rojas-Hernandez, Saul

    2013-10-01

    We analyzed the possible role of glycoconjugates containing α-D-mannose and α-D-glucose residues in adherence of trophozoites to mouse nasal epithelium. Trophozoites incubated with 20 μg of one of three different lectins which preferentially recognized these residues were inoculated intranasally in Balb/c mice. Mouse survival was 40% with Pisum sativum and Canavalia ensiformis and 20% with Galanthus nivalis amebic pretreatment, compared with 0% survival for control animals administered trophozoites without pretreatment. Possibly some of the glycoproteins found in Naegleria fowleri represent an adherence factor. Differences in the saccharide sequences of the Naegleria species, even on the same glycoconjugate structure, could explain the different results corresponding to the distinct pretreatments (C. ensiformis, G. nivalis, and P. sativum). We found a higher expression of glycoconjugates recognized by P. sativum in Naegleria lovaniensis than N. fowleri, probably due to the higher number of oligosaccharides containing an α-1,6-linked fucose moiety expressed on the former species.

  12. Generation of a KOR-Cre Knockin Mouse Strain to Study Cells Involved in Kappa Opioid Signaling

    PubMed Central

    Kuzirian, Marissa S.; Snyder, Lindsey M.; Matsushita, Megumi; Lee, Michael C.; Ferguson, Carolyn; Homanics, Gregg E.; Barth, Alison L.; Ross, Sarah E.

    2015-01-01

    The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non-neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR-Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR-Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR-Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR-Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR-Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR. PMID:26575788

  13. Inhibitory effect of genistein on mouse colon cancer MC-26 cells involved TGF-{beta}1/Smad pathway

    SciTech Connect

    Yu Zengli . E-mail: zengliy@yahoo.com.cn; Tang Yunan; Hu Dongsheng; Li Juan

    2005-08-05

    TGF-{beta}1/signaling has been shown to be associated with proapoptotic and antimitotic activities in epithelial tissues. Genistein, a major component of soybean isoflavone, has multiple functions resulting in anticancer proliferation. We herein showed that genistein dose-dependently increased TGF-{beta}1 mRNA expression in mouse colon cancer MC-26 cells. A mouse monoclonal anti-TGF-{beta}1 neutralizing antibody partially, but not completely, blocked the growth inhibition by genistein. By using adenoviral vector, we demonstrated that Smad7 overexpression attenuated genistein-induced growth inhibition and apoptosis as determined by MTT and apoptosis ELISA. Smad7 overexpression also inhibited upregulation of p21 and caspase-3 activity by geinistein. To further confirm inhibitory effect of genistein in MC-26 cells require TGF-{beta}1/Smad signaling, we employed Western blot and electrophoretic mobility shift assay to detect formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, respectively. Data revealed that genistein induced an evident formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, indicating increased TGF-{beta}1 signaling. Taken together, these findings first provided insights into possible molecular mechanisms of growth inhibition by genistein that required Smad signaling, which could aid in its evaluation for colon tumor prevention.

  14. Neurotoxic activity of venom from the Australian Eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating

    PubMed Central

    Rash, Lachlan D; Birinyi-Strachan, Liesl C; Nicholson, Graham M; Hodgson, Wayne C

    2000-01-01

    Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 μl ml−1 failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 μl ml−1) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations.In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 μl ml−1) caused rapid fasciculations and an increase in indirectly evoked twitches.Male venom (0.02 and 0.05 μl ml−1) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 μl ml−1) were attenuated by (+)-tubocurarine (100 μM) and by tetrodotoxin (TTX, 1 μM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml−1).In the unstimulated rat vas deferens, male venom (0.05 μl ml−1) caused contractions which were inhibited by a combination of prazosin (0.3 μM) and P2X-receptor desensitization (with α,β-methylene ATP 10 μM). In the rat stimulated vas deferens, male venom (0.05 μl ml−1) augmented indirectly evoked twitches.Male venom (0.1 μl ml−1) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons.These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the δ-atracotoxins from Australian funnel-web spiders. PMID:10952670

  15. Neurotoxic activity of venom from the Australian eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating.

    PubMed

    Rash, L D; Birinyi-Strachan, L C; Nicholson, G M; Hodgson, W C

    2000-08-01

    Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 microl ml(-1) failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 microl ml(-1)) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 microl ml(-1)) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 microl ml(-1)) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 microl ml(-1)) were attenuated by (+)-tubocurarine (100 microM) and by tetrodotoxin (TTX, 1 microM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml(-1)). In the unstimulated rat vas deferens, male venom (0.05 microl ml(-1)) caused contractions which were inhibited by a combination of prazosin (0.3 microM) and P(2X)-receptor desensitization (with alpha,beta-methylene ATP 10 microM). In the rat stimulated vas deferens, male venom (0.05 microl ml(-1)) augmented indirectly evoked twitches. Male venom (0.1 microl ml(-1)) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the delta-atracotoxins from Australian funnel-web spiders.

  16. Influence of histological degree of seminiferous tubular degeneration and stage of seminiferous cycle on the proliferation of spermatogonia in aged Syrian hamster (Mesocricetus auratus).

    PubMed

    Bernal-Mañas, C M; Cortes, S; Morales, E; Horn, R; Seco-Rovira, V; Beltran-Frutos, E; Ferrer, C; Canteras, M; Pastor, L M

    2014-08-01

    The ageing testis is associated with germ loss in the seminiferous epithelium and a decrease in spermatogonia proliferation. In this work, we study whether the stages of the seminiferous epithelium cycle and/or the degree of histological tubular degeneration resulting from ageing is related with this decrease in spermatogonia proliferation. Eleven hamsters were used, five aged 6 months and six aged 24 months. In both groups, the proliferative activity was studied by BrdU immunostaining. The number of BrdU-positive and BrdU-negative cells was measured, providing the overall proliferation index in adult and aged testes. The mean number of BrdU-positive cells was also determined for each degree of histological degeneration of seminiferous epithelium, and a spermatogonia proliferation index was obtained for each stage of the seminiferous cycle. Ageing caused an overall decrease in the BrdU-positive cell percentage and a decrease in the number of BrdU-positive cells in the tubular sections with hypospermatogenesis, the sloughing of germ cells and maturation arrest, these changes being similar in both young and old animals. The spermatogonia proliferation index was only seen to be significantly lower in ageing hamster in stages VII-VIII of the seminiferous epithelium cycle. In conclusion, the overall decrease in proliferation observed in aged seminiferous epithelium is correlated with an increase in the number of degenerated sections of the seminiferous tubules, and this decrease is a phenomenon which occurs in specific stages of the seminiferous cycle.

  17. Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types.

    PubMed

    Iliopoulos, Dimitrios; Hirsch, Heather A; Struhl, Kevin

    2011-05-01

    Metformin, the first-line drug for treating diabetes, selectively kills the chemotherapy resistant subpopulation of cancer stem cells (CSC) in genetically distinct types of breast cancer cell lines. In mouse xenografts, injection of metformin and the chemotherapeutic drug doxorubicin near the tumor is more effective than either drug alone in blocking tumor growth and preventing relapse. Here, we show that metformin is equally effective when given orally together with paclitaxel, carboplatin, and doxorubicin, indicating that metformin works together with a variety of standard chemotherapeutic agents. In addition, metformin has comparable effects on tumor regression and preventing relapse when combined with a four-fold reduced dose of doxorubicin that is not effective as a monotherapy. Finally, the combination of metformin and doxorubicin prevents relapse in xenografts generated with prostate and lung cancer cell lines. These observations provide further evidence for the CSC hypothesis for cancer relapse, an experimental rationale for using metformin as part of combinatorial therapy in a variety of clinical settings, and for reducing the chemotherapy dose in cancer patients.

  18. Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs.

    PubMed

    Connors, S A; Kanatsu-Shinohara, M; Schultz, R M; Kopf, G S

    1998-08-01

    Exocytosis of cortical granules in mouse eggs is required to produce the zona pellucida block to polyspermy. In this study, we examined the role of microfilaments and microtubules in the regulation of cortical granule movement toward the cortex during oocyte maturation and anchoring of cortical granules in the cortex. Fluorescently labeled cortical granules, microfilaments, and microtubules were visualized using laser-scanning confocal microscopy. It was observed that cortical granules migrate to the periphery of the oocyte during oocyte maturation. This movement is blocked by the treatment of oocytes with cytochalasin D, an inhibitor of microfilament polymerization, but not with nocodazole or colchicine, inhibitors of microtubule polymerization. Cortical granules, once anchored at the cortex, remained in the cortex following treatment of metaphase II-arrested eggs with each of these inhibitors; i.e., there was neither inward movement nor precocious exocytosis. Finally, the single cortical granule-free domain that normally becomes localized over the metaphase II spindle was not observed when the chromosomes become scattered following microtubule disruption with nocodazole or colchicine. In these instances a cortical granule-free domain was observed over each individual chromosome, suggesting that the chromosome or chromosome-associated material, and not the spindle, dictates the localization of the cortical granule-free domain.

  19. Involvement of NMDA receptors in the antidepressant-like effect of tramadol in the mouse forced swimming test.

    PubMed

    Ostadhadi, Sattar; Norouzi-Javidan, Abbas; Chamanara, Mohsen; Akbarian, Reyhaneh; Imran-Khan, Muhammad; Ghasemi, Mehdi; Dehpour, Ahmad-Reza

    2017-09-01

    Tramadol is an analgesic agent that is mainly used to treat moderate to severe pain. There is evidence that tramadol may have antidepressant property. However, the mechanisms underlying the antidepressant effects of tramadol have not been elucidated yet. Considering that fact that N-methyl-d-aspartate (NMDA) receptor signaling may play an important role in the pathophysiology of depression, the aim of the present study was to investigate the role of NMDA receptor signaling in the possible antidepressant-like effects of tramadol in the mouse forced swimming test (mFST). We found that tramadol exerted antidepressant-like effects at high dose (40mg/kg, intraperitoneally [i.p.]) in the mFST. Co-administration of non-effective doses of NMDA receptor antagonists (ketamine [1mg/kg, i.p.], MK-801 [0.05mg/kg, i.p.], or magnesium sulfate [10mg/kg, i.p.]) with sub-effective dose of tramadol (20mg/kg, i.p.) exerted significant antidepressant-like effects in the mFST. The antidepressant-like effects of tramadol (40mg/kg) was also inhibited by pre-treatment with non-effective dose of the NMDA receptor agonist NMDA (75mg/kg, i.p.). Our data suggest a role for NMDA receptor signaling in the antidepressant-like effects of tramadol in the mFST. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: Evidence for involvement of splicing regulatory proteins

    PubMed Central

    Huo, Qing; Kayikci, Melis; Odermatt, Philipp; Meyer, Kathrin; Michels, Olivia; Saxena, Smita; Ule, Jernej; Schümperli, Daniel

    2014-01-01

    Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations. PMID:25692239

  1. Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation.

    PubMed

    Konstantinidis, Diamantis G; Giger, Katie M; Risinger, Mary; Pushkaran, Suvarnamala; Zhou, Ping; Dexheimer, Phillip; Yerneni, Satwica; Andreassen, Paul; Klingmüller, Ursula; Palis, James; Zheng, Yi; Kalfa, Theodosia A

    2015-09-17

    RhoA GTPase has been shown in vitro in cell lines and in vivo in nonmammalian organisms to regulate cell division, particularly during cytokinesis and abscission, when 2 daughter cells partition through coordinated actomyosin and microtubule machineries. To investigate the role of this GTPase in the rapidly proliferating mammalian erythroid lineage, we developed a mouse model with erythroid-specific deletion of RhoA. This model was proved embryonic lethal as a result of severe anemia by embryonic day 16.5 (E16.5). The primitive red blood cells were enlarged, poikilocytic, and frequently multinucleated, but were able to sustain life despite experiencing cytokinesis failure. In contrast, definitive erythropoiesis failed and the mice died by E16.5, with profound reduction of maturing erythroblast populations within the fetal liver. RhoA was required to activate myosin-regulatory light chain and localized at the site of the midbody formation in dividing wild-type erythroblasts. Cytokinesis failure caused by RhoA deficiency resulted in p53 activation and p21-transcriptional upregulation with associated cell-cycle arrest, increased DNA damage, and cell death. Our findings demonstrate the role of RhoA as a critical regulator for efficient erythroblast proliferation and the p53 pathway as a powerful quality control mechanism in erythropoiesis. © 2015 by The American Society of Hematology.

  2. Regulation of Retinoid-Mediated Signaling Involved in Skin Homeostasis by RAR and RXR Agonists/Antagonists in Mouse Skin

    PubMed Central

    Gericke, Janine; Ittensohn, Jan; Mihály, Johanna; Álvarez, Susana; Álvarez, Rosana; Töröcsik, Dániel; de Lera, Ángel R.; Rühl, Ralph

    2013-01-01

    Endogenous retinoids like all-trans retinoic acid (ATRA) play important roles in skin homeostasis and skin-based immune responses. Moreover, retinoid signaling was found to be dysregulated in various skin diseases. The present study used topical application of selective agonists and antagonists for retinoic acid receptors (RARs) α and γ and retinoid-X receptors (RXRs) for two weeks on mouse skin in order to determine the role of retinoid receptor subtypes in the gene regulation in skin. We observed pronounced epidermal hyperproliferation upon application of ATRA and synthetic agonists for RARγ and RXR. ATRA and the RARγ agonist further increased retinoid target gene expression (Rbp1, Crabp2, Krt4, Cyp26a1, Cyp26b1) and the chemokines Ccl17 and Ccl22. In contrast, a RARα agonist strongly decreased the expression of ATRA-synthesis enzymes, of retinoid target genes, markers of skin homeostasis, and various cytokines in the skin, thereby markedly resembling the expression profile induced by RXR and RAR antagonists. Our results indicate that RARα and RARγ subtypes possess different roles in the skin and may be of relevance for the auto-regulation of endogenous retinoid signaling in skin. We suggest that dysregulated retinoid signaling in the skin mediated by RXR, RARα and/or RARγ may promote skin-based inflammation and dysregulation of skin barrier properties. PMID:23638129

  3. Analysis of PRICKLE1 in human cleft palate and mouse development demonstrates rare and common variants involved in human malformations

    PubMed Central

    Yang, Tian; Jia, Zhonglin; Bryant-Pike, Whitney; Chandrasekhar, Anand; Murray, Jeffrey C; Fritzsch, Bernd; Bassuk, Alexander G

    2014-01-01

    Palate development is shaped by multiple molecular signaling pathways, including the Wnt pathway. In mice and humans, mutations in both the canonical and noncanonical arms of the Wnt pathway manifest as cleft palate, one of the most common human birth defects. Like the palate, numerous studies also link different Wnt signaling perturbations to varying degrees of limb malformation; for example, shortened limbs form in mutations of Ror2,Vangl2looptail and, in particular, Wnt5a. We recently showed the noncanonical Wnt/planar cell polarity (PCP) signaling molecule Prickle1 (Prickle like 1) also stunts limb growth in mice. We now expanded these studies to the palate and show that Prickle1 is also required for palate development, like Wnt5a and Ror2. Unlike in the limb, the Vangl2looptail mutation only aggravates palate defects caused by other mutations. We screened Filipino cleft palate patients and found PRICKLE1 variants, both common and rare, at an elevated frequency. Our results reveal that in mice and humans PRICKLE1 directs palate morphogenesis; our results also uncouple Prickle1 function from Vangl2 function. Together, these findings suggest mouse and human palate development is guided by PCP-Prickle1 signaling that is probably not downstream of Vangl2. PMID:24689077

  4. Long-range comparison of human and mouse Sprr loci to identify conserved noncoding sequences involved in coordinate regulation

    PubMed Central

    Martin, Natalia; Patel, Satyakam; Segre, Julia A.

    2004-01-01

    Mammalian epidermis provides a permeability barrier between an organism and its environment. Under homeostatic conditions, epidermal cells produce structural proteins, which are cross-linked in an orderly fashion to form a cornified envelope (CE). However, under genetic or environmental stress, specific genes are induced to rapidly build a temporary barrier. Small proline-rich (SPRR) proteins are the primary constituents of the CE. Under stress the entire family of 14 Sprr genes is upregulated. The Sprr genes are clustered within the larger epidermal differentiation complex on mouse chromosome 3, human chromosome 1q21. The clustering of the Sprr genes and their upregulation under stress suggest that these genes may be coordinately regulated. To identify enhancer elements that regulate this stress response activation of the Sprr locus, we utilized bioinformatic tools and classical biochemical dissection. Long-range comparative sequence analysis identified conserved noncoding sequences (CNSs). Clusters of epidermal-specific DNaseI-hypersensitive sites (HSs) mapped to specific CNSs. Increased prevalence of these HSs in barrier-deficient epidermis provides in vivo evidence of the regulation of the Sprr locus by these conserved sequences. Individual components of these HSs were cloned, and one was shown to have strong enhancer activity specific to conditions when the Sprr genes are coordinately upregulated. PMID:15574822

  5. trans-Stilbene oxide induces expression of genes involved in metabolism and transport in mouse liver via CAR and Nrf2 transcription factors.

    PubMed

    Slitt, A L; Cherrington, N J; Dieter, M Z; Aleksunes, L M; Scheffer, G L; Huang, W; Moore, D D; Klaassen, C D

    2006-05-01

    trans-Stilbene oxide (TSO) induces drug metabolizing enzymes in rat and mouse liver. TSO is considered a phenobarbital-like compound because it induces Cyp2B mRNA expression in liver. Phenobarbital increases Cyp2B expression in liver via activation of the constitutive androstane receptor (CAR). The purpose of this study was to determine whether TSO induces gene expression in mouse liver via CAR activation. TSO increased CAR nuclear localization in mouse liver, activated the human Cyp2B6 promoter in liver in vivo, and activated a reporter plasmid that contains five nuclear receptor 1 (NR1) binding sites in HepG2 cells. TSO administration increased expression of Cyp2b10, NAD(P)H:quinone oxidoreductase (Nqo1), epoxide hydrolase, heme oxygenase-1, UDP-glucuronosyl-transferase (Ugt) 1a6 and 2b5, and multidrug resistance-associated proteins (Mrp) 2 and 3 mRNA in livers from male mice. Cyp2b10 and epoxide hydrolase induction by TSO was decreased in livers from CAR-null mice, compared with wild-type mice, suggesting CAR involvement. In contrast, TSO administration induced Nqo1 and Mrp3 mRNA expression equally in livers from wild-type and CAR-null mice, suggesting that TSO induces expression of some genes through a mechanism independent of CAR. TSO increased nuclear staining of the transcription factor Nrf2 in liver, and activated an antioxidant/electrophile response element luciferase reporter construct that was transfected into HepG2 cells. In summary, in mice, TSO increases Cyp2b10 and epoxide hydrolase expression in mice via CAR, and potentially induces Nqo1 and Mrp3 expression via Nrf2. Moreover, our data demonstrate that a single compound can activate both CAR and Nrf2 transcription factors in liver.

  6. Possible involvement of ATP-sensitive potassium channels in the antidepressant-like effects of gabapentin in mouse forced swimming test.

    PubMed

    Ostadhadi, Sattar; Akbarian, Reyhaneh; Norouzi-Javidan, Abbas; Nikoui, Vahid; Zolfaghari, Samira; Chamanara, Mohsen; Dehpour, Ahmad-Reza

    2017-07-01

    Gabapentin as an anticonvulsant drug also has beneficial effects in treatment of depression. Previously, we showed that acute administration of gabapentin produced an antidepressant-like effect in the mouse forced swimming test (FST) by a mechanism that involves the inhibition of nitric oxide (NO). Considering the involvement of NO in adenosine triphosphate (ATP)-sensitive potassium channels (KATP), in the present study we investigated the involvement of KATP channels in antidepressant-like effect of gabapentin. Gabapentin at different doses (5-10 mg/kg) and fluoxetine (20 mg/kg) were administrated by intraperitoneal route, 60 and 30 min, respectively, before the test. To clarify the probable involvement of KATP channels, mice were pretreated with KATP channel inhibitor or opener. Gabapentin at dose 10 mg/kg significantly decreased the immobility behavior of mice similar to fluoxetine (20 mg/kg). Co-administration of subeffective dose (1 mg/kg) of glibenclamide (inhibitor of KATP channels) with gabapentin (3 mg/kg) showed a synergistic antidepressant-like effect. Also, subeffective dose of cromakalim (opener of KATP channels, 0.1 mg/kg) inhibited the antidepressant-like effect of gabapentin (10 mg/kg). None of the treatments had any impact on the locomotor movement. Our study, for the first time, revealed that antidepressant-like effect of gabapentin in mice is mediated by blocking the KATP channels.

  7. Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

    PubMed

    Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

    2014-04-01

    The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

  8. Possible role of ZPAC, zygote-specific proteasome assembly chaperone, during spermatogenesis in the mouse.

    PubMed

    Shimizu, Natsumi; Ueno, Kimihiro; Kurita, Ena; Shin, Seung-Wook; Nishihara, Takuji; Amano, Tomoko; Anzai, Masayuki; Kishigami, Satoshi; Kato, Hiromi; Mitani, Tasuku; Hosoi, Yoshihiko; Matsumoto, Kazuya

    2014-01-01

    In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly chaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit α4/PSMA7 in the adult mouse testis. ZPAC and α4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of α4 persisted until step 12. We then examined the expression profile of ZPAC and α4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of α4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.

  9. The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.

    PubMed

    Narváez, Ana J; Voevodskaya, Nina; Thelander, Lars; Gräslund, Astrid

    2006-09-08

    Ribonucleotide reductase class I enzymes consist of two non-identical subunits, R1 and R2, the latter containing a diiron carboxylate center and a stable tyrosyl radical (Tyr*), both essential for catalysis. Catalysis is known to involve highly conserved amino acid residues covering a range of approximately 35 A and a concerted mechanism involving long range electron transfer, probably coupled to proton transfer. A number of residues involved in electron transfer in both the R1 and R2 proteins have been identified, but no direct model has been presented regarding the proton transfer side of the process. Arg265 is conserved in all known sequences of class Ia R2. In this study we have used site-directed mutagenesis to gain insight into the role of this residue, which lies close to the catalytically essential Asp266 and Trp103. Mutants to Arg265 included replacement by Ala, Glu, Gln, and Tyr. All mutants of Arg265 were found to have no or low catalytic activity with the exception of Arg265 to Glu, which shows approximately 40% of the activity of native R2. We also found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2. Our results, supported by molecular modeling, strongly suggest that Arg265 is involved in the proton-coupled electron transfer pathway and may act as a proton mediator during catalysis.

  10. Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development.

    PubMed

    Niederreither, Karen; Abu-Abed, Suzan; Schuhbaur, Brigitte; Petkovich, Martin; Chambon, Pierre; Dollé, Pascal

    2002-05-01

    Retinoic acid, the active derivative of vitamin A (retinol), is a hormonal signaling molecule that acts in developing and adult tissues. The Cyp26a1 (cytochrome p450, 26) protein metabolizes retinoic acid into more polar hydroxylated and oxidized derivatives. Whether some of these derivatives are biologically active metabolites has been debated. Cyp26a1(-/-) mouse fetuses have lethal morphogenetic phenotypes mimicking those generated by excess retinoic acid administration, indicating that human CYP26A1 may be essential in controlling retinoic acid levels during development. This hypothesis suggests that the Cyp26a1(-/-) phenotype could be rescued under conditions in which embryonic retinoic acid levels are decreased. We show that Cyp26a1(-/-) mice are phenotypically rescued by heterozygous disruption of Aldh1a2 (also known as Raldh2), which encodes a retinaldehyde dehydrogenase responsible for the synthesis of retinoic acid during early embryonic development. Aldh1a2 haploinsufficiency prevents the appearance of spina bifida and rescues the development of posterior structures (sacral/caudal vertebrae, hindgut, urogenital tract), while partly preventing cervical vertebral transformations and hindbrain pattern alterations in Cyp26a1(-/-) mice. Thus, some of these double-mutant mice can reach adulthood. This study is the first report of a mutation acting as a dominant suppressor of a lethal morphogenetic mutation in mammals. We provide genetic evidence that ALDH1A2 and CYP26A1 activities concurrently establish local embryonic retinoic acid levels that must be finely tuned to allow posterior organ development and to prevent spina bifida.

  11. Involvement of NADPH oxidase in A2A adenosine receptor-mediated increase in coronary flow in isolated mouse hearts.

    PubMed

    Zhou, Zhichao; Rajamani, Uthra; Labazi, Hicham; Tilley, Stephen L; Ledent, Catherine; Teng, Bunyen; Mustafa, S Jamal

    2015-06-01

    Adenosine increases coronary flow mainly through the activation of A2A and A2B adenosine receptors (ARs). However, the mechanisms for the regulation of coronary flow are not fully understood. We previously demonstrated that adenosine-induced increase in coronary flow is in part through NADPH oxidase (Nox) activation, which is independent of activation of either A1 or A3ARs. In this study, we hypothesize that adenosine-mediated increase in coronary flow through Nox activation depends on A2A but not A2BARs. Functional studies were conducted using isolated Langendorff-perfused mouse hearts. Hydrogen peroxide (H2O2) production was measured in isolated coronary arteries from WT, A2AAR knockout (KO), and A2BAR KO mice using dichlorofluorescein immunofluorescence. Adenosine-induced concentration-dependent increase in coronary flow was attenuated by the specific Nox2 inhibitor gp91 ds-tat or reactive oxygen species (ROS) scavenger EUK134 in both WT and A2B but not A2AAR KO isolated hearts. Similarly, the A2AAR selective agonist CGS-21680-induced increase in coronary flow was significantly blunted by Nox2 inhibition in both WT and A2BAR KO, while the A2BAR selective agonist BAY 60-6583-induced increase in coronary flow was not affected by Nox2 inhibition in WT. In intact isolated coronary arteries, adenosine-induced (10 μM) increase in H2O2 formation in both WT and A2BAR KO mice was attenuated by Nox2 inhibition, whereas adenosine failed to increase H2O2 production in A2AAR KO mice. In conclusion, adenosine-induced increase in coronary flow is partially mediated by Nox2-derived H2O2, which critically depends upon the presence of A2AAR.

  12. Involvement of Rictor/mTORC2 in cardiomyocyte differentiation of mouse embryonic stem cells in vitro

    PubMed Central

    Zheng, Bei; Wang, Jiadan; Tang, Leilei; Tan, Chao; Zhao, Zhe; Xiao, Yi; Ge, Renshan; Zhu, Danyan

    2017-01-01

    Rictor is a key regulatory/structural subunit of the mammalian target of rapamycin complex 2 (mTORC2) and is required for phosphorylation of Akt at serine 473. It plays an important role in cell survival, actin cytoskeleton organization and other processes in embryogenesis. However, the role of Rictor/mTORC2 in the embryonic cardiac differentiation has been uncovered. In the present study, we examined a possible link between Rictor expression and cardiomyocyte differentiation of the mouse embryonic stem (mES) cells. Knockdown of Rictor by shRNA significantly reduced the phosphorylation of Akt at serine 473 followed by a decrease in cardiomyocyte differentiation detected by beating embryoid bodies. The protein levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein) and α-Actinin (cardiomyocyte biomarker) decreased in Rictor knockdown group during cardiogenesis. Furthermore, knockdown of Rictor specifically inhibited the ventricular-like cells differentiation of mES cells with reduced level of ventricular-specific protein, MLC-2v. Meanwhile, patch-clamp analysis revealed that shRNA-Rictor significantly increased the number of cardiomyocytes with abnormal electrophysiology. In addition, the expressions and distribution patterns of cell-cell junction proteins (Cx43/Desmoplakin/N-cadherin) were also affected in shRNA-Rictor cardiomyocytes. Taken together, the results demonstrated that Rictor/mTORC2 might play an important role in the cardiomyocyte differentiation of mES cells. Knockdown of Rictor resulted in inhibiting ventricular-like myocytes differentiation and induced arrhythmias symptom, which was accompanied by interfering the expression and distribution patterns of cell-cell junction proteins. Rictor/mTORC2 might become a new target for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells. PMID:28123351

  13. Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17

    PubMed Central

    Lee, Mee-Hyun; Lim, Do Young; Kim, Myoung Ok; Lee, Sung-Young; Shin, Seung Ho; Kim, Jae Young; Kim, Sung-Hyun; Kim, Dong Joon; Jung, Sung Keun; Yao, Ke; Kundu, Joydeb Kumar; Lee, Hye Suk; Lee, Cheol-Jung; Dickinson, Sally E.; Alberts, David; Bowden, G.Timothy; Stratton, Steven; Curiel, Clara; Einspahr, Janine; Bode, Ann M.; Surh, Young-Joon; Dong, Zigang

    2015-01-01

    Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17. PMID:26271098

  14. Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17.

    PubMed

    Lee, Mee-Hyun; Lim, Do Young; Kim, Myoung Ok; Lee, Sung-Young; Shin, Seung Ho; Kim, Jae Young; Kim, Sung-Hyun; Kim, Dong Joon; Jung, Sung Keun; Yao, Ke; Kundu, Joydeb Kumar; Lee, Hye Suk; Lee, Cheol-Jung; Dickinson, Sally E; Alberts, David; Bowden, G Timothy; Stratton, Steven; Curiel, Clara; Einspahr, Janine; Bode, Ann M; Surh, Young-Joon; Cho, Yong-Yeon; Dong, Zigang

    2015-11-01

    Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Structural organization and function of mouse photoreceptor ribbon synapses involve the immunoglobulin protein synaptic cell adhesion molecule 1.

    PubMed

    Ribic, Adema; Liu, Xinran; Crair, Michael C; Biederer, Thomas

    2014-03-01

    Adhesive interactions in the retina instruct the developmental specification of inner retinal layers. However, potential roles of adhesion in the development and function of photoreceptor synapses remain incompletely understood. This contrasts with our understanding of synapse development in the CNS, which can be guided by select adhesion molecules such as the Synaptic Cell Adhesion Molecule 1 (SynCAM 1/CADM1/nectin-like 2 protein). This immunoglobulin superfamily protein modulates the development and plasticity of classical excitatory synapses. We show here by immunoelectron microscopy and immunoblotting that SynCAM 1 is expressed on mouse rod photoreceptors and their terminals in the outer nuclear and plexiform layers in a developmentally regulated manner. Expression of SynCAM 1 on rods is low in early postnatal stages (P3-P7) but increases after eye opening (P14). In support of functional roles in the photoreceptors, electroretinogram recordings demonstrate impaired responses to light stimulation in SynCAM 1 knockout (KO) mice. In addition, the structural integrity of synapses in the OPL requires SynCAM 1. Quantitative ultrastructural analysis of SynCAM 1 KO retina measured fewer fully assembled, triadic rod ribbon synapses. Furthermore, rod synapse ribbons are shortened in KO mice, and protein levels of Ribeye, a major structural component of ribbons, are reduced in SynCAM 1 KO retina. Together, our results implicate SynCAM 1 in the synaptic organization of the rod visual pathway and provide evidence for novel roles of synaptic adhesion in the structural and functional integrity of ribbon synapses.

  16. Mitofusin 2 expression dominates over mitofusin 1 exclusively in mouse dorsal root ganglia - a possible explanation for peripheral nervous system involvement in Charcot-Marie-Tooth 2A.

    PubMed

    Kawalec, Maria; Zabłocka, Barbara; Kabzińska, Dagmara; Neska, Jacek; Beręsewicz, Małgorzata

    2014-01-01

    Mitofusin 2 (Mfn2), a protein of the mitochondrial outer membrane, is essential for mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. Mutations in the mitofusin 2 gene cause axonal Charcot-Marie-Tooth type 2A (CMT2A), an inherited disease affecting peripheral nerve axons. The precise mechanism by which mutations in MFN2 selectively cause the degeneration of long peripheral axons is not known. There is a hypothesis suggesting the involvement of reduced expression of a homologous protein, mitofusin 1 (Mfn1), in the peripheral nervous system, and less effective compensation of defective mitofusin 2 by mitofusin 1. We therefore aimed to perform an analysis of the mitofusin 1 and mitofusin 2 mRNA and protein expression profiles in different mouse tissues, with special attention paid to dorsal root ganglia (DRGs), as parts of the peripheral nervous system. Quantitative measurement relating to mRNA revealed that expression of the Mfn2 gene dominates over Mfn1 mainly in mouse DRG, as opposed to other nervous system samples and other tissues studied. This result was further supported by Western blot evaluation. Both these sets of data confirm the hypothesis that the cellular consequences of mutations in the mitofusin 2 gene can mostly be manifested in the peripheral nervous system.

  17. Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis.

    PubMed

    Yamaguchi, Masayoshi; Zhu, Shijun; Zhang, Shumin; Wu, Daqing; Moore, Terry M; Snyder, James P; Shoji, Mamoru

    2014-07-01

    Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10(6) cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150 mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20 mg/kg body weight) once daily for 5 days per week for 7 weeks. After UBS109 administration for 7 weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150 mg/kg body weight) and i.p. treatment of UBS109 (10 and 20 mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow

  18. Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling

    SciTech Connect

    Kiukkonen, Anu . E-mail: anummela@mappi.helsinki.fi; Sahlberg, Carin; Partanen, Anna-Maija; Alaluusua, Satu; Pohjanvirta, Raimo; Tuomisto, Jouko; Lukinmaa, Pirjo-Liisa

    2006-05-01

    Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereomicroscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.

  19. IKAP Deficiency in an FD Mouse Model and in Oligodendrocyte Precursor Cells Results in Downregulation of Genes Involved in Oligodendrocyte Differentiation and Myelin Formation

    PubMed Central

    Cheishvili, David; Dietrich, Paula; Maayan, Channa; Even, Aviel; Weil, Miguel; Dragatsis, Ioannis; Razin, Aharon

    2014-01-01

    The splice site mutation in the IKBKAP gene coding for IKAP protein leads to the tissue-specific skipping of exon 20, with concomitant reduction in IKAP protein production. This causes the neurodevelopmental, autosomal-recessive genetic disorder - Familial Dysautonomia (FD). The molecular hallmark of FD is the severe reduction of IKAP protein in the nervous system that is believed to be the main reason for the devastating symptoms of this disease. Our recent studies showed that in the brain of two FD patients, genes linked to oligodendrocyte differentiation and/or myelin formation are significantly downregulated, implicating IKAP in the process of myelination. However, due to the scarcity of FD patient tissues, these results awaited further validation in other models. Recently, two FD mouse models that faithfully recapitulate FD were generated, with two types of mutations resulting in severely low levels of IKAP expression. Here we demonstrate that IKAP deficiency in these FD mouse models affects a similar set of genes as in FD patients' brains. In addition, we identified two new IKAP target genes involved in oligodendrocyte cells differentiation and myelination, further underscoring the essential role of IKAP in this process. We also provide proof that IKAP expression is needed cell-autonomously for the regulation of expression of genes involved in myelin formation since knockdown of IKAP in the Oli-neu oligodendrocyte precursor cell line results in similar deficiencies. Further analyses of these two experimental models will compensate for the lack of human postmortem tissues and will advance our understanding of the role of IKAP in myelination and the disease pathology. PMID:24760006

  20. IKAP deficiency in an FD mouse model and in oligodendrocyte precursor cells results in downregulation of genes involved in oligodendrocyte differentiation and myelin formation.

    PubMed

    Cheishvili, David; Dietrich, Paula; Maayan, Channa; Even, Aviel; Weil, Miguel; Dragatsis, Ioannis; Razin, Aharon

    2014-01-01

    The splice site mutation in the IKBKAP gene coding for IKAP protein leads to the tissue-specific skipping of exon 20, with concomitant reduction in IKAP protein production. This causes the neurodevelopmental, autosomal-recessive genetic disorder - Familial Dysautonomia (FD). The molecular hallmark of FD is the severe reduction of IKAP protein in the nervous system that is believed to be the main reason for the devastating symptoms of this disease. Our recent studies showed that in the brain of two FD patients, genes linked to oligodendrocyte differentiation and/or myelin formation are significantly downregulated, implicating IKAP in the process of myelination. However, due to the scarcity of FD patient tissues, these results awaited further validation in other models. Recently, two FD mouse models that faithfully recapitulate FD were generated, with two types of mutations resulting in severely low levels of IKAP expression. Here we demonstrate that IKAP deficiency in these FD mouse models affects a similar set of genes as in FD patients' brains. In addition, we identified two new IKAP target genes involved in oligodendrocyte cells differentiation and myelination, further underscoring the essential role of IKAP in this process. We also provide proof that IKAP expression is needed cell-autonomously for the regulation of expression of genes involved in myelin formation since knockdown of IKAP in the Oli-neu oligodendrocyte precursor cell line results in similar deficiencies. Further analyses of these two experimental models will compensate for the lack of human postmortem tissues and will advance our understanding of the role of IKAP in myelination and the disease pathology.

  1. Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ɛ to RACK2 in adult rat and mouse

    PubMed Central

    Fenton, Richard A.; Komatsu, Satoshi; Ikebe, Mitsuo; Shea, Lynne G.; Dobson, James G.

    2009-01-01

    Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ɛ to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ɛ induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ɛ and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ɛ to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ɛ co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ɛ with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ɛ translocation and association with RACK2. PMID:19525381

  2. Involvement of EZH2, SUV39H1, G9a and associated molecules in pathogenesis of urethane induced mouse lung tumors: Potential targets for cancer control

    SciTech Connect

    Pandey, Manuraj; Sahay, Satya; Tiwari, Prakash; Upadhyay, Daya S.; Sultana, Sarwat; Gupta, Krishna P.

    2014-10-15

    In the present study, we showed the correlation of EZH2, SUV39H1 or G9a expression and histone modifications with the urethane induced mouse lung tumorigenesis in the presence or absence of antitumor agent, inositol hexaphosphate (IP6). Tumorigenesis and the molecular events involved therein were studied at 1, 4, 12 or 36 weeks after the exposure. There were no tumors at 1 or 4 weeks but tumors started appearing at 12 weeks and grew further till 36 weeks after urethane exposure. Among the molecular events, upregulation of EZH2 and SUV39H1 expressions appeared to be time dependent, but G9a expression was altered significantly only at later stages of 12 or 36 weeks. Alteration in miR-138 expression supports the upregulation of its target, EZH2. H3K9me2, H3K27me3 or H4K20me3 was found to be altered at 12 or 36 weeks. However, ChIP analysis of p16 and MLH1 promoters showed their binding with H3K9me2 and H3K27me3 which was maximum at 36 weeks. Thus, histone modification and their interactions with gene promoter resulted in the reduced expression of p16 and MLH1. IP6 prevented the incidence and the size of urethane induced lung tumors. IP6 also prevented the urethane induced alterations in EZH2, SUV39H1, G9a expressions and histone modifications. Our results suggest that the alterations in the histone modification pathways involving EZH2 and SUV39H1 expressions are among the early events in urethane induced mouse lung tumorigenesis and could be exploited for cancer control. - Highlights: • Urethane induces mouse lung tumor in a time dependent manner. • EZH2, SUV39H1, G9a induced by urethane and progress with time • Downregulation of miRNA-138 supports the EZH2 upregulation. • Methylation of histones showed a consequence of upregulated EZH2, SUV39H1 and G9a. • IP6 inhibits urethane induced changes and prevents tumor development.

  3. Paradoxical effects of VEGF on synaptic activity partially involved in notch1 signaling in the mouse hippocampus.

    PubMed

    Yang, Jiajia; Yang, Chunxiao; Liu, Chunhua; Zhang, Tao; Yang, Zhuo

    2016-05-01

    It is well known that the neuronal effects of vascular endothelial growth factor (VEGF) include modulating learning and memory, plasticity of mature neurons, and synaptic transmission in addition to neurogenesis. However, there is conflicting evidence particularly of its role in the regulation of excitatory synaptic activity. In this study, application of the patch-clamp technique revealed that lower doses (10 and 50 ng/mL) of VEGF enhanced excitatory neurotransmission in hippocampal slices of mice through both presynaptic and postsynaptic mechanisms. However, the effects were reversed by higher doses of VEGF (>100 ng/mL), which inhibited excitatory neurotransmission via a presynaptic mechanism. These competing, concentration-dependent effects of VEGF suggested that different pathways were involved. The involvement of the Notch1 receptor was tested in the modulation of VEGF on synaptic activity by using heterozygous Notch1(+/-) mice. Notch1 knockdown did not influence the inhibitory effect of high VEGF doses (200 ng/mL) but reduced the enhancement effects of low concentration of VEGF (50 ng/mL) at the postsynaptic level, which might be due to the decreased level of VEGF receptor. The results indicate that the Notch1 receptor plays a role in VEGF-induced modulation of synaptic activity, which provides new insights into a complex VEGF/Notch signaling cross-talk. These findings set the groundwork for understanding new mechanisms of Notch signaling and the neurotrophic effects of VEGF, which is beneficial to develop new therapeutic targets to the VEGF/Notch axis and improve current treatments for neural diseases.

  4. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    PubMed

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.

  5. RIBONUCLEIC ACID SYNTHESIS DURING MITOSIS AND MEIOSIS IN THE MOUSE TESTIS

    PubMed Central

    Monesi, Valerio

    1964-01-01

    The pattern of ribonucleic acid synthesis during germ cell development, from the stem cell to the mature spermatid, was studied in the mouse testis, by using uridine-H3 or cytidine-H3 labeling and autoradiography. Incorporation of tritiated precursors into the RNA occurs in spermatogonia, resting primary spermatocytes (RPS), throughout the second half of pachytene stage up to early diplotene, and in the Sertoli cells. Cells in leptotene, zygotene, and in the first half of pachytene stage do not synthesize RNA. No RNA synthesis was detected in meiotic stages later than diplotene, with the exception of a very low rate of incorporation in a fraction of secondary spermatocytes and very early spermatids. At long intervals after administration of the tracer, as labeled cells develop to more mature stages, late stages of spermatogenesis also become labeled. The last structures to become labeled are the residual bodies of Regaud. Thus, the RNA synthesized during the active meiotic stages is partially retained within the cell during further development. The rate of RNA synthesis declines gradually with the maturation from type A to intermediate to type B spermatogonia and to resting primary spermatocytes. "Dormant" type A spermatogonia synthesize little or no RNA. The incorporation of RNA precursors occurs exclusively within the nucleus: at later postinjection intervals the cytoplasm also becomes labeled. In spermatogonia all mitotic stages, except metaphase and anaphase, were shown to incorporate uridine-H3. RNA synthesis is then a continuous process throughout the cell division cycle in spermatogonia (generation time about 30 hours), and stops only for a very short interval (1 hour) during metaphase and anaphase. PMID:14206420

  6. PI3K is involved in PDGF-beta receptor upregulation post-PDGF-BB treatment in mouse HSC.

    PubMed

    Lechuga, Carmen G; Hernández-Nazara, Zamira H; Hernández, Elizabeth; Bustamante, Marcia; Desierto, Gregory; Cotty, Adam; Dharker, Nachiket; Choe, Moran; Rojkind, Marcos

    2006-12-01

    Increased expression of PDGF-beta receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-beta receptor by cathepsin B, thus suggesting that the absence of PDGF-beta receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-beta receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).

  7. Obesity alters the gustatory perception of lipids in the mouse: plausible involvement of lingual CD36[S

    PubMed Central

    Chevrot, Michael; Bernard, Arnaud; Ancel, Déborah; Buttet, Marjorie; Martin, Céline; Abdoul-Azize, Souleymane; Merlin, Jean-François; Poirier, Hélène; Niot, Isabelle; Khan, Naim Akhtar; Passilly-Degrace, Patricia; Besnard, Philippe

    2013-01-01

    A relationship between orosensory detection of dietary lipids, regulation of fat intake, and body mass index was recently suggested. However, involved mechanisms are poorly understood. Moreover, whether obesity can directly modulate preference for fatty foods remains unknown. To address this question, exploration of the oral lipid sensing system was undertaken in diet-induced obese (DIO) mice. By using a combination of biochemical, physiological, and behavioral approaches, we found that i) the attraction for lipids is decreased in obese mice, ii) this behavioral change has an orosensory origin, iii) it is reversed in calorie-restricted DIO mice, revealing an inverse correlation between fat preference and adipose tissue size, iv) obesity suppresses the lipid-mediated downregulation of the lipid-sensor CD36 in circumvallate papillae, usually found during the refeeding of lean mice, and v) the CD36-dependent signaling cascade controlling the intracellular calcium levels ([Ca2+]i) in taste bud cells is decreased in obese mice. Therefore, obesity alters the lipid-sensing system responsible for the oral perception of dietary lipids. This phenomenon seems to take place through a CD36-mediated mechanism, leading to changes in eating behavior. PMID:23840049

  8. The basic domain of the lentiviral Tat protein is responsible for damages in mouse brain: involvement of cytokines.

    PubMed

    Philippon, V; Vellutini, C; Gambarelli, D; Harkiss, G; Arbuthnott, G; Metzger, D; Roubin, R; Filippi, P

    1994-12-01

    The HIV and visna lentiviruses induce an inflammatory reaction in the central nervous system (CNS) of the infected hosts leading to dysmyelination, demyelination, and neuronal loss. The basic domain of the transactivating Tat protein has been involved in CNS damage. Infusion of basic containing domain Tat peptides in the lateral ventricle (systemic injection) or in the grey matter, i.e., hippocampus and thalamus (local injection), induced an inflammatory process characterized by the formation of an edema and invasion of macrophage accompanied by reactive astrogliosis. Control peptides originating from either lentiviral proteins or irrelevant protein as ovalbumin did not lead to any inflammatory reaction or cell death. The inflammation led to the loss of ependymal cells in the lateral ventricles and neurons in the grey matter. RNA extracted from the Tat-injected hemisphere reacted with TNF-alpha, IL-1 alpha and beta, and IL-6 probes. The macrophage/microglia inducible nitric oxyde synthase was also expressed. Blockade of TNF-alpha by a pentoxifylline treatment led to the decrease of IL-1 and iNOS expression accompanied by a reduction of the volume of the lesions indicating that the Tat-induced lesions might be mediated by TNF production.

  9. Transcription factor CP2 is involved in activating mBMP4 in mouse mesenchymal stem cells.

    PubMed

    Kang, Ho Chul; Chae, Ji Hyung; Kim, Beom Sue; Han, Su Youne; Kim, Sung-Hyun; Auh, Chung-Kyoon; Yang, Sung-Il; Kim, Chul Geun

    2004-06-30

    CP2 is a member of a family of transcription factors that regulate genes involved in events from early development to terminal differentiation. In an effort to understand how it selects its target genes we carried out a database search, and located several CP2 binding motifs in the promoter region of bone morphogenetic protein-4 (BMP4). BMP4 is a key regulator of cell fate and body patterning throughout development. For the CP2 binding motifs in BMP4 promoter region to be relevant in vivo, CP2 and BMP4 should be expressed together. We found that CP2b and CP2c, two potent transcriptional activators, are expressed in a manner similar to BMP4 during osteoblast differentiation of C3H10T1/2 cells. In in vitro assays, the CP2 proteins bound to two CP2 binding motifs (-715 to -676 and -147 to -118) in the BMP4 promoter, and luciferase reporter assays indicated that this binding was essential for transcription of BMP4 during osteoblast differentiation. Taken together, our data indicate that CP2b and CP2c play important roles during bone development by activating BMP4 transcription.

  10. The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

    PubMed Central

    Manna, Pulak R.; Soh, Jae-Won; Stocco, Douglas M.

    2011-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a −83/−67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the

  11. Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling.

    PubMed

    Liu, Ming; Zhu, Jin-Gai; Yu, Zhang-Bin; Song, Gui-Xian; Shen, Ya-Hui; Liu, Yao-Qiu; Zhu, Chun; Qian, Ling-Mei

    2014-06-01

    Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration.

  12. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis.

    PubMed

    Culver, Brady P; Savas, Jeffrey N; Park, Sung K; Choi, Jeong H; Zheng, Shuqiu; Zeitlin, Scott O; Yates, John R; Tanese, Naoko

    2012-06-22

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.

  13. Involvement of FoxO1 in the effects of follicle-stimulating hormone on inhibition of apoptosis in mouse granulosa cells

    PubMed Central

    Shen, M; Liu, Z; Li, B; Teng, Y; Zhang, J; Tang, Y; Sun, S-C; Liu, H

    2014-01-01

    In mammalian ovaries, follicular atresia occurs periodically and destroys almost all the follicles in the ovary. Follicle-stimulating hormone (FSH) acts as the primary survival factor during follicular atresia by preventing apoptosis in granulosa cells. FoxO1 is a critical factor in promoting follicular atresia and granulosa cell apoptosis. FSH inhibits the induction of FoxO1. In this report, we investigated the role of FSH-FoxO1 pathway in mouse follicular atresia. FSH dampened stress-induced apoptosis and the expression of FoxO1 and pro-apoptosis genes in mouse granulosa cells (MGCs). In contrast, overexpression of FoxO1 inhibited the viability of MGCs and induced the expression of endogenous FoxO1. The signaling cascades involved in regulating FoxO1 activity upon FSH treatment were identified using FSH signaling antagonists. Blocking protein kinase A (PKA), phosphatidylinositol-3 kinase (PI3K) or protein kinase B (AKT) restored the upregulation of FoxO1 and apoptotic signals, which was suppressed by FSH. Moreover, inhibition of PKA or PI3K impaired FSH-induced AKT activity, but inactivation of PI3K or AKT had little effect on PKA activity in the presence of FSH. Correspondingly, constitutive activation of FoxO1 (all three AKT sites were replaced by alanines) also promoted MGC apoptosis despite FSH administration. Furthermore, both luciferase reporter assays and chromatin immunoprecipitation assays showed that FoxO1 directly bound to a FoxO-recognized element site within the FoxO1 promoter and contributed to the regulation of FoxO1 expression in response to FSH. Taken together, we propose a novel model in which FSH downregulates FoxO1-dependent apoptosis in MGCs by coordinating the PKA–PI3K–AKT–FoxO1 axis and FoxO1–FoxO1 positive feedback. PMID:25321482

  14. Proteomic Analysis of Wild-type and Mutant Huntingtin-associated Proteins in Mouse Brains Identifies Unique Interactions and Involvement in Protein Synthesis*

    PubMed Central

    Culver, Brady P.; Savas, Jeffrey N.; Park, Sung K.; Choi, Jeong H.; Zheng, Shuqiu; Zeitlin, Scott O.; Yates, John R.; Tanese, Naoko

    2012-01-01

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis. PMID:22556411

  15. Molecular networks involved in mouse cerebral corticogenesis and spatio-temporal regulation of Sox4 and Sox11 novel antisense transcripts revealed by transcriptome profiling.

    PubMed

    Ling, King-Hwa; Hewitt, Chelsee A; Beissbarth, Tim; Hyde, Lavinia; Banerjee, Kakoli; Cheah, Pike-See; Cannon, Ping Z; Hahn, Christopher N; Thomas, Paul Q; Smyth, Gordon K; Tan, Seong-Seng; Thomas, Tim; Scott, Hamish S

    2009-01-01

    Development of the cerebral cortex requires highly specific spatio-temporal regulation of gene expression. It is proposed that transcriptome profiling of the cerebral cortex at various developmental time points or regions will reveal candidate genes and associated molecular pathways involved in cerebral corticogenesis. Serial analysis of gene expression (SAGE) libraries were constructed from C57BL/6 mouse cerebral cortices of age embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4 to 6 months. Hierarchical clustering analysis of 561 differentially expressed transcripts showed regionalized, stage-specific and co-regulated expression profiles. SAGE expression profiles of 70 differentially expressed transcripts were validated using quantitative RT-PCR assays. Ingenuity pathway analyses of validated differentially expressed transcripts demonstrated that these transcripts possess distinctive functional properties related to various stages of cerebral corticogenesis and human neurological disorders. Genomic clustering analysis of the differentially expressed transcripts identified two highly transcribed genomic loci, Sox4 and Sox11, during embryonic cerebral corticogenesis. These loci feature unusual overlapping sense and antisense transcripts with alternative polyadenylation sites and differential expression. The Sox4 and Sox11 antisense transcripts were highly expressed in the brain compared to other mouse organs and are differentially expressed in both the proliferating and differentiating neural stem/progenitor cells and P19 (embryonal carcinoma) cells. We report validated gene expression profiles that have implications for understanding the associations between differentially expressed transcripts, novel targets and related disorders pertaining to cerebral corticogenesis. The study reports, for the first time, spatio-temporally regulated Sox4 and Sox11 antisense transcripts in the brain, neural stem/progenitor cells and P19 cells, suggesting they have an

  16. LPS-induced delayed preconditioning is mediated by Hsp90 and involves the heat shock response in mouse kidney.

    PubMed

    Kaucsár, Tamás; Bodor, Csaba; Godó, Mária; Szalay, Csaba; Révész, Csaba; Németh, Zalán; Mózes, Miklós; Szénási, Gábor; Rosivall, László; Sőti, Csaba; Hamar, Péter

    2014-01-01

    We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, i.p.) and subsequent lethal (L: 10 mg/kg, i.p.) doses of LPS alone or in combination with NB (100 mg/kg, i.p.). Controls received saline (C) or NB. Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning.

  17. LPS-Induced Delayed Preconditioning Is Mediated by Hsp90 and Involves the Heat Shock Response in Mouse Kidney

    PubMed Central

    Kaucsár, Tamás; Bodor, Csaba; Godó, Mária; Szalay, Csaba; Révész, Csaba; Németh, Zalán; Mózes, Miklós; Szénási, Gábor; Rosivall, László; Sőti, Csaba; Hamar, Péter

    2014-01-01

    Introduction We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. Methods Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, ip.) and subsequent lethal (L: 10 mg/kg, ip.) doses of LPS alone or in combination with NB (100 mg/kg, ip.). Controls received saline (C) or NB. Results Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. Conclusion LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning. PMID:24646925

  18. Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

    PubMed Central

    2009-01-01

    Background β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system. Results Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326. Conclusions Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1. PMID:20003456

  19. The mesolimbic nucleus accumbens is critically involved with the mediation of the motor inhibitory and facilitatory effects of dopamine agonists on mouse spontaneous climbing behaviour.

    PubMed

    Costall, B; Eniojukan, J F; Naylor, R J

    1983-12-23

    Mouse spontaneous climbing behaviour was dose-dependently inhibited by putative dopamine agonists administered subcutaneously (s.c.) or directly into the mesolimbic nucleus accumbens. ED50 values for s.c. administrations of apomorphine, bromocriptine, DPI, 3-PPP and DK118 (5-hydroxy-6-methyl-2-di-n-propylaminotetralin) were 0.01, 0.26, 0.17, 0.24 and 0.0004 mg/kg respectively, and for intra-accumbens apomorphine, DPI, bromocriptine, DK118 and 2-di-n-propylamino-5,6-dihydroxytetralin were 0.21, 0.22, 7.5, 0.00034 and 0.000034 micrograms respectively. Dose-dependent reduction in motor inhibitory potential/motor facilitation was also recorded for higher doses of apomorphine given s.c., or for apomorphine, DK118 and 2-di-n-propylamino-5,6-dihydroxytetralin given intra-accumbens (ED50 values to restore spontaneous climbing to control values were 4.1, 4.2 and 0.8 micrograms respectively). The motor inhibitory actions of apomorphine, 3-PPP, DK118 and 2-di-n-propylamino-5,6-dihydroxytetralin were antagonised by (-)-sulpiride, but not by yohimbine or prazosin. The actions of DPI were yohimbine-sensitive. Whilst the motor inhibition caused by s.c. bromocriptine was neuroleptic-sensitive, that observed on intra-accumbens injection was resistant to all antagonists. The intra-accumbens effectiveness of the dopamine agonists could not be mimicked by injections above the nucleus accumbens (into the head of the caudate-putamen complex) or below the nucleus accumbens (into the tuberculum olfactorium) (with the exception of the effectiveness of bromocriptine administered into the tuberculum olfactorium). It is suggested that the actions of 'dopamine agonist' to both inhibit and facilitate mouse spontaneous climbing behaviour involves an action in the mesolimbic nucleus accumbens. Whilst the mechanisms involved are generally neuroleptic-sensitive, alpha 2-adrenoceptor (for DPI) and other unidentified mechanisms (for bromocriptine) may also be important.

  20. Genetic and molecular analysis of chlorambucil-induced germ-line mutations in the mouse.

    PubMed

    Rinchik, E M; Bangham, J W; Hunsicker, P R; Cacheiro, N L; Kwon, B S; Jackson, I J; Russell, L B

    1990-02-01

    Eighteen variants recovered from specific locus mutation rate experiments involving the mutagen chlorambucil were subjected to several genetic and molecular analyses. Most mutations were found to be homozygous lethal. Because lethality is often presumptive evidence for multilocus-deletion events, 10 mutations were analyzed by Southern blot analysis with probes at, or closely linked to, several of the specific locus test markers, namely, albino (c), brown (b), and dilute (d). All eight mutations (two c; three b; two d; and one dilute-short ear [Df(d se)]) that arose in post-spermatogonial germ cells were deleted for DNA sequences. No evidence for deletion of two d-se region probes was obtained for the remaining two d mutations that arose in stem-cell spermatogonia. Six of the primary mutants also produced low litter sizes ("semisterility"). Karyotypic analysis has, to date, confirmed the presence of reciprocal translocations in four of the six. The high frequency of deletions and translocations among the mutations induced in post-spermatogonial stages by chlorambucil, combined with its overall high efficiency in inducing mutations in these stages, should make chlorambucil mutagenesis useful for generating experimentally valuable germ-line deletions throughout the mouse genome.

  1. Effects of 50 Hz magnetic fields on mouse spermatogenesis monitored by flow cytometric analysis

    SciTech Connect

    Vita, R. de; Cavallo, D.; Raganella, L.; Eleuteri, P.; Grollino, M.G.; Calugi, A.

    1995-12-01

    Flow cytometry (FCM) was performed to monitor the cellular effects of extremely-low-frequency magnetic field on mouse spermatogenesis. Groups of five male hybrid F1 mice aged 8--10 weeks were exposed to 50 Hz magnetic field. The strength of the magnetic field was 1.7 mT. Exposure times of 2 and 4 h were chosen. FCM measurements were performed 7, 14, 21, 28, 35, and 42 days after treatment. For each experimental point, a sham-treated group was used as a control. The possible effects were studied by analyzing the DNA content distribution of the different cell types involved in spermatogenesis and using the elongated spermatids as the reference population. The relative frequencies of the various testicular cell types were calculated using specific software. In groups exposed for 2 h, no effects were observed. In groups exposed for 4 h, a statistically significant (P < 0.001) decrease in elongated spermatids was observed at 28 days after treatment. This change suggests a possible cytotoxic and/or cytostatic effect on differentiating spermatogonia. However, further studies are being carried out to investigate the effects of longer exposure times.

  2. Serotype-specific anti-Dengue virus NS1 mouse antibodies cross-react with prM and are potentially involved in virus production.

    PubMed

    Masrinoul, Promsin; Omokoko, Magot Diata; Pambudi, Sabar; Ikuta, Kazuyoshi; Kurosu, Takeshi

    2013-08-01

    Dengue virus (DENV) infection induces a strong B-cell immune response against the viral nonstructural protein 1 (NS1). Anti-NS1 antibodies (Abs) may affect virus production because they coexist with the virus in the patients' blood. The present study examined whether ten mouse monoclonal antibodies (MAbs) raised against NS1 affected production of the DENV-2. Three MAbs, 4C2, 4G11, and 4E5, showed weak neutralizing activity in a focus reduction assay. In addition, two serotype-specific MAbs, 4C2 and 4G11, protected suckling mice from lethal infection with DENV-2. An immunoprecipitation assay with DENV-2 showed that these MAbs, which were specific for the NS1 of DENV-4 and DENV-1, cross-reacted with the DENV-2 pre-membrane (prM) protein, but not with DENV-2 NS1. Interestingly, high concentrations of MAb 4G11 showed antibody-dependent enhancement of DENV-2 infection in human monocyte THP-1 cells. Taken together, these observations suggest that serotype-specific anti-NS1 MAbs are potentially involved in virus production.

  3. Interleukin-6 and interleukin-6 receptor expression, localization, and involvement in pain-sensing neuron activation in a mouse intervertebral disc injury model.

    PubMed

    Sainoh, Takeshi; Orita, Sumihisa; Miyagi, Masayuki; Sakuma, Yoshihiro; Yamauchi, Kazuyo; Suzuki, Miyako; Kubota, Go; Oikawa, Yasuhiro; Inage, Kazuhide; Sato, Jun; Fujimoto, Kazuki; Shiga, Yasuhiro; Inoue, Gen; Aoki, Yasuchika; Takahashi, Kazuhisa; Ohtori, Seiji

    2015-10-01

    The pathological mechanism of intractable low back pain is unclear. However, intervertebral disc (IVD) degeneration is a primary cause of low back pain, and pain-related mediators, such as interleukin-6 (IL-6), have been correlated with discogenic pain. The objective of this study is to elucidate the mechanism of local IL-6 and IL-6 receptor (IL-6R) expression after IVD injury as well as determine the involvement of IL-6/IL-6 signaling in discogenic pain. To do this, quantitative and immunohistological analyses in a mouse model of IVD injury were performed. Firstly, we measured the local expression levels of IL-6 and IL-6R in IVDs by enzyme-linked immunosorbent assay (ELISA). Secondly, we immunohistochemically confirmed their localization in injured IVDs. Lastly, we evaluated the effects of intradiscal injection of an IL-6 inhibitor by evaluating pain-related protein, calcitonin gene-related peptide (CGRP), expression in dorsal root ganglia (DRG) neurons that innervate IVDs. Injured IVDs showed increased production of IL-6 and IL-6R. IL-6 and IL-6R expression in the injured IVD were predominantly localized in the annulus fibrosus and endplate, and intradiscal injection of the IL-6 inhibitor suppressed CGRP expression in the DRG neurons. These results show that IL-6 and IL-6R expression levels are responsive to IVD injury and that inhibition of IL-6/IL-6R signaling may be a promising analgesic treatment for degenerative disc diseases.

  4. Molecular mechanisms involved in the non-monotonic effect of bisphenol-a on ca2+ entry in mouse pancreatic β-cells.

    PubMed

    Villar-Pazos, Sabrina; Martinez-Pinna, Juan; Castellano-Muñoz, Manuel; Alonso-Magdalena, Paloma; Marroqui, Laura; Quesada, Ivan; Gustafsson, Jan-Ake; Nadal, Angel

    2017-09-18

    In regulatory toxicology, the dose-response relationship is a key element towards fulfilling safety assessments and satisfying regulatory authorities. Conventionally, the larger the dose, the greater the response, following the dogma "the dose makes the poison". Many endocrine disrupting chemicals, including bisphenol-A (BPA), induce non-monotonic dose response (NMDR) relationships, which are unconventional and have tremendous implications in risk assessment. Although several molecular mechanisms have been proposed to explain NMDR relationships, they are largely undemonstrated. Using mouse pancreatic β-cells from wild-type and oestrogen receptor ERβ-/- mice, we found that exposure to increasing doses of BPA affected Ca(2+) entry in an NMDR manner. Low doses decreased plasma membrane Ca(2+) currents after downregulation of Cav2.3 ion channel expression, in a process involving ERβ. High doses decreased Ca(2+) currents through an ERβ-mediated mechanism and simultaneously increased Ca(2+) currents via oestrogen receptor ERα. The outcome of both molecular mechanisms explains the NMDR relationship between BPA and Ca(2+) entry in β-cells.

  5. The Histone Demethylase FBXL10 Regulates the Proliferation of Spermatogonia and Ensures Long-Term Sustainable Spermatogenesis in Mice.

    PubMed

    Ozawa, Manabu; Fukuda, Tsuyoshi; Sakamoto, Reiko; Honda, Hiroaki; Yoshida, Nobuaki

    2016-04-01

    The F-box and leucine-rich repeat protein 10 (Fbxl10) gene encodes a protein that catalyzes demethylation of H3K4 and H3K36. In this study, we show the important roles of FBXL10 as a histone demethylase in sustainable sperm production using mice in which the JmjC domain of Fbxl10 was deleted (Fbxl10(DeltaJ/DeltaJ)). In histological analysis, testis sections from 10-wk-old Fbxl10(DeltaJ/DeltaJ) mice appeared normal. On the other hand, testes from 7-mo-old Fbxl10(DeltaJ/DeltaJ) mice contained a greater ratio of seminiferous tubules exhibiting degeneration of spermatogenesis. Further analysis using an in vitro spermatogonia culture system, that is, germline stem cells (GSCs), revealed that Fbxl10(DeltaJ/DeltaJ) GSCs expressed a significantly higher level of P21 and P19 mRNA, cyclin-dependent kinase inhibitors and also known as cellular senescence markers, than wild-type (WT) GSCs. Furthermore, the ratio of Fbxl10(DeltaJ/DeltaJ) GSCs in G0/G1 phase was higher and the ratios in S and G2/M phases were lower than the corresponding ratios of WT GSCs, and the doubling speed of Fbxl10(DeltaJ/DeltaJ) GSCs was significantly slower than that of WT GSCs. In addition to these in vitro results, an in vivo study indicated that recovery of spermatogenesis after a transient reduction in the number of testicular germ cells by busulfan treatment was significantly slower in Fbxl10(DeltaJ/DeltaJ) mice than in WT mice. These data suggest that Fbxl10 plays important roles in long-term sustainable spermatogenesis via regulating cell cycle.

  6. Expression of genes involved in mouse lung cell differentiation/regulation after acute exposure to photons and protons with or without low-dose preirradiation.

    PubMed

    Tian, Jian; Zhao, WeiLing; Tian, Sisi; Slater, James M; Deng, Zhiyong; Gridley, Daila S

    2011-11-01

    The goal of this study was to compare the effects of acute 2 Gy irradiation with photons (0.8 Gy/min) or protons (0.9 Gy/min), both with and without pre-exposure to low-dose/low-dose-rate γ rays (0.01 Gy at 0.03 cGy/h), on 84 genes involved in stem cell differentiation or regulation in mouse lungs on days 21 and 56. Genes with a ≥1.5-fold difference in expression and P < 0.05 compared to 0 Gy controls are emphasized. Two proteins specific for lung stem cells/progenitors responsible for local tissue repair were also compared. Overall, striking differences were present between protons and photons in modulating the genes. More genes were affected by protons than by photons (22 compared to 2 and 6 compared to 2 on day 21 and day 56, respectively) compared to 0 Gy. Preirradiation with low-dose-rate γ rays enhanced the acute photon-induced gene modulation on day 21 (11 compared to 2), and all 11 genes were significantly downregulated on day 56. On day 21, seven genes (aldh2, bmp2, cdc2a, col1a1, dll1, foxa2 and notch1) were upregulated in response to most of the radiation regimens. Immunoreactivity of Clara cell secretory protein was enhanced by all radiation regimens. The number of alveolar type 2 cells positive for prosurfactant protein C in irradiated groups was higher on day 56 (12.4-14.6 cells/100) than on day 21 (8.5-11.2 cells/100) (P < 0.05). Taken together, these results showed that acute photons and protons induced different gene expression profiles in the lungs and that pre-exposure to low-dose-rate γ rays sometimes had modulatory effects. In addition, proteins associated with lung-specific stem cells/progenitors were highly sensitive to radiation.

  7. Involvement of commensal bacteria may lead to dysregulated inflammatory and autoimmune responses in a mouse model for chronic nonsuppurative destructive cholangitis.

    PubMed

    Haruta, Ikuko; Kikuchi, Ken; Nakamura, Minoru; Hirota, Katsuhiko; Kato, Hidehito; Miyakawa, Hiroshi; Shibata, Noriyuki; Miyake, Yoichiro; Hashimoto, Etsuko; Shiratori, Keiko; Yagi, Junji

    2012-10-01

    We previously reported a mouse model of primary biliary cirrhosis (PBC)-like chronic nonsuppurative destructive cholangitis (CNSDC), in which frequent injections of Streptococcus intermedius induced CNSDC and autoantibody production. The present study was performed to verify the model by examining 1) the reappearance of the PBC-like CNSDC after lymphocyte transfer from model to naïve mice, 2) the involvement of autophagy, and 3) the influence of the strain difference. Mice were inoculated with S. intermedius weekly for 8 weeks, then sacrificed to obtain samples. Spleen cells obtained from S. intermedius-inoculated mice were transferred to RAG2(-/-) mice. CNSDC and elevated serum level of anti-gp210 titers were observed in S. intermedius-inoculated C57BL/6 mice, similar to the results of our previous report using BALB/c mice. Portal inflammation was induced in the livers of RAG2(-/-) mice by the transfer of spleen cells from S. intermedius-inoculated C57BL/6 mice. Among the inflammatory cells in the RAG2(-/-) mice, CD3-positive cells were predominant. Autophagosome-like structures were detected histologically, in the cytoplasm of infiltrated cells around the bile ducts in the livers of S. intermedius-inoculated both C57BL/6 and BALB/c mice. In S. intermedius-inoculated C3H/HeJ mice, inflammation in the portal area was less extensive than that in the hepatic parenchyma. Bacterial component(s) and sequentially upregulated innate and acquired immune responses, accompanied by autophagy, might trigger CNSDC, via autoimmune mechanisms. Throughout the generation of bacteria-triggered PBC-like CNSDC, strain difference may influence the response to S. intermedius-inoculation in the liver.

  8. Amyloid Core Formed of Full-Length Recombinant Mouse Prion Protein Involves Sequence 127–143 but Not Sequence 107–126

    PubMed Central

    Chatterjee, Biswanath; Lee, Chung-Yu; Lin, Chen; Chen, Eric H.-L.; Huang, Chao-Li; Yang, Chien-Chih; Chen, Rita P.-Y.

    2013-01-01

    The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrPC) into its disease-causing isoform, PrPSc. This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high β-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23–230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107–143), mPrP(107–126), and mPrP(127–143). Our results showed that the amyloid fibrils formed from mPrP(107–143) and mPrP(127–143), but not those formed from mPrP(107–126), were able to seed the amyloidogenesis of mPrP(23–230), showing that the segment residing in sequence 127–143 was used to form the amyloid core in the fibrillization of mPrP(23–230). PMID:23844138

  9. Involvement of capsaicin-sensitive afferents and the Transient Receptor Potential Vanilloid 1 Receptor in xylene-induced nocifensive behaviour and inflammation in the mouse.

    PubMed

    Sándor, Katalin; Helyes, Zsuzsanna; Elekes, Krisztián; Szolcsányi, János

    2009-02-27

    The inflammatory actions of xylene, an aromatic irritant and sensitizing agent, were described to be predominantly neurogenic in the rat, but the mechanism and the role of the Transient Receptor Potential Vanilloid 1 (TRPV1) capsaicin receptor localized on a subpopulation of sensory nerves has not been elucidated. This paper characterizes the involvement of capsaicin-sensitive afferents and the TRPV1 receptor in nociceptive and acute inflammatory effects of xylene in the mouse. Topical application of xylene on the paw induced a short, intensive nocifensive behaviour characterized by paw liftings and shakings, which was more intensive in Balb/c than in C57Bl/6 mice. Genetic deletion of the TRPV1 receptor as well as destroying capsaicin-sensitive nerve terminals with resiniferatoxin (RTX) pretreatment markedly reduced, but did not abolish nocifensive behaviours. In respect to the xylene-induced plasma protein extravasation detected by Evans blue leakage, significant difference was neither observed between the Balb/c and C57Bl/6 strains, nor the ear and the dorsal paw skin. These inflammatory responses were diminished in the RTX pretreated group, but not in the TRPV1 gene-deleted one. Injection of the antioxidant N-acetylcysteine 15min prior to xylene smearing significantly reduced plasma protein extravasation at both sites. These results demonstrate that xylene-induced acute nocifensive behaviour is mediated by capsaicin-sensitive afferents via TRPV1 receptor activation in mice. Neurogenic inflammatory components play an important role in xylene-induced plasma protein extravasation, but independently of the TRPV1 ion channel. Reactive oxygen or carbonyl species participate in this process presumably via stimulation of the TRPA1 channel.

  10. A brain site for the antigonadal action of melatonin in the white-footed mouse (Peromyscus leucopus): involvement of the immunoreactive GnRH neuronal system.

    PubMed

    Glass, J D; Knotts, L K

    1987-06-01

    Quantitative assessment of immunocytochemical staining for gonadotropin-releasing hormone (GnRH) was undertaken to determine the effects of an intracranial implant of melatonin on the GnRH neuronal system in the male white-footed mouse (Peromyscus leucopus). Melatonin-containing pellets stereotaxically placed in the anterior hypothalamic area (AH) caused a 60% reduction in testes weight relative to control mice with melatonin-free pellets in the AH (p less than 0.01). Subcutaneous melatonin-containing implants had little effect on reproductive state (p less than 0.8). Melatonin pellets in the AH increased significantly both the optical density (OD) for immunostaining of cell bodies in the medial preoptic area and AH (p less than 0.04), and the percentage of area covered by GnRH fibers and beads in the median eminence (p less than 0.01). The melatonin-induced increase in OD of the GnRH cell bodies was independent of the distance of the cells from the melatonin implant, and there was little apparent effect of melatonin on the size and morphology of the GnRH cell bodies, or the trajectories of their fiber pathways. These results support the hypothesis that the antigonadal action of melatonin in the brain involves suppression of the release, rather than the synthesis of GnRH. Also, this effect may not be mediated via a direct action of melatonin on GnRH neurons. The finding that the brain site and time course for melatonin's antigonadal action in male. P. leucopus is similar to that found previously in the female is evidence that melatonin may induce gonadal regression, in part, by helping to suppress the tonic secretion of gonadotropins.

  11. p75(NTR) activation of NF-kappaB is involved in PrP106-126-induced apoptosis in mouse neuroblastoma cells.

    PubMed

    Bai, Yu; Li, Qiang; Yang, Jianmin; Zhou, Xiangmei; Yin, Xiaomin; Zhao, Deming

    2008-09-01

    Neuronal death is a pathological hallmark of prion diseases. Synthetic prion peptide PrP106-126 can convert PrP(C) into protease-resistant aggregates, which can cause neurotoxicity in vivo and in vitro. Various cell surface proteins can participate in the infection process of prions. p75(NTR) can interact with PrP106-126 and has a neurotoxic effect on neurons. However, for p75(NTR) lacking intrinsic catalytic activity domain in cytoplasm, p75(NTR) -associated signaling molecular and the signaling events in cytoplasm in p75(NTR)-mediated apoptosis responding to PrP106-126 remain still unknown. Thus p75(NTR) -associated NF-kappaB signaling pathway was investigated in this study. Herein PrP106-126-induced apoptosis in mouse neuroblastoma cell line N2a, PrP106-126 significantly up-regulated p75(NTR) expression on mRNA and protein levels. For the first time we found that PrP106-126 induced activation of NF-kappaB by Western blot assay, and blocking the interaction of p75(NTR) with PrP106-126 by p75(NTR) polyclonal antibody sc-6189 or pretreatment with inhibitor NF-kappaB SN50 reduced the activation of NF-kappaB and attenuated the apoptotic effect by PrP106-126. This study offers a possible interpretation that NF-kappaB signaling pathway was activated by the interaction of PrP106-126 with p75(NTR), and NF-kappaB activity showed the pro-apoptotic effect in PrP106-126-induced apoptosis in N2a cells. Involvement of NF-kappaB signaling pathway in p75(NTR)-mediated apoptosis may partially account for the PrP106-126-induced neurotoxicity in N2a cells.

  12. Involvement of insulin-like growth factor 1 receptor signaling in the amyloid-β peptide oligomers-induced p75 neurotrophin receptor protein expression in mouse hippocampus.

    PubMed

    Ito, Shingo; Ménard, Michel; Atkinson, Trevor; Gaudet, Chantal; Brown, Leslie; Whitfield, James; Chakravarthy, Balu

    2012-01-01

    The p75 neurotrophin receptor (p75NTR) has been thought to play a critical role in amyloid-β peptide (Aβ)-mediated neurodegeneration and Aβ metabolism in Alzheimer's disease (AD) brains. Our previous report showed that membrane-associated p75NTR protein expression was significantly increased in the hippocampi of two different strains of transgenic AD mice and was associated with the age-dependent elevation of Aβ1-42 levels. Here, we provide evidence that the Aβ1-42 oligomers known as ADDLs (Aβ-derived diffusible ligands) induce p75NTR protein expression through insulin-like growth factor 1 receptor (IGF-1R) phosphorylation in SH-SY5Y human neuroblastoma cells. An in vivo microinjection study demonstrated that microinjected ADDLs increased the p75NTR protein expression by 1.4-fold in the ipsilateral hippocampus compared to the contralateral hippocampus. In addition, ADDLs microinjected into mouse hippocampi facilitated IGF-1R phosphorylation within 30 min and the co-administration of picropodophyllin, an IGF-1R kinase inhibitor, blocked ADDLs-induced p75NTR expression. We examined the possible involvement of IGF-1R in the increased p75NTR protein expression in the hippocampi of 6-month-old AβPPswe/PS1dE9 AD model mice that had accumulated significant amounts of Aβ1-42 and showed significantly higher p75NTR expression than age-matched wild-type mice. We found that IGF-1R phosphorylation in these transgenic mice was higher than that in the wild-type mice. These findings indicate that Aβ1-42 oligomers stimulate the p75NTR protein expression in the hippocampus through IGF-1R signaling. Thus, Aβ1-42 oligomers-mediated IGF-1R activation may trigger an increase in p75NTR protein expression in the hippocampus of AD brain during the early stages of disease development.

  13. In-Vivo Characterization of Mammalian Polarity Genes as Novel Tumor Suppressors Involved in Breast Cancer Development and Progression in a Mouse Model

    DTIC Science & Technology

    2006-03-01

    Spring Harbor Laboratory Cold Spring Harbor , New York 11724 REPORT DATE: March...PERFORMING ORGANIZATION REPORT NUMBER Cold Spring Harbor Laboratory Cold Spring Harbor , New York 11724...Development and Progression in a Mouse Model PRINCIPAL INVESTIGATOR: Avi Z. Rosenberg CONTRACTING

  14. MyD88-dependent responses involving toll-like receptor 2 are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires' disease.

    PubMed

    Archer, Kristina A; Roy, Craig R

    2006-06-01

    Legionella pneumophila is a gram-negative facultative intracellular parasite of macrophages. Although L. pneumophila is the causative agent of a severe pneumonia known as Legionnaires' disease, it is likely that most infections caused by this organism are cleared by the host innate immune system. It is predicted that host pattern recognition proteins belonging to the Toll-like receptor (TLR) family are involved in the protective innate immune responses. We examined the role of TLR-mediated responses in L. pneumophila detection and clearance using genetically altered mouse hosts in which the macrophages are permissive for L. pneumophila intracellular replication. Our data demonstrate that cytokine production by bone marrow-derived macrophages (BMMs) in response to L. pneumophila infection requires the TLR adapter protein MyD88 and is reduced in the absence of TLR2 but not in the absence of TLR4. Bacterial growth ex vivo in BMMs from MyD88-deficient mice was not enhanced compared to bacterial growth ex vivo in BMMs from heterozygous littermate controls. Wild-type mice were able to clear L. pneumophila from the lung, whereas respiratory infection of MyD88-deficient mice caused death that resulted from robust bacterial replication and dissemination. In contrast to an infection with virulent L. pneumophila, MyD88-deficient mice were able to clear infections with L. pneumophila dotA mutants, indicating that MyD88-independent responses in the lung are sufficient to clear bacteria that are unable to replicate intracellularly. In vivo growth of L. pneumophila was enhanced in the lungs of TLR2-deficient mice, which resulted in a delay in bacterial clearance. No significant differences were observed in the growth and clearance of L. pneumophila in the lungs of TLR4-deficient mice and heterozygous littermate control mice. Our data indicate that MyD88 is crucial for eliciting a protective innate immune response against virulent L. pneumophila and that TLR2 is one of the pattern

  15. Exacerbation of innate immune response in mouse primary cultured sertoli cells caused by nanoparticulate TiO2 involves the TAM/TLR3 signal pathway.

    PubMed

    Wu, Nan; Hong, Fashui; Zhou, Yingjun; Wang, Yajing

    2017-01-01

    Sertoli cells provide appropriate mitogens, differentiation factors and sources of energy for developing germ cells throughout the lifetime of males, and protect these germ cells from harmful agents and from the host's own immune system. Therefore, reductions in the rate and quality of spermatogenesis caused by nanoparticulate titanium dioxide (nano-TiO2 ) may be closely involved in the immunoregulation of Sertoli cells. However, the underlying mechanism of this response is still unclear. To address this issue, we used mouse primary cultured Sertoli cells to examine the toxic effects of nano-TiO2 via alterations in morphology, cell viability, and activation of the TAM/TLR3 signal pathway. The results demonstrated that nano-TiO2 could cross the cytomembrane into the cytoplasm or nucleus, decrease Sertoli cell viability, damage morphology (such as elongated fusiform, cellular and nuclear shrinkage) and induce the expression of various immune mediators and inflammatory cytokines, including TLR3(+0.31-fold to +0.81-fold), IL-lβ(+0.33-fold to +5.0-fold), NF-κB(+0.22-fold to +3.65-fold), IL-6(+0.47-fold to +3.53-fold), TNF-α(+0.14-fold to +2.44-fold), IFN-α(+0.17-fold to +2.27-fold), and IFN-β(+0.09-fold to +2.29-fold), and suppress the expression of Tyro3(-9.33% to -61.93%), Axl(-19.03% to -60.67%), Mer(-8.04% to -59.16%), and IκB(-34.35% to -86.59%) in primary cultured Sertoli cells. These results suggest that testicular innate immune responses to pathogens caused by nano-TiO2 may be involved in the regulatory mechanisms of TAM/TLR3 signaling in testicular Sertoli cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 198-208, 2017.

  16. Comparison of the genetic effects of equimolar doses of ENU and MNU: While the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

    SciTech Connect

    Russell, Liane B; Hunsicker, Patricia R; Russell, William

    2007-03-01

    Mutagenoic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem cell spermatogonial (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonial (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75 mg MNU/kg during this peak interval is, to date, the highest induced by any single- xposure mutagenic treatment chemical or radiation that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonial in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite

  17. Sin3a is required by sertoli cells to establish a niche for undifferentiated spermatogonia, germ cell tumors, and spermatid elongation.

    PubMed

    Payne, Christopher J; Gallagher, Shannon J; Foreman, Oded; Dannenberg, Jan Hermen; Depinho, Ronald A; Braun, Robert E

    2010-08-01

    Microenvironments support the maintenance of stem cells and the growth of tumors through largely unknown mechanisms. While cell-autonomous chromatin modifications have emerged as important determinants for self-renewal and differentiation of stem cells, a role for non-cell autonomous epigenetic contributions is not well established. Here, we genetically ablated the chromatin modifier Swi-independent 3a (Sin3a) in fetal Sertoli cells, which partly comprise the niche for male germline stem cells, and investigated its impact on spermatogenic cell fate and teratoma formation in vivo. Sertoli cell-specific Sin3a deletion resulted in the formation of few undifferentiated spermatogonia after birth while initially maintaining spermatogenic differentiation. Stem cell-associated markers Plzf, Gfra1, and Oct4 were downregulated in the mutant fetal gonad, while Sertoli cell markers Steel and Gdnf, which support germ cells, were not diminished. Following birth, markers of differentiating spermatogonia, Kit and Sohlh2, exhibited normal levels, but chemokine-signaling molecules chemokine (C-X-C motif) ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF1) and chemokine (C-X-C motif) receptor 4 (CXCR4), expressed in Sertoli cells and germ cells, respectively, were not detected. In the juvenile, mutant testes exhibited a progressive loss of differentiating spermatogonia and a block in spermatid elongation, followed by extensive germ cell degeneration. Sertoli cell-specific Sin3a deletion also suppressed teratoma formation by fetal germ cells in an in vivo transplantation assay. We conclude that the epigenome of Sertoli cells influences the establishment of a niche for germline stem cells as well as for tumor initiating cells.

  18. Retinoic acid metabolism links the periodical differentiation of germ cells with the cycle of Sertoli cells in mouse seminiferous epithelium.

    PubMed

    Sugimoto, Ryo; Nabeshima, Yo-ichi; Yoshida, Shosei

    2012-01-01

    Homeostasis of tissues relies on the regulated differentiation of stem cells. In the epithelium of mouse seminiferous tubules, the differentiation process from undifferentiated spermatogonia (A(undiff)), which harbor the stem cell functions, to sperm occurs in a periodical manner, known as the "seminiferous epithelial cycle". To identify the mechanism underlying this periodic differentiation, we investigated the roles of Sertoli cells (the somatic supporting cells) and retinoic acid (RA) in the seminiferous epithelial cycle. Sertoli cells cyclically change their functions in a coordinated manner with germ cell differentiation and support the entire process of spermatogenesis. RA is known to play essential roles in this periodic differentiation, but its precise mode of action and its regulation remains largely obscure. We showed that an experimental increase in RA signaling was capable of both inducing A(undiff) differentiation and resetting the Sertoli cell cycle to the appropriate stage. However, these actions of exogenous RA signaling on A(undiff) and Sertoli cells were strongly interfered by the differentiating germ cells of intimate location. Based on the expression of RA metabolism-related genes among multiple cell types - including germ and Sertoli cells - and their regulation by RA signaling, we propose here that differentiating germ cells play a primary role in modulating the local RA metabolism, which results in the timed differentiation of A(undiff) and the appropriate cycling of Sertoli cells. Similar regulation by differentiating progeny through the modulation of local environment could also be involved in other stem cell systems.

  19. Involvement of L-arginine-nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of bis selenide in the mouse tail suspension test.

    PubMed

    Jesse, Cristiano R; Wilhelm, Ethel A; Bortolatto, Cristiani F; Rocha, João B T; Nogueira, Cristina W

    2010-06-10

    The present study investigated a possible antidepressant-like effect of bis selenide by using the forced swimming and the tail suspension tests. The involvement of the l-arginine-nitric oxide-cyclic guanosine monophosphate signaling pathway in the antidepressant-like action of bis selenide was investigated. Bis selenide, given by oral route at doses of 0.5-5mg/kg, decreased the immobility time in the forced swimming and tail suspension tests. Pretreatment with l-arginine (750mg/kg, intraperitoneal, i.p., a nitric oxide precursor), sildenafil (5mg/kg, i.p., a phosphodiesterase 5 inhibitor) or S-nitroso-N-acetyl-penicillamine (25microg/site, intracerebroventricular, i.c.v., a nitric oxide donor) reversed the reduction in the immobility time elicited by bis selenide (1mg/kg, p.o.) in the tail suspension test. Bis selenide (0.1mg/kg, p.o., a subeffective dose) produced a synergistic antidepressant-like effect with N(G)-nitro-L-arginine (0.3mg/kg, i.p., an inhibitor of nitric oxide synthase) or 7-nitroindazole (25mg/kg, i.p., a specific neuronal nitric oxide synthase inhibitor) in the tail suspension test. Pretreatment of animals with methylene blue (10mg/kg, i.p., an inhibitor of nitric oxide synthase and soluble guanylate cyclase) or 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (30pmol, i.c.v., a specific inhibitor of soluble guanylate cyclase), at subeffective doses, caused a synergistic effect with bis selenide in the tail suspension test. Bis selenide (1mg/kg, p.o.), at an effective dose in the forced swimming and tail suspension tests, caused a significant decrease in the mouse cerebral nitrate/nitrite levels. The antidepressant-like effect of bis selenide in the tail suspension test is dependent on the inhibition of the L-arginine-nitric oxide-cyclic guanosine monophosphate pathway.

  20. Organic cation transporter 1 (OCT1) is involved in pentamidine transport at the human and mouse blood-brain barrier (BBB)

    PubMed Central

    Georgian, Ana R.; Sanderson, Lisa; Vizcay-Barrena, Gema; Brown, Rachel C.; Muresan, Paula; Fleck, Roland A.; Thomas, Sarah A.

    2017-01-01

    Pentamidine is an effective trypanocidal drug used against stage 1 Human African Trypanosomiasis (HAT). At the blood-brain barrier (BBB), it accumulates inside the endothelial cells but has limited entry into the brain. This study examined transporters involved in pentamidine transport at the human and mouse BBB using hCMEC/D3 and bEnd.3 cell lines, respectively. Results revealed that both cell lines expressed the organic cation transporters (OCT1, OCT2 and OCT3), however, P-gp was only expressed in hCMEC/D3 cells. Polarised expression of OCT1 was also observed. Functional assays found that ATP depletion significantly increased [3H]pentamidine accumulation in hCMEC/D3 cells (***p<0.001) but not in bEnd.3 cells. Incubation with unlabelled pentamidine significantly decreased accumulation in hCMEC/D3 and bEnd.3 cells after 120 minutes (***p<0.001). Treating both cell lines with haloperidol and amantadine also decreased [3H]pentamidine accumulation significantly (***p<0.001 and **p<0.01 respectively). However, prazosin treatment decreased [3H]pentamidine accumulation only in hCMEC/D3 cells (*p<0.05), and not bEnd.3 cells. Furthermore, the presence of OCTN, MATE, PMAT, ENT or CNT inhibitors/substrates had no significant effect on the accumulation of [3H]pentamidine in both cell lines. From the data, we conclude that pentamidine interacts with multiple transporters, is taken into brain endothelial cells by OCT1 transporter and is extruded into the blood by ATP-dependent mechanisms. These interactions along with the predominant presence of OCT1 in the luminal membrane of the BBB contribute to the limited entry of pentamidine into the brain. This information is of key importance to the development of pentamidine based combination therapies which could be used to treat CNS stage HAT by improving CNS delivery, efficacy against trypanosomes and safety profile of pentamidine. PMID:28362799

  1. Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation.

    PubMed

    Dubrova, Y E; Plumb, M; Brown, J; Fennelly, J; Bois, P; Goodhead, D; Jeffreys, A J

    1998-05-26

    Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose-response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.

  2. Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPARα and CAR

    EPA Science Inventory

    A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear recep...

  3. Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for Involvement of the Nuclear Receptors PPARα and CAR

    EPA Science Inventory

    A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects related to liver carcinogenesis through the nucle...

  4. Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPARα and CAR

    EPA Science Inventory

    A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear recep...

  5. Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for Involvement of the Nuclear Receptors PPARα and CAR

    EPA Science Inventory

    A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects related to liver carcinogenesis through the nucle...

  6. Involvement of AMPA receptors in the antidepressant-like effects of lithium in the mouse tail suspension test and forced swim test.

    PubMed

    Gould, Todd D; O'Donnell, Kelley C; Dow, Eliot R; Du, Jing; Chen, Guang; Manji, Husseini K

    2008-03-01

    In addition to its clinical antimanic effects, lithium also has efficacy in the treatment of depression. However, the mechanism by which lithium exerts its antidepressant effects is unclear. Our objective was to further characterize the effects of peripheral and central administration of lithium in mouse models of antidepressant efficacy as well as to investigate the role of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in these behaviors. We utilized the mouse forced swim test (FST) and tail suspension test (TST), intracerebroventricular (ICV) lithium administration, AMPA receptor inhibitors, and BS3 crosslinking followed by Western blot. Both short- and long-term administration of lithium resulted in robust antidepressant-like effects in the mouse FST and TST. Using ICV administration of lithium, we show that these effects are due to actions of lithium on the brain, rather than to peripheral effects of the drug. Both ICV and rodent chow (0.4% LiCl) administration paradigms resulted in brain lithium concentrations within the human therapeutic range. The antidepressant-like effects of lithium in the FST and TST were blocked by administration of AMPA receptor inhibitors. Additionally, administration of lithium increased the cell surface expression of GluR1 and GluR2 in the mouse hippocampus. Collectively, these data show that lithium exerts centrally mediated antidepressant-like effects in the mouse FST and TST that require AMPA receptor activation. Lithium may exert its antidepressant effects in humans through AMPA receptors, thus further supporting a role of targeting AMPA receptors as a therapeutic approach for the treatment of depression.

  7. Involvement of AMPA Receptors in the Antidepressant-Like Effects of Lithium in the Mouse Tail Suspension Test and Forced Swim Test

    PubMed Central

    Gould, Todd D.; O’Donnell, Kelley C.; Dow, Eliot R.; Du, Jing; Chen, Guang; Manji, Husseini K.

    2008-01-01

    In addition to its clinical antimanic effects, lithium also has efficacy in the treatment of depression. However, the mechanism by which lithium exerts its antidepressant effects is unclear. Our objective was to further characterize the effects of peripheral and central administration of lithium in mouse models of antidepressant efficacy as well as to investigate the role of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in these behaviors. We utilized the mouse forced swim test (FST) and tail suspension test (TST), intracerebroventricular (ICV) lithium administration, AMPA receptor inhibitors, and BS3 crosslinking followed by western blot. Both short- and long-term administration of lithium resulted in robust antidepressant-like effects in the mouse FST and TST. Using ICV administration of lithium, we show that these effects are due to actions of lithium on the brain, rather than to peripheral effects of the drug. Both ICV and rodent chow (0.4% LiCl) administration paradigms resulted in brain lithium concentrations within the human therapeutic range. The effects of lithium to decrease immobility in the FST and TST were blocked by administration of AMPA receptor inhibitors. Additionally, administration of lithium increased the cell surface expression of GluR1 and GluR2 in the mouse hippocampus. Collectively, these data show that lithium exerts centrally mediated antidepressant-like effects in the mouse FST and TST that require AMPA receptor activation. Lithium may exert its antidepressant effects in humans through AMPA receptors, thus further supporting a role of targeting AMPA receptors as a therapeutic approach for the treatment of depression. PMID:18096191

  8. Induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia: effects of low doses and low dose rates

    SciTech Connect

    van Buul, P.P.; Richardson, J.F. Jr.; Goudzwaard, J.H.

    1986-01-01

    The induction of reciprocal translocation in rhesus monkey spermatogonial stem cells was studied following exposure to low doses of acute X rays (0.25 Gy, 300 mGy/min) or to low-dose-rate X rays (1 Gy, 2 mGy/min) and gamma rays (1 Gy, 0.2 mGy/min). The results obtained at 0.25 Gy of X rays fitted exactly the linear extrapolation down from the 0.5 and 1.0 Gy points obtained earlier. Extension of X-ray exposure reduced the yield of translocations similar to that in the mouse by about 50%. The reduction to 40% of translocation rate after chronic gamma exposure was clearly less than the value of about 80% reported for the mouse over the same range of dose rates. Differential cell killing with ensuing differential elimination of aberration-carrying cells is the most likely explanation for the differences between mouse and monkey.

  9. Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing

    PubMed Central

    1996-01-01

    The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome- specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere- specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8- specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed. PMID:8794855

  10. Functional Comparison of Mx1 from Two Different Mouse Species Reveals the Involvement of Loop L4 in the Antiviral Activity against Influenza A Viruses

    PubMed Central

    Verhelst, Judith; Spitaels, Jan; Nürnberger, Cindy; De Vlieger, Dorien; Ysenbaert, Tine; Staeheli, Peter; Fiers, Walter

    2015-01-01

    ABSTRACT The interferon-induced Mx1 gene is an important part of the mammalian defense against influenza viruses. Mus musculus Mx1 inhibits influenza A virus replication and transcription by suppressing the polymerase activity of viral ribonucleoproteins (vRNPs). Here, we compared the anti-influenza virus activity of Mx1 from Mus musculus A2G with that of its ortholog from Mus spretus. We found that the antiviral activity of M. spretus Mx1 was less potent than that of M. musculus Mx1. Comparison of the M. musculus Mx1 sequence with the M. spretus Mx1 sequence revealed 25 amino acid differences, over half of which were present in the GTPase domain and 2 of which were present in loop L4. However, the in vitro GTPase activity of Mx1 from the two mouse species was similar. Replacement of one of the residues in loop L4 in M. spretus Mx1 by the corresponding residue of A2G Mx1 increased its antiviral activity. We also show that deletion of loop L4 prevented the binding of Mx1 to influenza A virus nucleoprotein and, hence, abolished the antiviral activity of mouse Mx1. These results indicate that loop L4 of mouse Mx1 is a determinant of antiviral activity. Our findings suggest that Mx proteins from different mammals use a common mechanism to inhibit influenza A viruses. IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and inhibit a wide range of viruses. Still, the exact details of their antiviral mechanisms remain largely unknown. Functional comparison of the Mx genes from two species that diverged relatively recently in evolution can provide novel insights into these mechanisms. We show that both Mus musculus A2G Mx1 and Mus spretus Mx1 target the influenza virus nucleoprotein. We also found that loop L4 in mouse Mx1 is crucial for its antiviral activity, as was recently reported for primate MxA. This indicates that human and mouse Mx proteins, which have diverged by 75 million years of evolution, recognize and inhibit influenza A viruses by a common

  11. Auditory function in the Tc1 mouse model of down syndrome suggests a limited region of human chromosome 21 involved in otitis media.

    PubMed

    Kuhn, Stephanie; Ingham, Neil; Pearson, Selina; Gribble, Susan M; Clayton, Stephen; Steel, Karen P; Marcotti, Walter

    2012-01-01

    Down syndrome is one of the most common congenital disorders leading to a wide range of health problems in humans, including frequent otitis media. The Tc1 mouse carries a significant part of human chromosome 21 (Hsa21) in addition to the full set of mouse chromosomes and shares many phenotypes observed in humans affected by Down syndrome with trisomy of chromosome 21. However, it is unknown whether Tc1 mice exhibit a hearing phenotype and might thus represent a good model for understanding the hearing loss that is common in Down syndrome. In this study we carried out a structural and functional assessment of hearing in Tc1 mice. Auditory brainstem response (ABR) measurements in Tc1 mice showed normal thresholds compared to littermate controls and ABR waveform latencies and amplitudes were equivalent to controls. The gross anatomy of the middle and inner ears was also similar between Tc1 and control mice. The physiological properties of cochlear sensory receptors (inner and outer hair cells: IHCs and OHCs) were investigated using single-cell patch clamp recordings from the acutely dissected cochleae. Adult Tc1 IHCs exhibited normal resting membrane potentials and expressed all K(+) currents characteristic of control hair cells. However, the size of the large conductance (BK) Ca(2+) activated K(+) current (I(K,f)), which enables rapid voltage responses essential for accurate sound encoding, was increased in Tc1 IHCs. All physiological properties investigated in OHCs were indistinguishable between the two genotypes. The normal functional hearing and the gross structural anatomy of the middle and inner ears in the Tc1 mouse contrast to that observed in the Ts65Dn model of Down syndrome which shows otitis media. Genes that are trisomic in Ts65Dn but disomic in Tc1 may predispose to otitis media when an additional copy is active.

  12. Possible involvement of type 1 inositol 1,4,5-trisphosphate receptors up-regulated by dopamine D1 and D2 receptors in mouse nucleus accumbens neurons in the development of methamphetamine-induced place preference.

    PubMed

    Kurokawa, K; Mizuno, K; Ohkuma, S

    2012-12-27

    Little is known about regulatory mechanisms of type 1 inositol-1,4,5-triphosphate receptor (IP(3)R-1) expression in conditioned place preference by methamphetamine (METH), though significant enhancement of IP(3)R-1 expression in the mouse frontal cortex and limbic forebrain by intermittent administration of cocaine is reported. The present study investigated the role and regulation of IP(3)R-1 in mice with METH-induced place preference. Injection of IP(3)R antagonists with different chemical structures, 2-aminophenoxyethane-borate and xestospongin C, into the mouse nucleus accumbens (NAcc) dose-dependently inhibited METH-induced place preference. The levels of IP(3)R-1 protein in the NAcc of METH-conditioned mice significantly increased, which was completely abolished by microinjection of SCH23390 and raclopride, selective dopamine D1-like and D2-like receptor (D1 and D2DR) antagonists respectively, into the mouse NAcc. Immunohistochemical assessment revealed co-localization of immunoreactivity for IP(3)R-1 and those for D1 and D2DRs in the NAcc. These findings suggest that IP(3)R-1 could be involved in the development of METH-induced place preference and that D1 and D2DRs in the NAcc of mice showing METH-induced place preference play possible regulatory roles in IP(3)R-1 expression.

  13. Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical.

    PubMed

    Schmidt, P P; Rova, U; Katterle, B; Thelander, L; Gräslund, A

    1998-08-21

    Class I ribonucleotide reductases consist of two subunits, R1 and R2. The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr.), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-A distance between Tyr. in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway. The diferric/Tyr. site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr. site. Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with >/=4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr. formation in D266A and W103Y is on average 20 times slower than in wild type R2. More strikingly, Tyr. formation is completely suppressed in W103F. No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H. Finally, the formation of Tyr. is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr. and the iron center in mouse R2. A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E. coli R2, was detected only

  14. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.

    PubMed

    Osuru, Hari Prasad; Pramoonjago, Patcharin; Abhyankar, Mayuresh M; Swanson, Eric; Roker, LaToya Ann; Cathro, Helen; Reddi, Prabhakara P

    2017-08-01

    TAR DNA-binding protein of 43 kDa (TDP-43) is an evolutionarily conserved, ubiquitously expressed, multi-functional DNA/RNA-binding protein with roles in gene transcription, mRNA splicing, stability, transport, micro RNA biogenesis, and suppression of transposons. Aberrant expression of TDP-43 in testis and sperm was recently shown to be associated with male infertility, which highlights the need to understand better the expression of TDP-43 in the testis. We previously cloned TDP-43 from a mouse testis cDNA library, and showed that it functions as a transcriptional repressor and regulates the precise spatiotemporal expression of the Acrv1 gene, which encodes the acrosomal protein SP-10, during spermatogenesis. Here, we performed immunoblotting and immunohistochemistry of the mouse testis using four separate antibodies recognizing the amino and carboxyl termini of TDP-43. TDP-43 is present in the nuclei of germ cells as well as Sertoli cells. TDP-43 expression begins in type B/intermediate spermatogonia, peaks in preleptotene spermatocytes, and becomes undetectable in leptotene and zygotene spermatocytes. Pachytene spermatocytes and early round spermatids again express TDP-43, but its abundance diminishes later in spermatids (at steps 5-8). Interestingly, two of the four antibodies showed TDP-43 expression in spermatids at steps 9-10, which coincides with the initial phase of the histone-to-protamine transition. Immunoreactivity patterns observed in the study suggest that TDP-43 assumes different conformational states at different stages of spermatogenesis. TDP-43 pathology has been extensively studied in the context of neurodegenerative diseases; its role in spermatogenesis warrants further detailed investigation of the involvement of TDP-43 in male infertility. © 2017 Wiley Periodicals, Inc.

  15. Characterization and expression analysis during embryo development of the mouse ortholog of MLL3.

    PubMed

    Brun, Marie-Elisabeth; Gasca, Stéphan; Girard, Cyrille; Bouton, Katia; De Massy, Bernard; De Sario, Albertina

    2006-04-12

    We characterized the mouse ortholog of the human MLL3 gene and a 10.6 kb-Mll3 transcript. The mouse Mll3 gene comprises 60 exons that encompass 226 kb in chromosome 5. The predicted protein of 3464 amino acids contains two PHD domains, an ATPase alpha_beta signature, an HMG, and a SET domain. We analyzed the expression of the Mll3 gene during the embryonic development of the mouse by whole-mount in situ hybridization. Low levels of expression throughout the embryo were first detected at 8.0 dpc. At this stage, the signal was already stronger in the forebrain neuroepithelium and absent in the heart. Next, expression outlined the ventral neural tube, the somites, the limbs, and the eye lens remaining at low levels throughout the embryo. By 13.0 dpc, expression became stronger in the spinal cord, in hand/foot plates, and in gonads. RT-PCR confirmed that Mll3 is expressed early during gametogenesis. We suggest that Mll3 is expressed early in pre-spermatogonia and then in spermatogonia.

  16. Tests for urethane induction of germ-cell mutations and germ-cell killing in the mouse.

    PubMed

    Russell, L B; Hunsicker, P R; Oakberg, E F; Cummings, C C; Schmoyer, R L

    1987-08-01

    Urethane, a chemical that has given varied results in mutagenesis assays, was tested in the mouse specific-locus test, and its effect on germ-cell survival was explored. Altogether 32,828 offspring were observed from successive weekly matings of males exposed to the maximum tolerated i.p. dose of 1750 mg urethane/kg. The combined data rule out (at the 5% significance level) an induced mutation rate greater than 1.7 times the historical control rate. For spermatogonial stem cells alone, the multiple ruled out is 3.2, and for poststem-cell stages, 3.5. Litter sizes from successive conceptions made in any of the first 7 weeks give no indication of induced dominant lethality, confirming results of past dominant-lethal assays. That urethane (or an active metabolite) reaches germ cells is indicated by SCE induction in spermatogonia demonstrated by other investigators. Cytotoxic effects in spermatogonia are suggested by our finding of a slight reduction in numbers of certain types of spermatogonia in seminiferous tubule cross-sections and of a borderline decrease in the number of litters conceived during the 8th and 9th posttreatment weeks. The negative results for induction of gene mutations as well as clastogenic damage are at variance with Nomura's reports of dominant effects (F1 cancers and malformations) produced by urethane.

  17. The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

    PubMed

    Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

    2016-07-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo.

  18. Immunotherapy of melanoma by GPI-anchored IL-21 tumour vaccine involves down-regulating regulatory T cells in mouse model.

    PubMed

    Wang, J; Zhao, F; Dou, J; He, X F; Chu, L; Cao, M; Liu, C; Li, Y; Gu, N

    2011-02-01

    In this study, we developed a tumour cell vaccine expressing a glycosylphosphatidylinositol (GPI)-anchored IL-21 to test the effect of immunotherapy of melanoma in mouse model. The results indicated that the tumour vaccine was functional, exhibiting delayed tumour growth and prolonging longevity of tumour bearing mice. The immunotherapeutic effect was associated with decreasing the numbers of CD4(+) CD25(+) Foxp3(+) Treg (Tregs) cells, increasing IFN-γ level and promoting lymphocyte-infiltration in tumour tissues. Overall, our data demonstrate that the GPI-anchored IL-21 tumour vaccine regulates immune responses at least in part by down-regulating Tregs and reveals enhanced efficacy of tumour vaccine therapy of melanoma. © 2010 Blackwell Publishing Ltd.

  19. Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors

    PubMed Central

    Viana da Silva, Silvia; Haberl, Matthias Georg; Zhang, Pei; Bethge, Philipp; Lemos, Cristina; Gonçalves, Nélio; Gorlewicz, Adam; Malezieux, Meryl; Gonçalves, Francisco Q.; Grosjean, Noëlle; Blanchet, Christophe; Frick, Andreas; Nägerl, U Valentin; Cunha, Rodrigo A.; Mulle, Christophe

    2016-01-01

    Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients. PMID:27312972

  20. Involvement of Phospholipase Cγ1 in Mouse Egg Activation Induced by a Truncated Form of the C-kit Tyrosine Kinase Present in Spermatozoa

    PubMed Central

    Sette, Claudio; Bevilacqua, Arturo; Geremia, Raffaele; Rossi, Pellegrino

    1998-01-01

    Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit– induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267–2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the γ1 isoform of PLC (PLCγ1) competitively inhibits tr-kit– induced egg activation. A GST fusion protein containing the SH3 domain of PLCγ1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit–induced egg activation, showing that the effect of the SH3 domain of PLCγ1 is specific. Tr-kit–induced egg activation is also suppressed by co-injection of antibodies raised against the PLCγ1 SH domains, but not against the PLCγ1 COOH-terminal region. In transfected COS cells, coexpression of PLCγ1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCγ1 with respect to cells expressing PLCγ1 alone. These data indicate that tr-kit activates PLCγ1, and that the SH3 domain of PLCγ1 is essential for tr-kit–induced egg activation. PMID:9722617

  1. High frequency of mosaic mutants produced by N-ethyl-N-nitrosourea exposure of mouse zygotes.

    PubMed

    Russell, L B; Bangham, J W; Stelzner, K F; Hunsicker, P R

    1988-12-01

    Mouse zygotes containing one multiple-recessive parental genome (a, b; p cch; d se; s) and the corresponding wild-type alleles in the other were exposed to N-ethyl-N-nitrosourea (ENU) at various stages in vivo. At weaning age, the resulting mice were examined for mutations at the marked loci as well as at others producing externally visible phenotypes. Because of viability problems in one of two reciprocal crosses, the bulk of the mutagenesis data are derived from the cross that detects recessive mutations in the maternal genome. The mutation rate was approximately 8 times higher in groups treated 2.5-3 hr postmating (sperm entry, completion of second meiotic division) than in those injected 5-6 hr postmating (pronuclear formation). In the former more sensitive zygote population, the mutation rate is about an order of magnitude greater than that induced by the same ENU exposure (50 mg/kg) to spermatogonial stem cells. Of 11 mutants recovered, 8 were mosaics. Progeny tests have demonstrated germ-line involvement for most of the mosaics, and the average fraction of the germ line carrying the mutation is close to 50%. The nature of the mutations indicates (i) that the mosaicism results not from misassortment at the first cleavage but from mutation affecting one DNA strand of the maternal chromosome, and (ii) that the mutations are intragenic lesions rather than multilocus deletions, thus resembling ENU-induced mutations in spermatogonia. The finding that mosaicism for presumed point mutations is readily inducible by ENU treatment of zygotes may provide a means of generating genetic materials that can be of use for developmental studies.

  2. Potential Involvement of Impaired BKCa Channel Function in Sensory Defensiveness and Some Behavioral Disturbances Induced by Unfamiliar Environment in a Mouse Model of Fragile X Syndrome.

    PubMed

    Carreno-Munoz, Maria Isabel; Martins, Fabienne; Medrano, Maria Carmen; Aloisi, Elisabetta; Pietropaolo, Susanna; Dechaud, Corentin; Subashi, Enejda; Bony, Guillaume; Ginger, Melanie; Moujahid, Abdelmalik; Frick, Andreas; Leinekugel, Xavier

    2017-07-19

    In fragile X syndrome (FXS), sensory hypersensitivity and impaired habituation is thought to result in attention overload and various behavioral abnormalities in reaction to the excessive and remanent salience of environment features that would normally be ignored. This phenomenon, termed sensory defensiveness, has been proposed as the potential cause of hyperactivity, hyperarousal, and negative reactions to changes in routine that are often deleterious for FXS patients. However, the lack of tools for manipulating sensory hypersensitivity has not allowed the experimental testing required to evaluate the relevance of this hypothesis. Recent work has shown that BMS-204352, a BKCa channel agonist, was efficient to reverse cortical hyperexcitability and related sensory hypersensitivity in the Fmr1-KO mouse model of FXS. In the present study, we report that exposing Fmr1-KO mice to novel or unfamiliar environments resulted in multiple behavioral perturbations, such as hyperactivity, impaired nest building and excessive grooming of the back. Reversing sensory hypersensitivity with the BKCa channel agonist BMS-204352 prevented these behavioral abnormalities in Fmr1-KO mice. These results are in support of the sensory defensiveness hypothesis, and confirm BKCa as a potentially relevant molecular target for the development of drug medication against FXS/ASD.Neuropsychopharmacology advance online publication, 16 August 2017; doi:10.1038/npp.2017.149.

  3. The laminin binding protein p40 is involved in inducing limb abnormality of mouse fetuses as the effects of methoxyacetic acid treatment.

    PubMed

    Ruyani, Aceng; Sudarwati, Sri; Sutasurya, Lien A; Sumarsono, Sony H; Gloe, Torsten

    2003-09-01

    This study is intended to characterize a protein that is linked with mouse limb teratogenicity as the effects of methoxyacetic acid (MAA) treatment. A single dose of MAA (10 mmol/kg body weight) was given by gavage on gestation day (GD) 11, whereas the control group were administered vehicle only. The pregnant mice were killed at 4 h after MAA treatment, and forelimb buds were isolated from both the control and treated group embryos. Proteins from forelimb buds GD 11 + 4 h, which were precipitated out using 40-60% ammonium sulfate, then were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) technique. The 2-D gels reveal one protein with 41.6 kDa and pI 6.4, which expression was downregulated after MAA treatment. Tentative protein identification via peptide mass database search and definitive protein identification via a primary sequence database search indicate that the protein matches exactly to 34/67 kDa laminin binding protein (LBP; P14206, SwissProt), which is encoded by p40 gene (MGI:105381). The identity was further verified by Western blotting with an antibody against the 67 kDa LBP. The results suggest that MAA treatment to pregnant mice downregulates the LBP-p40 in the forelimb buds.

  4. The involvement of Cav3.2/α1H T-type calcium channels in excitability of mouse embryonic primary vestibular neurones

    PubMed Central

    Autret, Laurence; Mechaly, Ilana; Scamps, Frédérique; Valmier, Jean; Lory, Philippe; Desmadryl, Gilles

    2005-01-01

    Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between −65 and −45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation. PMID:15961427

  5. The involvement of Cav3.2/alpha1H T-type calcium channels in excitability of mouse embryonic primary vestibular neurones.

    PubMed

    Autret, Laurence; Mechaly, Ilana; Scamps, Frédérique; Valmier, Jean; Lory, Philippe; Desmadryl, Gilles

    2005-08-15

    Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between -65 and -45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.

  6. Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG family) involved in the negative control of the cell cycle.

    PubMed

    Guéhenneux, F; Duret, L; Callanan, M B; Bouhas, R; Hayette, S; Berthet, C; Samarut, C; Rimokh, R; Birot, A M; Wang, Q; Magaud, J P; Rouault, J P

    1997-03-01

    It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a non-conserved 20-25 amino acid sequence.

  7. Effects of realgar on GSH synthesis in the mouse hippocampus: Involvement of system XAG(-), system XC(-), MRP-1 and Nrf2.

    PubMed

    Wang, Yanlei; Chen, Mo; Zhang, Yinghua; Huo, Taoguang; Fang, Ying; Jiao, Xuexin; Yuan, Mingmei; Jiang, Hong

    2016-10-01

    Realgar is a type of mineral drug that contains arsenic and has neurotoxicity. Glutathione (GSH), which is the main antioxidant in the central nervous system, plays a key role in antioxidant defenses and the detoxification of arsenic. However, whether realgar interferes with the synthesis of GSH in the brain and the molecular mechanisms underlying its effects are largely unknown. Here, we used mouse models of exposure to realgar to show that realgar affects the synthesis of GSH in the hippocampus, leading to ultrastructural changes in hippocampal neurons and synapses and deficiencies in cognitive abilities, and that the mechanisms that cause this effect may be associated with alterations in the expression of system XAG(-), system XC(-), multidrug resistance-associated protein 1(MRP-1), nuclear factor E2-related factor 2 (Nrf2), γ-glutamylcysteine synthetase (γ-GCS), and the levels of glutamate (Glu) and cysteine (Cys) in the extracellular fluid. These findings provide a theoretical basis for preventing the drug-induced chronic arsenic poisoning in the nervous system that is triggered by realgar.

  8. Enhancement of β-amyloid oligomer accumulation after intracerebroventricular injection of streptozotocin, which involves central insulin signaling in a transgenic mouse model.

    PubMed

    Lin, Fangju; Jia, Jianping; Qin, Wei

    2014-11-12

    The β-amyloid (Aβ) oligomer rather than fibrillar Aβ has become the important focus of recent studies on the pathogenesis of Alzheimer's disease (AD). Insulin signaling plays important roles in cognitive disease, such as AD. However, in-vivo evidence for the link between central insulin signaling and the Aβ oligomer are lacking, and the mechanisms underlying the effect of central insulin signaling on AD are still elusive. Our team has established the Presenilin-1 Val97Leu mutant transgenic (PS1V97L) AD mouse model with the intraneuronal Aβ oligomer as the potential initiator for other pathologies, but without extracellular amyloid plaque formation. Using this model, we investigated the roles of disturbed central insulin signaling induced by intracerebroventricular injection of streptozotocin (STZ) in the progression of AD. We observed that PS1V97L mice after intracerebroventricular injection of STZ showed increased Aβ oligomer accumulation and aggravated spatial learning and memory deficit in the absence of diabetes symptoms. Furthermore, STZ administration inhibited the activation of the insulin receptor and enhanced the activation of c-Jun NH2-terminal kinase, which was accompanied by increased production of carboxy-terminal fragments from the amyloid precursor protein, in the brain of PS1V97L mice. Overall, our study provided in-vivo evidence for a role of central insulin signaling in AD progression.

  9. Neuroprotective Effects of Salidroside in the MPTP Mouse Model of Parkinson's Disease: Involvement of the PI3K/Akt/GSK3β Pathway

    PubMed Central

    He, Hong; Song, Hujie; Zhao, Junjie; Li, Tao; Wu, Leitao

    2016-01-01

    The degenerative loss through apoptosis of dopaminergic neurons in the substantia nigra pars compacta plays a primary role in the progression of Parkinson's disease (PD). Our in vitro experiments suggested that salidroside (Sal) could protect against 1-methyl-4-phenylpyridine-induced cell apoptosis in part by regulating the PI3K/Akt/GSK3β pathway. The current study aims to increase our understanding of the protective mechanisms of Sal in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine- (MPTP-) induced PD mouse model. We found that pretreatment with Sal could protect against MPTP-induced increase of the time of turning downwards and climbing down to the floor. Sal also prevented MPTP-induced decrease of locomotion frequency and the increase of the immobile time. Sal provided a protection of in MPTP-induced loss of tyrosine hydroxylase-positive neurons in SNpc and the level of DA, DOPAC, and HVA in the striatum. Furthermore, Sal could increase the phosphorylation level of Akt and GSK3β, upregulate the ratio of Bcl-2/Bax, and inhibit the activation of caspase-3, caspase-6, and caspase-9. These results show that Sal prevents the loss of dopaminergic neurons and the PI3K/Akt/GSK3β pathway signaling pathway may have mediated the protection of Sal against MPTP, suggesting that Sal may be a potential candidate in neuroprotective treatment for PD. PMID:27738547

  10. Up-regulation of immunomodulatory effects of mouse bone-marrow derived mesenchymal stem cells by tetrahydrocannabinol pre-treatment involving cannabinoid receptor CB2

    PubMed Central

    Xie, Junran; Xiao, Dongju; Xu, Yun; Zhao, Jinning; Jiang, Li; Hu, Xuming; Zhang, Yaping; Yu, Lina

    2016-01-01

    Chronic pain is commonly and closely correlated with inflammation. Both cannabinoid signaling and mesenchymal stem cells (MSCs) have been demonstrated to reduce inflammatory pain. Although cannabinoid signaling is essential for mesenchymal stem cell survival and differentiation, little is known about its role in modulatory effect of MSCs on inflammation and pain sensitivity. Here we showed that mouse bone-marrow derived MSCs (BM-MSCs) expressed both cannabinoid receptor type 1 and 2 (CB1 and CB2). CB2 expression level in BM-MSCs increased with their maturation. In addition, we found that tetrahydrocannabinol (THC) activated CB2 receptor and ERK signaling, consequently enhancing the modulation of MSCs on inflammation-associated cytokine release from lipopolysaccharides-stimulated microglia. Consistent with in vitro data, THC pretreatment enhanced the immunomodulatory effects of BM-MSC on thermal hyperalgesia and mechanical allodynia in chronic constriction injury model, by decreasing the release of pro-inflammation cytokines. Our study revealed the crucial role of THC in promoting the immunomodulatory effects of MSCs and proposed a new strategy to alleviate pain based on stem cells therapy. PMID:26824325

  11. Inhibition of interleukin-12 production in lipopolysaccharide-activated mouse macrophages by parthenolide, a predominant sesquiterpene lactone in Tanacetum parthenium: involvement of nuclear factor-kappaB.

    PubMed

    Kang, B Y; Chung, S W; Kim, T S

    2001-07-02

    Pharmacological control of interleukin-12 (IL-12) production may be a key therapeutic strategy for modulating immunological diseases dominated by type-1 cytokine responses. In this study, we investigated the effects of parthenolide, an anti-inflammatory sesquiterpene, on the production of IL-12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Parthenolide potently inhibited the LPS-induced IL-12 production in a dose-dependent manner. The effect of parthenolide on IL-12 p40 promoter activation was analyzed by transfecting RAW264.7 monocytic cells with p40 promoter/luciferase constructs. The repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (p40-kappaB). Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the kappaB site, which significantly decreased upon addition of parthenolide. These results suggest that parthenolide-induced inhibition of IL-12 production in macrophages may explain some of the biological effects of parthenolide including its anti-inflammatory activity.

  12. Expression of murine Asb-9 during mouse spermatogenesis.

    PubMed

    Lee, Man Ryul; Kim, Soo Kyoung; Kim, Jong Soo; Rhim, Si Youn; Kim, Kye-Seong

    2008-12-31

    We previously showed that Asb-4 and Asb-17 is uniquely expressed in developing male germ cells. A recent report showed that Asb-9 is specifically expressed in the kidney and testes; however, detailed expression patterns in developing germ cells have not been shown. Northern blot analysis in various tissues demonstrated that mAsb-9 was strongly expressed in the testes. Expression analysis by RT-PCR and Northern blot in developing mouse testes indicates that mAsb-9 is expressed from the fourth week after birth to adulthood, with the highest expression in round spermatids. Expression sites were further localized by in situ hybridization in the testes. Pachytene spermatocytes and spermatids expressed mAsb-9 but spermatogonia and generated spermatozoa did not. This study reveals that mAsb-9 could be a specific marker of active spermatogenesis and would be useful for studies of male germ cell development.

  13. Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues.

    PubMed

    Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine; Iturrioz, Xavier

    2017-01-01

    Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis.

  14. Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues

    PubMed Central

    Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine

    2017-01-01

    Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. PMID:28877217

  15. Involvement of the MyD88-independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7.

    PubMed

    Tangsudjai, S; Pudla, M; Limposuwan, K; Woods, D E; Sirisinha, S; Utaisincharoen, P

    2010-05-01

    Burkholderia pseudomallei is a facultative intracellular Gram-negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O-antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88-dependent (ikappabzeta, il-6 and tnf-alpha), but not of the MyD88-independent (inos, ifn-beta and irg-1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant-infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88-independent pathway in B. pseudomallei-infected macrophages, the expression of TBK1, an essential protein in the MyD88-independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant-infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88-independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN-gamma upregulated gene expression downstream of the MyD88-independent pathway, and interfered with intracellular survival in both wild type and tbk1-knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88-independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN-gamma regulates gene expression through the TBK1-independent pathway.

  16. Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation.

    PubMed

    Gao, Wanxia; Zhao, Jie; Gao, Zhonghong; Li, Hailing

    2017-01-01

    It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction.

  17. The Same Major Histocompatibility Complex Polymorphism Involved in Control of HIV Influences Peptide Binding in the Mouse H-2Ld System*

    PubMed Central

    Narayanan, Samanthi; Kranz, David M.

    2013-01-01

    Single-site polymorphisms in human class I major histocompatibility complex (MHC) products (HLA-B) have recently been shown to correlate with HIV disease progression or control. An identical single-site polymorphism (at residue 97) in the mouse class I product H-2Ld influences stability of the complex. To gain insight into the human polymorphisms, here we examined peptide binding, stability, and structures of the corresponding Ld polymorphisms, Trp97 and Arg97. Expression of LdW97 and LdR97 genes in a cell line that is antigen-processing competent showed that LdR97 was expressed at higher levels than LdW97, consistent with enhanced stability of self-peptide·LdR97 complexes. To further examine peptide-binding capacities of these two allelic variants, we used a high affinity pep-Ld specific probe to quantitatively examine a collection of self- and foreign peptides that bind to Ld. LdR97 bound more effectively than LdW97 to most peptides, although LdW97 bound more effectively to two peptides. The results support the view that many self-peptides in the Ld system (or the HLA-B system) would exhibit enhanced binding to Arg97 alleles compared with Trp97 alleles. Accordingly, the self-peptide·MHC-Arg97 complexes would influence T-cell selection behavior, impacting the T-cell repertoire of these individuals, and could also impact peripheral T cell activity through effects of self-peptide·Ld interacting with TCR and/or CD8. The structures of several peptide·LdR97 and peptide·LdW97 complexes provided a framework of how this single polymorphism could impact peptide binding. PMID:24064213

  18. Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation

    PubMed Central

    Gao, Wanxia; Zhao, Jie; Gao, Zhonghong

    2017-01-01

    It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction. PMID:28103293

  19. Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.

    PubMed Central

    Le Ricousse, S; Gouilleux, F; Fortin, D; Joulin, V; Richard-Foy, H

    1996-01-01

    We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643531

  20. Three-dimensional inversion recovery manganese-enhanced MRI of mouse brain using super-resolution reconstruction to visualize nuclei involved in higher brain function.

    PubMed

    Poole, Dana S; Plenge, Esben; Poot, Dirk H J; Lakke, Egbert A J F; Niessen, Wiro J; Meijering, Erik; van der Weerd, Louise

    2014-07-01

    The visualization of activity in mouse brain using inversion recovery spin echo (IR-SE) manganese-enhanced MRI (MEMRI) provides unique contrast, but suffers from poor resolution in the slice-encoding direction. Super-resolution reconstruction (SRR) is a resolution-enhancing post-processing technique in which multiple low-resolution slice stacks are combined into a single volume of high isotropic resolution using computational methods. In this study, we investigated, first, whether SRR can improve the three-dimensional resolution of IR-SE MEMRI in the slice selection direction, whilst maintaining or improving the contrast-to-noise ratio of the two-dimensional slice stacks. Second, the contrast-to-noise ratio of SRR IR-SE MEMRI was compared with a conventional three-dimensional gradient echo (GE) acquisition. Quantitative experiments were performed on a phantom containing compartments of various manganese concentrations. The results showed that, with comparable scan times, the signal-to-noise ratio of three-dimensional GE acquisition is higher than that of SRR IR-SE MEMRI. However, the contrast-to-noise ratio between different compartments can be superior with SRR IR-SE MEMRI, depending on the chosen inversion time. In vivo experiments were performed in mice receiving manganese using an implanted osmotic pump. The results showed that SRR works well as a resolution-enhancing technique in IR-SE MEMRI experiments. In addition, the SRR image also shows a number of brain structures that are more clearly discernible from the surrounding tissues than in three-dimensional GE acquisition, including a number of nuclei with specific higher brain functions, such as memory, stress, anxiety and reward behavior.

  1. Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P(+) mouse epidermal cells.

    PubMed

    Cheng, Pai-Shan; Hu, Chao-Chin; Wang, Chau-Jong; Lee, Yean-Jang; Chung, Wei-Chia; Tseng, Tsui-Hwa

    2017-02-25

    Chemoprevention has been acknowledged as an important and practical strategy for managing cancer. We have previously synthesized morusin, a prenylated flavonoid that exhibits anti-cancer progression activity. In the present study, we evaluated the anti-cancer promotion potential of morusin by using the mouse epidermal JB6 P(+) cell model. Extensive evidence shows that tumor promotion by phorbol esters is due to the stimulation of reactive oxygen species (ROS). Therefore, the effect of morusin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ROS production was assessed. Noncytotoxic concentrations of morusin were found to dose-dependently reduce TPA-induced ROS production. Moreover, morusin inhibited TPA-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activation, which can mediate cell proliferation and malignant transformation. Furthermore, morusin inhibited the TPA upregulation of cyclooxygenase 2 (COX-2), which may be regulated by AP-1 and NF-κB. In addition, noncytotoxic concentrations of morusin reduced the TPA-promoted cell growth of JB6 P(+) cells and inhibited TPA-induced malignant properties, such as cytoskeletal rearrangement and cell migration of JB6 P(+) cells. Similar to the effects of glutathione (GSH) pretreatment, morusin inhibited TPA-induced expression of N-cadeherin and vimentin, which are malignant cell surface proteins. Finally, morusin treatment dose-dependently suppressed the TPA-induced anchorage-independent cell transformation of JB6 P(+) cells. In conclusion, our results evidence that morusin possesses anti-cancer promotion potential because of its antioxidant property, which mediates multiple transformation-associated gene expression.

  2. SKAP, an outer kinetochore protein, is required for mouse germ cell development

    PubMed Central

    Grey, Corinne; Espeut, Julien; Ametsitsi, Rachel; Kumar, Rajeev; Luksza, Malgorzata; Brun, Christine; Verlhac, Marie-Hélene; Suja, José Angél; de Massy, Bernard

    2016-01-01

    In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes. PMID:26667018

  3. The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea.

    PubMed

    Shibuya, T; Murota, T; Horiya, N; Matsuda, H; Hara, T

    1993-12-01

    A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC. ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.

  4. Enhanced Genetic Integrity in Mouse Germ Cells1

    PubMed Central

    Murphey, Patricia; McLean, Derek J.; McMahan, C. Alex; Walter, Christi A.; McCarrey, John R.

    2012-01-01

    ABSTRACT Genetically based diseases constitute a major human health burden, and de novo germline mutations represent a source of heritable genetic alterations that can cause such disorders in offspring. The availability of transgenic rodent systems with recoverable, mutation reporter genes has been used to assess the occurrence of spontaneous point mutations in germline cells. Previous studies using the lacI mutation reporter transgenic mouse system showed that the frequency of spontaneous mutations is significantly lower in advanced male germ cells than in somatic cell types from the same individuals. Here we used this same mutation reporter transgene system to show that female germ cells also display a mutation frequency that is lower than that in corresponding somatic cells and similar to that seen in male germ cells, indicating this is a common feature of germ cells in both sexes. In addition, we showed that statistically significant differences in mutation frequencies are evident between germ cells and somatic cells in both sexes as early as mid-fetal stages in the mouse. Finally, a comparison of the mutation frequency in a general population of early type A spermatogonia with that in a population enriched for Thy-1-positive spermatogonia suggests there is heterogeneity among the early spermatogonial population such that a subset of these cells are predestined to form true spermatogonial stem cells. Taken together, these results support the disposable soma theory, which posits that genetic integrity is normally maintained more stringently in the germ line than in the soma and suggests that this is achieved by minimizing the initial occurrence of mutations in early germline cells and their subsequent gametogenic progeny relative to that in somatic cells. PMID:23153565

  5. In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.

    PubMed

    Sato, Takuya; Katagiri, Kumiko; Kojima, Kazuaki; Komeya, Mitsuru; Yao, Masahiro; Ogawa, Takehiko

    2015-01-01

    Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

  6. Extracellular Ca2+-induced force restoration in K+-depressed skeletal muscle of the mouse involves an elevation of [K+]i: implications for fatigue.

    PubMed

    Cairns, Simeon P; Leader, John P; Loiselle, Denis S; Higgins, Amanda; Lin, Wei; Renaud, Jean-Marc

    2015-03-15

    We examined whether a Ca(2+)-K(+) interaction was a potential mechanism operating during fatigue with repeated tetani in isolated mouse muscles. Raising the extracellular Ca(2+) concentration ([Ca(2+)]o) from 1.3 to 10 mM in K(+)-depressed slow-twitch soleus and/or fast-twitch extensor digitorum longus muscles caused the following: 1) increase of intracellular K(+) activity by 20-60 mM (raised intracellular K(+) content, unchanged intracellular fluid volume), so that the K(+)-equilibrium potential increased by ∼10 mV and resting membrane potential repolarized by 5-10 mV; 2) large restoration of action potential amplitude (16-54 mV); 3) considerable recovery of excitable fibers (∼50% total); and 4) restoration of peak force with the peak tetanic force-extracellular K(+) concentration ([K(+)]o) relationship shifting rightward toward higher [K(+)]o. Double-sigmoid curve-fitting to fatigue profiles (125 Hz for 500 ms, every second for 100 s) showed that prior exposure to raised [K(+)]o (7 mM) increased, whereas lowered [K(+)]o (2 mM) decreased, the rate and extent of force loss during the late phase of fatigue (second sigmoid) in soleus, hence implying a K(+) dependence for late fatigue. Prior exposure to 10 mM [Ca(2+)]o slowed late fatigue in both muscle types, but was without effect on the extent of fatigue. These combined findings support our notion that a Ca(2+)-K(+) interaction is plausible during severe fatigue in both muscle types. We speculate that a diminished transsarcolemmal K(+) gradient and lowered [Ca(2+)]o contribute to late fatigue through reduced action potential amplitude and excitability. The raised [Ca(2+)]o-induced slowing of fatigue is likely to be mediated by a higher intracellular K(+) activity, which prolongs the time before stimulation-induced K(+) efflux depolarizes the sarcolemma sufficiently to interfere with action potentials.

  7. Involvement of neuronal and glial activities in control of the extracellular d-serine concentrations by the AMPA glutamate receptor in the mouse medial prefrontal cortex.

    PubMed

    Ishiwata, Sayuri; Umino, Asami; Nishikawa, Toru

    2017-09-28

    It has been well accepted that d-serine may be an exclusive endogenous coagonist for the N-methyl-d-aspartate (NMDA)-type glutamate receptor in mammalian forebrain regions. We have recently found by using an in vivo dialysis method that an intra-medial prefrontal cortex infusion of S-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (S-AMPA), a selective AMPA-type glutamate receptor agonist, causes a reduction in the extracellular levels of d-serine in a calcium-permeable AMPA receptor antagonist-sensitive manner. The inhibitory influence by the AMPA receptor on the extracellular d-serine, however, contradicts the data obtained from in vitro experiments that the AMPA receptor stimulation leads to facilitation of the d-serine liberation. This discrepancy appears to be due to the different cell setups between the in vivo and in vitro preparations. From the viewpoints of the previous reports indicating (1) the neuronal presence of d-serine synthesizing enzyme, serine racemase, and d-serine-like immunoreactivity and (2) the same high tissue concentrations of d-serine in the glia-enriched white matter and in the neuron-enriched gray matter of the mammalian neocortex, we have now investigated in the mouse medial prefrontal cortex, the effects of attenuation of neuronal and glial activities, by tetrodotoxin or fluorocitrate, respectively, on the S-AMPA-induced downregulation of the extracellular d-serine contents. In vivo dialysis studies revealed that a local infusion of tetrodotoxin or fluorocitrate eliminated the ability of S-AMPA given intra-cortically to cause a significant decrease in the dialysate concentrations of d-serine without affecting the elevating effects of S-AMPA on those of glycine, another intrinsic coagonist for the NMDA receptor. These findings suggest that the control by the AMPA receptor of the extracellular d-serine levels could be modulated by the neuronal and glial activities in the prefrontal cortex. It cannot be excluded that

  8. Extracellular Ca2+-induced force restoration in K+-depressed skeletal muscle of the mouse involves an elevation of [K+]i: implications for fatigue

    PubMed Central

    Leader, John P.; Loiselle, Denis S.; Higgins, Amanda; Lin, Wei; Renaud, Jean-Marc

    2015-01-01

    We examined whether a Ca2+-K+ interaction was a potential mechanism operating during fatigue with repeated tetani in isolated mouse muscles. Raising the extracellular Ca2+ concentration ([Ca2+]o) from 1.3 to 10 mM in K+-depressed slow-twitch soleus and/or fast-twitch extensor digitorum longus muscles caused the following: 1) increase of intracellular K+ activity by 20–60 mM (raised intracellular K+ content, unchanged intracellular fluid volume), so that the K+-equilibrium potential increased by ∼10 mV and resting membrane potential repolarized by 5–10 mV; 2) large restoration of action potential amplitude (16–54 mV); 3) considerable recovery of excitable fibers (∼50% total); and 4) restoration of peak force with the peak tetanic force-extracellular K+ concentration ([K+]o) relationship shifting rightward toward higher [K+]o. Double-sigmoid curve-fitting to fatigue profiles (125 Hz for 500 ms, every second for 100 s) showed that prior exposure to raised [K+]o (7 mM) increased, whereas lowered [K+]o (2 mM) decreased, the rate and extent of force loss during the late phase of fatigue (second sigmoid) in soleus, hence implying a K+ dependence for late fatigue. Prior exposure to 10 mM [Ca2+]o slowed late fatigue in both muscle types, but was without effect on the extent of fatigue. These combined findings support our notion that a Ca2+-K+ interaction is plausible during severe fatigue in both muscle types. We speculate that a diminished transsarcolemmal K+ gradient and lowered [Ca2+]o contribute to late fatigue through reduced action potential amplitude and excitability. The raised [Ca2+]o-induced slowing of fatigue is likely to be mediated by a higher intracellular K+ activity, which prolongs the time before stimulation-induced K+ efflux depolarizes the sarcolemma sufficiently to interfere with action potentials. PMID:25571990

  9. Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: involvement of RhoA, Ca(2+)/CaM, and ERK.

    PubMed

    Park, Jae Hong; Ryu, Jung Min; Yun, Seung Pil; Kim, Mi Ok; Han, Ho Jae

    2012-10-01

    Extracellular matrix (ECM) components and intracellular pH (pH(i)) may serve as regulators of cell migration in various cell types. The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na(+)/H(+) exchanger (NHE)-1 activity was evaluated by measuring pH(i) and [(22)Na(+)] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed. ECM components (FN, laminin, fibrinogen, and collagen type I) increased [(22)Na(+)] uptake, pH(i), and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca(2+) influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca(2+)/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [(22)Na(+)] uptake and pH(i). Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration. FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca(2+)/CaM signaling pathways. The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Involvement of the renin-angiotensin system in the development of nephrogenic systemic fibrosis-like lesions in the RenTag mouse model.

    PubMed

    Sexton, Sandra; Tulowitzki, Ryan; Jones, Craig A; Shah, Silvi; Hajduczok, George; Gross, Kenneth W; Panesar, Mandip

    2016-04-01

    Renin-angiotensin system (RAS) activation increases angiotensin II production stimulating profibrotic factors, especially in the setting of chronic kidney disease. Nephrogenic systemic fibrosis (NSF) has been associated with gadolinium (Gd) exposure and renal failure. RAS involvement in NSF is unclear compared to transforming growth factor beta and Smad. RenTag mice were chosen to investigate the role of RAS in NSF-like dermal fibrosis because they demonstrated dermal fibrosis at birth, perturbations of RAS in subcutaneous tissue, and renal failure within 4 weeks of age. Wild-type and RenTag mice were injected weekly with a supratherapeutic dose of intravenous gadodiamide (3.0 mmol/kg body weight) and killed at 12 weeks of age for skin and kidney histology. RenTag mice had elevated BUN levels, pitted kidneys, and glomerular damage. RenTag mice skin revealed an increased density of fibroblasts, no mucopolysaccharide deposits, and increased collagen fibril density regardless of Gd exposure. Skin and kidney histopathology of wild-type mice were normal regardless of Gd exposure. CD34 positivity was higher in RenTag compared to wild-type. Since RenTag dermal lesions remained unchanged after gadolinium exposure in the setting of renal failure, this animal model suggests perturbations of subcutaneous RAS may be involved in Gd-naïve dermal fibrosis.

  11. Melatonin promotes goat spermatogonia stem cells (SSCs) proliferation by stimulating glial cell line-derived neurotrophic factor (GDNF) production in Sertoli cells

    PubMed Central

    Niu, Bowen; Li, Bo; Wu, Chongyang; Wu, Jiang; Yan, Yuan; Shang, Rui; Bai, Chunling; Li, Guangpeng; Hua, Jinlian

    2016-01-01

    Melatonin has been reported to be an important endogenous hormone for regulating neurogenesis, immunityand the biological clock. Recently, the effects of melatonin on neural stem cells (NSCs), mesenchymal stem cells(MSCs), and induced pluripotent stem cells(iPSCs) have been reported; however, the effects of melatonin on spermatogonia stem cells (SSCs) are not clear. Here, 1μM and 1nM melatonin was added to medium when goat SSCs were cultured in vitro, the results showed that melatonin could increase the formation and size of SSC colonies. Real-time quantitative PCR (QRT-PCR) and western blot analysis showed that the expression levels of SSC proliferation and self-renewal markers were up-regulated. Meanwhile, QRT-PCR results showed that melatonin inhibit the mRNA expression level of SSC differentiation markers. ELISA analysis showed an obvious increase in the concentration of GDNF (a niche factor secreted by Sertoli cells) in the medium when treated with melatonin. Meanwhile, the phosphorylation level of AKT, a downstream of GDNF-GFRa1-RET pathway was activated. In conclusion, melatonin promotes goat SSC proliferation by stimulating GDNF production in Sertoli cells. PMID:27769051

  12. NRG1 and KITL Signal Downstream of Retinoic Acid in the Germline to Support Soma-Free Syncytial Growth of Differentiating Spermatogonia.

    PubMed

    Chapman, Karen M; Medrano, Gerardo A; Chaudhary, Jaideep; Hamra, F Kent

    Defined culture systems supporting spermatogonial differentiation will provide experimental platforms to study spermatogenesis. However, germline-intrinsic signaling mechanisms sufficient to support spermatogonial differentiation without somatic cells remain largely undefined. Here, we analyzed EGF superfamily receptor and ligand diversity in rat testis cells, and delineated germline-intrinsic signaling via an ERBB3 co-transducer, ERBB2, as essential for retinoic acid-induced syncytial growth by differentiating spermatogonia. Like the ERBB2/3 agonist NRG1, we found KIT Ligand (KITL) robustly supported spermatogonial differentiation without serum or somatic cells. ERBB2 inhibitors failed to disrupt KITL-dependent spermatogonial development, and, KITL prevented ERBB3-deficient spermatogonial degeneration upon differentiation. Thus, we report NRG1 and KITL activate alternative pathways downstream of retinoic acid signaling in the germline that are essential for stem cells to undergo pre-meiotic steps of spermatogenesis in culture. Robust serum/soma-free spermatogonial differentiation opens new doors to study mammalian germ cell biology in culture, which will facilitate the discovery of spermatogenic factors that can drive meiotic progression in vitro.

  13. Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis.

    PubMed

    van Lent, Peter L E M; Blom, Arjen B; Schelbergen, Rik F P; Slöetjes, Annet; Lafeber, Floris P J G; Lems, Willem F; Cats, Hans; Vogl, Thomas; Roth, Johannes; van den Berg, Wim B

    2012-05-01

    To investigate whether alarmins S100A8 and S100A9 are involved in mediating cartilage destruction during murine and human osteoarthritis (OA). Two different murine models of OA that differed in terms of synovial activation were compared. Cartilage destruction was measured histologically. Synovial biopsy and serum samples from OA patients were derived from the Cohort Hip and Cohort Knee (CHECK) patients with symptomatic early OA. Expression of mediators in the synovium was measured by reverse transcription-polymerase chain reaction analysis and immunolocalization. In collagenase-induced OA, which showed marked synovial activation, interleukin-1β was expressed at significant levels only during the early stages of disease, whereas S100A8 and S100A9 expression remained high for a prolonged period of time (up to day 21 after induction). In S100A9-knockout mice, we found a major impact of S100A8 and S100A9 on synovial activation (62% inhibition) and OA cartilage destruction (45-73% inhibition) as compared to wild-type controls. In contrast, in the surgically induced destabilized medial meniscus model, in which synovial involvement is scant, we found no role of S100A8 and S100A9 in the focal OA cartilage destruction. Examination of arthroscopic synovial biopsy samples from patients in the early symptomatic OA CHECK cohort revealed substantial levels of S100A8 and S100A9 messenger RNA and protein, which correlated significantly with synovial lining thickness, cellularity in the subintima, and joint destruction. Levels of S100A8/A9 serum protein were significantly enhanced (19%) at baseline in patients who had pronounced progression of joint destruction after 2 years. Our data suggest that the S100A8 and S100A9 proteins are crucially involved in synovial activation and cartilage destruction during OA and that high levels may predict joint destruction in humans with OA. Copyright © 2012 by the American College of Rheumatology.

  14. Evidence for the involvement of the GABAergic, but not serotonergic transmission in the anxiolytic-like effect of bisabolol in the mouse elevated plus maze.

    PubMed

    Tabari, Mohaddeseh Abouhosseini; Tehrani, Mohammad Amir Bagherpour

    2017-07-20

    Bisabolol (α-(-)-bisabolol) is a sesquiterpene which is a part of the essential oil of a variety of plants, but its common source is German chamomile. Several bioactivities including anti-inflammatory, anti-nociceptive, and anti-tumor effects were attributed to bisabolol. However, the neuropharmacological properties of bisabolol have not yet been reported. The present study evaluated behavioral effects of bisabolol using elevated plus maze (EPM), open field test (OFT), and rotarod test. Moreover, this study also examined whether the 5-HT1A and GABAA-benzodiazepine receptor systems are involved in the anxiolytic-like effects of bisabolol. After acute intraperitoneal treatment with bisabolol at the doses of 0.5, 1, 2, 5, and 10 mg/kg, OFT, EPM, and rotarod were utilized for investigating behavioral effects. Flumazenil, a benzodiazepine receptor antagonist, and WAY-100635, a 5-HT1A receptor antagonist, were used to determine the action mechanism in the EPM. Bisabolol especially at the dose of 1 mg/kg was effective in increasing the total number of entries and time spent in the open arms of EPM while number of rearing and grooming in OFT was decreased in comparison to the control. In the rotarod, permanence time was decreased in the mice treated with the high doses of bisabolol. Pretreatment with flumazenil, but not WAY-100635, was able to reverse the effect of bisabolol 1 mg/kg in the EPM, indicating that the anxiolytic-like activity of bisabolol occurs via the GABAergic but not serotonergic transmission. The present study supports the idea that bisabolol may mediate its anxiolytic-like and sedative mechanisms involving GABAA receptors.

  15. Relationship between Rgs2 gene expression level and anxiety and depression-like behaviour in a mutant mouse model: serotonergic involvement.

    PubMed

    Lifschytz, Tzuri; Broner, Esther Channah; Zozulinsky, Polina; Slonimsky, Alexandra; Eitan, Renana; Greenbaum, Lior; Lerer, Bernard

    2012-10-01

    RGS2 is a member of a family of proteins that negatively modulate G-protein coupled receptor transmission. Variations in the RGS2 gene were found to be associated in humans with anxious and depressive phenotypes. We sought to study the relationship of Rgs2 expression level to depression and anxiety-like behavioural features, sociability and brain 5-HT1A and 5-HT1B receptor expression. We studied male mice carrying a mutation that causes lower Rgs2 gene expression, employing mice heterozygous (Het) or homozygous (Hom) for this mutation, or wild-type (WT). Mice were subjected to behavioural tests reflecting depressive-like behaviour [forced swim test (FST), novelty suppressed feeding test (NSFT)], elevated plus maze (EPM) for evaluation of anxiety levels and the three-chamber sociability test. The possible involvement of raphe nucleus 5-HT1A receptors in these behavioural features was examined by 8-OH-DPAT-induced hypothermia. Expression levels of 5-HT1A and 5-HT1B receptors in the cortex, raphe nucleus and hypothalamus were compared among mice of the different Rgs2 genotype groups. NSFT results demonstrated that Hom mice showed more depressive-like features than Rgs2 Het and WT mice. A trend for such a relationship was also suggested by the FST results. EPM and sociability test results showed Hom and Het mice to be more anxious and less sociable than WT mice. In addition Hom and Het mice were characterized by lower basal body temperature and demonstrated less 8-OH-DPAT-induced hypothermia than WT mice. Finally, Hom and Het mice had significantly lower 5-HT1A and 5-HT1B receptor expression levels in the raphe than WT mice. Our findings demonstrate a relationship between Rgs2 gene expression level and a propensity for anxious and depressive-like behaviour and reduced social interaction that may involve changes in serotonergic receptor expression.

  16. Proteinase activated receptor-2-mediated dual oxidase-2 up-regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma.

    PubMed

    Nadeem, Ahmed; Alharbi, Naif O; Vliagoftis, Harissios; Tyagi, Manoj; Ahmad, Sheikh F; Sayed-Ahmed, Mohamed M

    2015-07-01

    Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.

  17. An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis.

    PubMed

    Tanaka, Niina; Goto, Miki; Kawasaki, Azusa; Sasano, Takashi; Eto, Ko; Nishi, Ryotaro; Sugasawa, Kaoru; Abe, Shinichi; Saitoh, Hisato

    2010-10-01

    A multifunctional calcium-binding protein, centrin-1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin-1 as a protein interacting with SUMO-2/3 using yeast two-hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin-1 and SUMO-2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl₂. immunostaining of seminiferous tubules in 35-day-old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO-2/3 with centrin-1 in cytoplasmic spots. Identification of centrin-1 as the EGTA-sensitive SUMO-2/3-interacting protein indicates the possible role of calcium in modulating the centrin-1-SUMO-2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.

  18. Distinct expression patterns predict differential roles of the miRNA-binding proteins, Lin28 and Lin28b, in the mouse testis: studies during postnatal development and in a model of hypogonadotropic hypogonadism.

    PubMed

    Gaytan, Francisco; Sangiao-Alvarellos, Susana; Manfredi-Lozano, María; García-Galiano, David; Ruiz-Pino, Francisco; Romero-Ruiz, Antonio; León, Silvia; Morales, Concepción; Cordido, Fernando; Pinilla, Leonor; Tena-Sempere, Manuel

    2013-03-01

    Lin28 (also termed Lin28a) and Lin28b are related RNA-binding proteins, involved in the control of microRNA synthesis, especially of the let-7 family, with putative functions in early (embryo) development. However, their roles during postnatal maturation remain ill defined. Despite the general assumption that Lin28 and Lin28b share similar targets and functions, conclusive demonstration of such redundancy is still missing. In addition, recent observations suggest a role of Lin28 proteins in mammalian reproduction, which is yet to be defined. We document herein the patterns of RNA expression and protein distribution of Lin28 and Lin28b in mouse testis during postnatal development and in a model of hypogonadotropic hypogonadism as a result of inactivation of the kisspeptin receptor, Gpr54. Lin28 and Lin28b mRNAs were expressed in mouse testis across postnatal maturation, but their levels disparately varied between neonatal and pubertal periods, with peak Lin28 levels in infantile testes and sustained elevation of Lin28b mRNA in young adult male gonads, where relative levels of let-7a and let-7b miRNAs were significantly suppressed. In addition, Lin28 peptides displayed totally different patterns of cellular distribution in mouse testis: Lin28 was located in undifferentiated and type-A1 spermatogonia, whereas Lin28b was confined to spermatids and interstitial Leydig cells. These profiles were perturbed in Gpr54 null mouse testis, which showed preserved but irregular Lin28 signal and absence of Lin28b peptide, which was rescued by administration of gonadotropins, mainly hCG (as super-agonist of LH). In addition, increased relative levels of Lin28, but not Lin28b, mRNA and of let-7a/let-7b miRNAs were observed in Gpr54 KO mouse testes. Altogether, our data are the first to document the divergent patterns of cellular distribution and mRNA expression of Lin28 and Lin28b in the mouse testis along postnatal maturation and their alteration in a model of congenital

  19. Involvement of PPAR-γ in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP model of Parkinson's disease

    PubMed Central

    2012-01-01

    Background Several recent studies have shown that angiotensin type 1 receptor (AT1) antagonists such as candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of Parkinson's disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1 blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate peroxisome proliferator-activated receptor gamma (PPAR γ). PPAR-γ activation inhibits inflammation, and may be responsible for neuroprotective effects, independently of AT1 blocking actions. Methods We have investigated whether oral treatment with telmisartan (the most potent PPAR-γ activator among AT1 blockers) provides neuroprotection against dopaminergic cell death and neuroinflammation, and the possible role of PPAR-γ activation in any such neuroprotection. We used a mouse model of parkinsonism induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and co-administration of the PPAR-γ antagonist GW9662 to study the role of PPAR-γ activation. In addition, we used AT1a-null mice lesioned with MPTP to study whether deletion of AT1 in the absence of any pharmacological effect of AT1 blockers provides neuroprotection, and investigated whether PPAR-γ activation may also be involved in any such effect of AT1 deletion by co-administration of the PPAR-γ antagonist GW9662. Results We observed that telmisartan protects mouse dopaminergic neurons and inhibits the microglial response induced by administration of MPTP. The protective effects of telmisartan on dopaminergic cell death and microglial activation were inhibited by co-administration of GW9662. Dopaminergic cell death and microglial activation were significantly lower in AT1a-null mice treated with MPTP than in mice not subjected to AT1a deletion. Interestingly, the protective effects of AT1 deletion were also inhibited by co-administration of GW9662

  20. Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage

    PubMed Central

    2011-01-01

    Background Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known. Results Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically. Conclusions These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin. PMID:21489308

  1. The involvement of NMDA receptor/NO/cGMP pathway in the antidepressant like effects of baclofen in mouse force swimming test.

    PubMed

    Khan, Muhammad Imran; Ostadhadi, Sattar; Zolfaghari, Samira; Ejtemaei Mehr, Shahram; Hassanzadeh, Gholamreza; Dehpour, Ahmad-Reza

    2016-01-26

    In the current study, the involvement of N-methyl-d-aspartate receptor (NMDAR) and nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system in the antidepressant-like effects of baclofen was evaluated by using animal model in forced swimming test. Followed by an open field test for the evaluation of locomotor activity, the immobility time for mice in force swimming test was recorded. Only the last four min was analyzed. Administration of Baclofen (0.5 and 1mg/kg, i.p.) reduced the immobility interval in the FST. Prior administration of l-arginine (750mg/kg, i.p.,) a nitric oxide synthase substrate or sildenafil (5mg/kg, i.p.) a phosphodiesterase 5 into mice suppressed the antidepressant-like activity of baclofen (1mg/kg, i.p.).Co-treatment of 7-nitroindazole (50mg/kg, i.p.,) an inhibitor of neuronal nitric oxide synthase, L-NAME (10mg/kg, i.p.,) a non-specific inhibitor of nitric oxide synthase or MK-801 (0.05mg/kg, i.p.) an NMDA receptor antagonist with subeffective dose of baclofen (0.1mg/kg, i.p.), reduced the immobility time in the FST as compared to the drugs when used alone. Co-administrated of lower doses of MK-801 (0.01mg/kg) or l-NAME (1mg/kg) failed to effect immobility time however, simultaneous administration of these two agents in same dose with subeffective dose of baclofen (0.1mg/kg, i.p.), minimized the immobility time in the FST. Thus, our results support the role of NMDA receptors and l-arginine-NO-GMP pathway in the antidepressant-like action of baclofen.

  2. Effects of Melatonin on Early Pregnancy in Mouse: Involving the Regulation of StAR, Cyp11a1, and Ihh Expression

    PubMed Central

    Guan, Shengyu; Xie, Lu; Ma, Teng; Lv, Dongying; Jing, Wang; Tian, Xiuzhi; Song, Yukun; Liu, Zhiping; Xiao, Xianghong

    2017-01-01

    To test whether melatonin plays an important role in the process of early pregnancy, melatonin was given in drinking water to pregnant mice at different gestation stages. These included mice who were given melatonin 14 days prior to their successful mating (confirmed by vaginal plug) (Group A), after successful mating (Group B), and 14 days prior to and until after successful mating (Group C). Melatonin administration significantly enhanced serum as well as ovarian melatonin levels in the mice. It was observed that melatonin did not affect the natural estrous of mice. On day 0.5 of gestation (D0.5), melatonin not only elevated progesterone (P) secretion, but also upregulated expressions of StAR and Cyp11a1, the two marker genes of corpus luteum in ovaries (p < 0.05). Group A had a significantly lower estradiol (E2) secretion and a higher number of implantation sites as well as litter size than controls (p < 0.05) and also had an increased Ihh expression in endometrium of D7.5 gestation. Melatonin treatment after successful mating improved the progesterone (P) secretion at D7.5 of gestation (p < 0.05) and significantly induced leukaemia inhibitory factor (LIF) expression (p < 0.05). Our study indicates that melatonin treatment up-regulated the genes involved in pregnenolone synthesis in ovary and Ihh expression in uterine endometrium. The mechanisms of melatonin to improve embryo implantation related to their actions on promoting the development of corpus luteum before gestation and helping to specify uterine receptivity in early pregnant mice. PMID:28749439

  3. Effects of Melatonin on Early Pregnancy in Mouse: Involving the Regulation of StAR, Cyp11a1, and Ihh Expression.

    PubMed

    Guan, Shengyu; Xie, Lu; Ma, Teng; Lv, Dongying; Jing, Wang; Tian, Xiuzhi; Song, Yukun; Liu, Zhiping; Xiao, Xianghong; Liu, Guoshi

    2017-07-27

    To test whether melatonin plays an important role in the process of early pregnancy, melatonin was given in drinking water to pregnant mice at different gestation stages. These included mice who were given melatonin 14 days prior to their successful mating (confirmed by vaginal plug) (Group A), after successful mating (Group B), and 14 days prior to and until after successful mating (Group C). Melatonin administration significantly enhanced serum as well as ovarian melatonin levels in the mice. It was observed that melatonin did not affect the natural estrous of mice. On day 0.5 of gestation (D0.5), melatonin not only elevated progesterone (P) secretion, but also upregulated expressions of StAR and Cyp11a1, the two marker genes of corpus luteum in ovaries (p < 0.05). Group A had a significantly lower estradiol (E₂) secretion and a higher number of implantation sites as well as litter size than controls (p < 0.05) and also had an increased Ihh expression in endometrium of D7.5 gestation. Melatonin treatment after successful mating improved the progesterone (P) secretion at D7.5 of gestation (p < 0.05) and significantly induced leukaemia inhibitory factor (LIF) expression (p < 0.05). Our study indicates that melatonin treatment up-regulated the genes involved in pregnenolone synthesis in ovary and Ihh expression in uterine endometrium. The mechanisms of melatonin to improve embryo implantation related to their actions on promoting the development of corpus luteum before gestation and helping to specify uterine receptivity in early pregnant mice.

  4. Effect of Diet on Expression of Genes Involved in Lipid Metabolism, Oxidative Stress, and Inflammation in Mouse Liver–Insights into Mechanisms of Hepatic Steatosis

    PubMed Central

    Renaud, Helen J.; Cui, Julia Y.; Lu, Hong; Klaassen, Curtis D.

    2014-01-01

    Nutritional intake is a fundamental determinant of health. Many studies have correlated excess caloric intake, as well as a high ratio of n-6:n-3 fatty acids, with detrimental health outcomes, such as the metabolic syndrome. In contrast, low-calorie diets have beneficial health effects. Despite these associations, our understanding of the causal relationship between diet and health remains largely elusive. The present study examined the molecular changes elicited by nine diets with varying fat, sugar, cholesterol, omega-3 fatty acids, omega-6 fatty acids, and calories in C57BL/6 male mice. Microarray analyses were conducted on liver samples from three mice per diet and detected 20,449 genes of which 3,734 were responsive to changes in dietary components. Principal component analysis showed that diet restriction correlated the least with the other diets and also affected more genes than any other diet. Interestingly, Gene Set Enrichment Analysis (GSEA) identified gene sets involved in glutathione metabolism, immune response, fatty acid metabolism, cholesterol metabolism, ABC transporters, and oxidative phosphorylation as being highly responsive to changes in diet composition. On the gene level, this study reveals novel findings such as the induction of the drug efflux pump Abcb1a (p-glycoprotein) by diet restriction and an atherogenic diet, as well as the suppression of the rate limiting step of bile acid synthesis, Cyp7a1, by a high fructose diet. This study provides considerable insight into the molecular changes incurred by a variety of diets and furthers our understanding of the causal relationships between diet and health. PMID:24551121

  5. Genetic ablation of Slc4a10 alters the expression pattern of transporters involved in solute movement in the mouse choroid plexus.

    PubMed

    Damkier, Helle Hasager; Praetorius, Jeppe

    2012-05-15

    Mutational changes of one transporter can have deleterious effects on epithelial function leaving the cells with the options of either compensating for the loss of function or dedifferentiating. Previous studies have shown that the choroid plexus epithelium (CPE) from mice lacking the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger (NCBE) encoded by Slc4a10 leads to retargeting of the Na(+)/H(+) exchanger 1 (NHE1) from the luminal to the basolateral plasma membrane. We hypothesized that disruption of NCBE, the main basolateral Na(+) importer in the CPE, would lead to a compensatory increase in the abundance of other important transport proteins in this tissue. Aquaporin-1 (AQP1) abundance was 42.7% lower and Na,K-ATPase 36.4% lower in the CPE of Slc4a10 knockout mice, respectively. The NHE1 binding ezrin cytoskeleton appeared disrupted in Slc4a10 knockout mice, whereas no changes were observed in cellular polarization with respect to claudin-2 and appearance of luminal surface microvilli. The renal proximal tubule constitutes a leaky epithelium with high transport rate similar to CPE. Here, Slc4a10 knockout did not affect Na,K-ATPase or AQP1 expression. CPE from AQP1 knockout mice has a secretory defect similar to Slc4a10 mice. However, neither NCBE nor Na,K-ATPase expression was affected in CPE from AQP1 knockout mice. By contrast, the abundance of Na,K-ATPase and NBCe1 was decreased by 23 and 31.7%, respectively, in AQP1 knockout proximal tubules, while the NHE3 abundance was unchanged. In conclusion, CPE lacking NCBE seems to spare the molecular machinery involved in CSF secretion rather than compensate for the loss of the Na(+) loader. Slc4a10 knockout seems to be more deleterious to CPE than AQP1 knockout.

  6. CIBZ, a Novel BTB Domain-Containing Protein, Is Involved in Mouse Spinal Cord Injury via Mitochondrial Pathway Independent of p53 Gene

    PubMed Central

    Yang, Shiyong; Li, Ping; Zhang, Xuan; Liu, Honglin

    2012-01-01

    Spinal cord injury (SCI) induces both primary uncontrollable mechanical injury and secondary controllable degeneration, which further results in the activation of cell death cascades that mediate delayed tissue damage. To alleviate its impairments and seek for an effective remedy, mRNA differential display was used to investigate gene mRNA expression profiling in mice following SCI. A specific Zinc finger and BTB domain-containing protein, CIBZ, was discovered to implicate in the SCI process for the first time. Further researches indicated that CIBZ was extensively distributed in various tissues, and the expression level was highest in muscle, followed by spinal cord, large intestine, kidney, spleen, thymus, lung, cerebrum, stomach, ovary and heart, respectively. After injury, the CIBZ expression decreased dramatically and reached the lowest level at 8 h, but it gradually increased to the maximal level at 7 d. Caspase-3 and C-terminal-binding protein (CtBP), two CIBZ-related proteins, showed similar tendency. Interestingly, p53 expression remained constant in all groups. Via flow cytometry (FCM) analysis, it was found that the cell death rate in SCI group markedly increased and reached the highest value 1 d after surgery and the mitochondrial transmembrane potential (ΔΨm) at 1 d was the lowest in all groups. Taken together, it is suggested that: (i) in the presence of CtBP, CIBZ gene is involved in secondary injury process and trigger the activation of apoptotic caspase-3 and bax genes independent of p53; (ii) abrupt down-regulation of CtBP at 8 h is a sign of mitochondria dysfunction and the onset of cell death; (iii) it could be used as an inhibitor or target drug of caspase-3 gene to improve spinal cord function. PMID:22427977

  7. Delta(9)-tetrahydrocannabinol prolongs the immobility time in the mouse forced swim test: involvement of cannabinoid CB(1) receptor and serotonergic system.

    PubMed

    Egashira, Nobuaki; Matsuda, Tomomi; Koushi, Emi; Higashihara, Fuminori; Mishima, Kenichi; Chidori, Shozo; Hasebe, Nobuyoshi; Iwasaki, Katsunori; Nishimura, Ryoji; Oishi, Ryozo; Fujiwara, Michihiro

    2008-07-28

    In the present study, we investigated the effect of Delta(9)-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, on immobility time during the forced swim test. THC (2 and 6 mg/kg, i.p.) significantly prolonged the immobility time. In addition, THC at the same doses did not significantly affect locomotor activity in the open-field test. The selective cannabinoid CB(1) receptor antagonist rimonabant (3 mg/kg, i.p.) significantly reduced the enhancement of immobility by THC (6 mg/kg). Similarly, the selective serotonin (5-HT) reuptake inhibitor (SSRI) citalopram (10 mg/kg, i.p.) and 5-HT(1A/7) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.3 mg/kg, i.p.) significantly reduced this THC-induced effect. Moreover, the selective 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide dihydrochloride (WAY100635, 1 mg/kg, i.p.) and the postsynaptic 5-HT(1A) receptor antagonist MM-77 (0.1 mg/kg, i.p.) reversed this reduction effect of 8-OH-DPAT (0.3 mg/kg). In contrast, the selective 5-HT(7) receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl]phenol hydrochloride (SB269970) had no effect on this reduction effect of 8-OH-DPAT. WAY100635 (1 mg/kg) also reversed the reduction effect of citalopram (10 mg/kg). These findings suggest that the 5-HT(1A) receptors are involved in THC-induced enhancement of immobility.

  8. Involvement of D1 and D2 dopamine receptors in the antidepressant-like effects of selegiline in maternal separation model of mouse.

    PubMed

    Amiri, Shayan; Amini-Khoei, Hossein; Mohammadi-Asl, Ali; Alijanpour, Sakineh; Haj-Mirzaian, Arya; Rahimi-Balaei, Maryam; Razmi, Ali; Olson, Carl O; Rastegar, Mojgan; Mehdizadeh, Mehdi; Zarrindast, Mohammad-Reza

    2016-09-01

    Mother-infant interactions are known to be associated with the psychological well-being of an individual in adulthood. It is well accepted that emotional stress in early life, such as maternal separation (MS), leads to alterations in the neurotransmission systems of various brain regions, especially the mesolimbic dopaminergic system, and subsequently can increase the risk for development of psychiatric disorders including depression in adulthood. Selegiline is an irreversible monoamine oxidase (MAO) type B inhibitor which increases striatal dopamine levels and exerts an antidepressant effect. In this study, 180min of MS stress was applied to mice at postnatal day (PND) 2-14 followed by behavioral tests for determining depressive-like behaviors, such as forced swimming test (FST), splash test and sucrose preference test (SPT) in adult mice (PND 50). The open field test (OFT) also was applied to validate FST results. We used SCH23390 (D1 antagonist) and sulpiride (D2 antagonist) in order to determine the role of D1 and D2 dopamine receptors in antidepressant-like effects of selegiline. Our results revealed that MS provoked depressive-like behaviors in adult male mice, and the administration of selegiline attenuated depressive-like behaviors in MS mice. Our findings showed that D1 dopamine receptors facilitate the positive effects of selegiline on the passive behavior in the FST. Furthermore, antidepressant effects of selegiline on hedonic difficulties are mediated via D2 receptor in the SPT. The results of the splash test revealed that both D1 and D2 receptors mediate the protective effect of selegiline against motivational and self-care problems. Based on our results, we conclude that both D1 and D2 dopamine receptors are involved in mediating the antidepressant-like effect of selegiline. We found that D1 receptors mediate an effect on despair behavior, D2 receptors mediate an effect on anhedonia, and both D1 and D2 receptors contribute to the protective effects of

  9. Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse.

    PubMed

    La Salle, Sophie; Trasler, Jacquetta M

    2006-08-01

    In the male germ line, sequence-specific methylation patterns are initially acquired prenatally in diploid gonocytes and are further consolidated after birth during spermatogenesis. It is still unclear how DNA methyltransferases are involved in establishing and/or maintaining these patterns in germ cells, or how their activity is regulated. We compared the temporal expression patterns of the postulated de novo DNA methyltransferases DNMT3a and DNMT3b in murine male germ cells. Mitotic, meiotic and post-meiotic male germ cells were isolated, and expression of various transcript variants and isoforms of Dnmt3a and Dnmt3b was examined using Quantitative RT-PCR and Western blotting. We found that proliferating and differentiating male germ cells were marked by distinctive expression profiles. Dnmt3a2 and Dnmt3b transcripts were at their highest levels in type A spermatogonia, decreased dramatically in type B spermatogonia and preleptotene spermatocytes and rose again in leptotene/zygotene spermatocytes, while Dnmt3a expression was mostly constant, except in type B spermatogonia where it increased. In all cases, expression declined as pachynema progressed. At the protein level, DNMT3a was the predominant isoform in type B spermatogonia, while DNMT3a2, DNMT3b2, and DNMT3b3 were expressed throughout most of spermatogenesis, except in pachytene spermatocytes. We also detected DNMT3a2 and DNMT3b2 in round spermatids. Taken together, these data highlight the tightly regulated expression of these genes during spermatogenesis and provide evidence that DNMTs may be contributing differentially to the establishment and/or maintenance of methylation patterns in male germ cells.

  10. Differential involvement of the sigma1 (σ1) receptor in the anti-amnesic effect of neuroactive steroids, as demonstrated using an in vivo antisense strategy in the mouse

    PubMed Central

    Maurice, Tangui; Phan, Vân-Ly; Urani, Alexandre; Guillemain, Isabelle

    2001-01-01

    The sigma1 (σ1) receptor cDNA was cloned in several animal species. Molecular tools are now available to identify its endogenous effectors, such as neuroactive steroids, and to establish its precise physiological role. In particular, the σ1 receptor is involved in memory processes, as observed in pharmacological and pathological rodent models of amnesia. In order to establish the involvement of σ1 receptors in memory, a 16-mer oligodeoxynucleotide antisense to the σ1 receptor cDNA (aODN), and its mismatched control (mODN) were prepared and centrally administered into the mouse brain. The anti-amnesic effects induced by the selective σ1 agonist PRE-084 and the steroid dehydroepiandrosterone (DHEA) sulphate or pregnenolone sulphate were examined in ODN-treated animals. The aODN treatment failed to affect the dissociation constant (Kd) but significantly decreased the number of σ1 sites (Bmax) labelled with [3H]-(+)-SKF-10,047 in the hippocampus and cortex. In these structures, the in vivo binding levels were also diminished, according to the dose and number of injections, as compared with control animals injected with saline or mODN. Cannulation and injections failed to affect the open-field behaviour of the animals. However, the anti-amnesic effects of PRE-084 and DHEA sulphate against the dizocilpine-induced impairments were blocked after aODN treatment in the short- and long-term memory tests. The anti-amnesic effects of pregnenolone sulphate remained unchanged. These observations bring a molecular basis to the modulatory role of σ1 receptors in memory, and reveal that the anti-amnesic action of neuroactive steroids may not similarly involve an interaction with σ1 receptors. PMID:11739250

  11. Functional evidence that ATP or a related purine is an inhibitory NANC neurotransmitter in the mouse jejunum: study on the identity of P2X and P2Y purinoceptors involved

    PubMed Central

    De Man, Joris G; De Winter, Benedicte Y; Seerden, Tom C; De Schepper, Heiko U; Herman, Arnold G; Pelckmans, Paul A

    2003-01-01

    neurotransmitter in the mouse jejunum, activating P2 but not P1 purinoceptors. Relaxations to the purinergic NANC neurotransmitter mainly involve P2Y receptors of the P2Y1 subtype that are located postjunctionally. Purinergic NANC neurotransmission also involves P2X receptors, most likely of the P2X1 and P2X3 subtype, located pre- and/or postjunctionally. PMID:14530212

  12. Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation.

    PubMed

    Teng, Hui; Sui, Xuesong; Zhou, Cheng; Shen, Cong; Yang, Ye; Zhang, Pang; Guo, Xuejiang; Huo, Ran

    2016-01-01

    Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca(2+)/CamKII/5'-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation.

  13. Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation

    PubMed Central

    Teng, Hui; Sui, Xuesong; Zhou, Cheng; Shen, Cong; Yang, Ye; Zhang, Pang; Guo, Xuejiang; Huo, Ran

    2016-01-01

    ABSTRACT Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca2+/CamKII/5′-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation. PMID:26716399

  14. Cyclosporin A induced toxicity in mouse liver slices is only slightly aggravated by Fxr-deficiency and co-occurs with upregulation of pro-inflammatory genes and downregulation of genes involved in mitochondrial functions.

    PubMed

    Szalowska, Ewa; Pronk, Tessa E; Peijnenburg, Ad Acm

    2015-10-20

    The transcription factor farnesoid X receptor (FXR) governs bile acid and energy homeostasis, is involved in inflammation, and has protective functions in the liver. In the present study we investigated the effect of Fxr deficiency in mouse precision cut liver slices (PCLS) exposed to a model hepatotoxicant cyclosporin A (CsA). It was anticipated that Fxr deficiency could aggravate toxicity of CsA in PCLS and pinpoint to novel genes/processes regulated by FXR. To test this hypothesis, PCLS obtained from livers of wild type mice (WT-PCLS) and Fxr-knockout mice (FXRKO-PCLS) were treated with 40 μM CsA for 24 h and 48 h. ATP and histological assays were applied to assess the viability of PCLS. DNA microarrays combined with bioinformatics analysis were used to identify genes and processes that were affected by CsA in WT-PCLS and/or FXRKO-PCLS. In addition, WT-PCLS and FXRKO-PCLS were exposed to the endogenous FXR ligand chenodeoxycholic acid (CDCA) and subjected to q-PCR to determine whether subsets of known FXR-targets and the identified genes were regulated upon FXR activation in an FXR-dependent manner. No difference in viability was observed between WT-PCLS and FXRKO-PCLS upon CsA treatment. Transcriptomics data analysis revealed that CsA significantly upregulated stress-response and inflammation and significantly downregulated processes involved in lipid and glucose metabolism in WT-PCLS and FXRKO-PCLS. However, only in FXRKO-PCLS, CsA upregulated additional pro-inflammatory genes and downregulated genes related to mitochondrial functions. Furthermore, only in WT-PCLS, CDCA upregulated a subset of known FXR-target genes as well as the regulator of inflammation and mitochondrial functions peroxisome proliferator- activated receptor delta (Ppar delta). Although FXR governs energy metabolism, no major differences in response to CsA could be observed between WT-PCLS and FXRKO-PCLS in regulation of processes involved in lipid and glucose metabolism. This finding

  15. Obesity in Aging Exacerbates Blood–Brain Barrier Disruption, Neuroinflammation, and Oxidative Stress in the Mouse Hippocampus: Effects on Expression of Genes Involved in Beta-Amyloid Generation and Alzheimer’s Disease

    PubMed Central

    Tucsek, Zsuzsanna; Toth, Peter; Sosnowska, Danuta; Gautam, Tripti; Mitschelen, Matthew; Koller, Akos; Szalai, Gabor; Sonntag, William E.; Csiszar, Anna

    2014-01-01

    There is growing evidence that obesity has deleterious effects on the brain and cognitive function in the elderly population. However, the specific mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. To test the hypothesis that aging exacerbates obesity-induced cerebromicrovascular damage and neuroinflammation, we compared young (7 months) and aged (24 months) high fat diet–fed obese C57BL/6 mice. Aging exacerbated obesity-induced systemic inflammation and blood–brain barrier disruption, as indicated by the increased circulating levels of proinflammatory cytokines and increased presence of extravasated immunoglobulin G in the hippocampus, respectively. Obesity-induced blood–brain barrier damage was associated with microglia activation, upregulation of activating Fc-gamma receptors and proinflammatory cytokines, and increased oxidative stress. Treatment of cultured primary microglia with sera derived from aged obese mice resulted in significantly more pronounced microglia activation and oxidative stress, as compared with treatment with young sera. Serum-induced activation and oxidative stress were also exacerbated in primary microglia derived from aged animals. Hippocampal expression of genes involved in regulation of the cellular amyloid precursor protein–dependent signaling pathways, beta-amyloid generation, and the pathogenesis of tauopathy were largely unaffected by obesity in aged mice. Collectively, obesity in aging is associated with a heightened state of systemic inflammation, which exacerbates blood–brain barrier disruption. The resulting neuroinflammation and oxidative stress in the mouse hippocampus likely contribute to the significant cognitive decline observed in aged obese animals. PMID:24269929

  16. A missense mutation in the transcription factor ETV5 leads to sterility, increased embryonic and perinatal death, postnatal growth restriction, renal asymmetry and polydactyly in the mouse.

    PubMed

    Jamsai, Duangporn; Clark, Brett J; Smith, Stephanie J; Whittle, Belinda; Goodnow, Christopher C; Ormandy, Christopher J; O'Bryan, Moira K

    2013-01-01

    ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility.

  17. Sulfur mustard analog, 2-chloroethyl ethyl sulfide-induced skin injury involves DNA damage and induction of inflammatory mediators, in part via oxidative stress, in SKH-1 hairless mouse skin.

    PubMed

    Jain, Anil K; Tewari-Singh, Neera; Gu, Mallikarjuna; Inturi, Swetha; White, Carl W; Agarwal, Rajesh

    2011-09-10

    Bifunctional alkyalating agent, sulfur mustard (SM)-induced cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 mg or 4 mg CEES for 9-48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and matrix metalloproteinase-9 (MMP-9) levels, indicating the involvement of DNA damage and inflammation in CEES-induced skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-related DNA damage and the induction of inflammatory molecules. Oral GSH (300 mg/kg) administration 1h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injury involves DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injury in humans by SM. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Acrylamide-induced carcinogenicity in mouse lung involves mutagenicity: cII gene mutations in the lung of big blue mice exposed to acrylamide and glycidamide for up to 4 weeks.

    PubMed

    Manjanatha, Mugimane G; Guo, Li-Wu; Shelton, Sharon D; Doerge, Daniel R

    2015-06-01

    Potential health risks for humans from exposure to acrylamide (AA) and its epoxide metabolite glycidamide (GA) have garnered much attention lately because substantial amounts of AA are present in a variety of fried and baked starchy foods. AA is tumorigenic in rodents, and a large number of in vitro and in vivo studies indicate that AA is genotoxic. A recent cancer bioassay on AA demonstrated that the lung was one of the target organs for tumor induction in mice; however, the mutagenicity of AA in this tissue is unclear. Therefore, to investigate whether or not gene mutation is involved in the etiology of AA- or GA-induced mouse lung carcinogenicity, we screened for cII mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19-111 mg/kg bw/days). Both doses of AA and GA produced significant increases in cII MFs, with the high doses producing responses 2.7-5.6-fold higher than the corresponding controls (P ≤ 0.05; control MFs = 17.2 ± 2.2 and 15.8 ± 3.5 × 10(-6) in males and females, respectively). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P ≤ 0.01). The predominant types of mutations in the lung cII gene from AA- and GA-treated mice were A:T → T:A, and G:C → C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted. Environ. Mol. Mutagen. 56:446-456, 2015. © 2015 Wiley Periodicals, Inc.

  19. Estimation of the distribution of low-intensity ultrasound mechanical index as a parameter affecting the proliferation of spermatogonia stem cells in vitro.

    PubMed

    Moghaddam, Zeinab Hormozi; Mokhtari-Dizaji, Manijhe; Movahedin, Mansoureh; Ravari, Mohammad Ehsan

    2017-07-01

    Considering the use of physical and mechanical stimulation, such as low-intensity ultrasound for proliferation and differentiation of stem cells, it is essential to understand the physical and acoustical mechanisms of acoustic waves in vitro. Mechanical index is used for quantifying acoustic cavitation and the relationship between acoustic pressure and the frequency. In this study, modeling of the mechanical index was applied to provide treatment protocol and to understand the effective physical processes on reproducibility of stem cells. Due to low intensity of ultrasound, Rayleigh integral model has been used for acoustic pressure computation. The acoustic pressure and mechanical index equations are modeled and solved to estimate optimal mechanical index for 28, 40, 150kHz and 1MHz frequencies. This model are solved in different intensities and distances from transducer in cylindrical coordinates. Based on the results of the mechanical index, regions with threshold mechanical index of 0.7 were identified for extracting of radiation arrangement to cell medium. Acoustic pressure distribution along the axial and radial was extracted. In order to validate the results of the modeling, the acoustic pressure in the water and near field depth was measured by a piston hydrophone. Results of modeling and experiments show that the model is consistent well to experimental results with 0.91 and 0.90 correlation of coefficient (p<0.05) for 1MHz and 40kHz. Low-intensity ultrasound with 0.40 mechanical index is more effective on enhancing the proliferation rate of the spermatogonia stem cells during the seven days of culture. In contrast, higher mechanical index has a harmful effect on the spermatogonial stem cells. Thus, considering cavitation threshold of different materials is necessary to find effective mechanical index ranges on proliferation for the used frequencies. This acoustic propagation model and ultrasound mechanical index assessments can be used with acceptable

  20. Mouse phenotyping.

    PubMed

    Fuchs, Helmut; Gailus-Durner, Valérie; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Da Silva-Buttkus, Patricia; Neff, Frauke; Götz, Alexander; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kastenmüller, Gabi; Kemter, Elisabeth; Lengger, Christoph; Maier, Holger; Matloka, Mikolaj; Möller, Gabriele; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Römisch-Margl, Werner; Rozman, Jan; Wang-Sattler, Rui; Schrewe, Anja; Stöger, Claudia; Tost, Monica; Adamski, Jerzy; Aigner, Bernhard; Beckers, Johannes; Behrendt, Heidrun; Busch, Dirk H; Esposito, Irene; Graw, Jochen; Illig, Thomas; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Kremmer, Elisabeth; Mempel, Martin; Neschen, Susanne; Ollert, Markus; Schulz, Holger; Suhre, Karsten; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Hrabě de Angelis, Martin

    2011-02-01

    Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]). Copyright © 2010 Elsevier Inc. All rights reserved.

  1. DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline.

    PubMed

    Hiura, Hitoshi; Komiyama, Junichi; Shirai, Motomu; Obata, Yayoi; Ogawa, Hidehiko; Kono, Tomohiro

    2007-04-03

    Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.

  2. Obesity in aging exacerbates blood-brain barrier disruption, neuroinflammation, and oxidative stress in the mouse hippocampus: effects on expression of genes involved in beta-amyloid generation and Alzheimer's disease.

    PubMed

    Tucsek, Zsuzsanna; Toth, Peter; Sosnowska, Danuta; Gautam, Tripti; Mitschelen, Matthew; Koller, Akos; Szalai, Gabor; Sonntag, William E; Ungvari, Zoltan; Csiszar, Anna

    2014-10-01

    There is growing evidence that obesity has deleterious effects on the brain and cognitive function in the elderly population. However, the specific mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. To test the hypothesis that aging exacerbates obesity-induced cerebromicrovascular damage and neuroinflammation, we compared young (7 months) and aged (24 months) high fat diet-fed obese C57BL/6 mice. Aging exacerbated obesity-induced systemic inflammation and blood-brain barrier disruption, as indicated by the increased circulating levels of proinflammatory cytokines and increased presence of extravasated immunoglobulin G in the hippocampus, respectively. Obesity-induced blood-brain barrier damage was associated with microglia activation, upregulation of activating Fc-gamma receptors and proinflammatory cytokines, and increased oxidative stress. Treatment of cultured primary microglia with sera derived from aged obese mice resulted in significantly more pronounced microglia activation and oxidative stress, as compared with treatment with young sera. Serum-induced activation and oxidative stress were also exacerbated in primary microglia derived from aged animals. Hippocampal expression of genes involved in regulation of the cellular amyloid precursor protein-dependent signaling pathways, beta-amyloid generation, and the pathogenesis of tauopathy were largely unaffected by obesity in aged mice. Collectively, obesity in aging is associated with a heightened state of systemic inflammation, which exacerbates blood-brain barrier disruption. The resulting neuroinflammation and oxidative stress in the mouse hippocampus likely contribute to the significant cognitive decline observed in aged obese animals. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Protective effects of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the MPTP-induced mouse model of Parkinson's disease: Involvement of reactive oxygen species-mediated JNK, P38 and mitochondrial pathways.

    PubMed

    He, Hong; Wang, Songhai; Tian, Jiyu; Chen, Lei; Zhang, Wei; Zhao, Junjie; Tang, Haifeng; Zhang, Xiaojun; Chen, Jianzong

    2015-11-15

    Parkinson's disease (PD) is characterized by the selective death of dopaminergic neurons in the substantia nigra pars compacta. Oxidative stress-induced neuron loss is thought to play a crucial role in the pathogenesis of PD. Previous work from our group suggests that 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from a traditional Chinese herb, Polygonum multiflorum thunb, can attenuate 1-methyl-4-phenyl pyridium-induced apoptosis in the neuronal cell line PC12, by inhibiting reactive oxygen species generation and modulating c-Jun N-terminal kinases (JNK) activation. Here, we investigated the protective effects of TSG against 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP)-induced loss of tyrosine hydroxylase positive cells in mice and the underlying mechanisms. The results showed that MPTP-induced loss of tyrosine hydroxylase positive cells and reactive oxygen species generation were prevented by TSG in a dose-dependent manner. The reactive oxygen species scavenger N-acetylcysteine could also mitigate reactive oxygen species generation. Moreover, JNK and P38 were activated by MPTP, but extracellular signal-regulated protein kinases phosphorylation did not change after MPTP treatment. TSG at different doses blocked the activation of JNK and P38. The protective effect of TSG was also associated with downregulation of the bax/bcl-2 ratio, reversed the release of cytochrome c and smac, and inhibited the activation of caspase-3, -6, and -9 induced by MPTP. In conclusion, our studies demonstrated that the protective effects of TSG in the MPTP-induced mouse model of PD are involved, at least in part, in controlling reactive oxygen species-mediated JNK, P38, and mitochondrial pathways.

  4. Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

    PubMed Central

    Ponzetto-Zimmerman, C; Wolgemuth, D J

    1984-01-01

    The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis. Images PMID:6324127

  5. Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling.

    PubMed

    Toocheck, Corey; Clister, Terri; Shupe, John; Crum, Chelsea; Ravindranathan, Preethi; Lee, Tae-Kyung; Ahn, Jung-Mo; Raj, Ganesh V; Sukhwani, Meena; Orwig, Kyle E; Walker, William H

    2016-01-01

    Testosterone acts though the androgen receptor in Sertoli cells to support germ cell development (spermatogenesis) and male fertility, but the molecular and cellular mechanisms by which testosterone acts are not well understood. Previously, we found that in addition to acting through androgen receptor to directly regulate gene expression (classical testosterone signaling pathway), testosterone acts through a nonclassical pathway via the androgen receptor to rapidly activate kinases that are known to regulate spermatogenesis. In this study, we provide the first evidence that nonclassical testosterone signaling occurs in vivo as the MAP kinase cascade is rapidly activated in Sertoli cells within the testis by increasing testosterone levels in the rat. We find that either classical or nonclassical signaling regulates testosterone-mediated Rhox5 gene expression in Sertoli cells within testis explants. The selective activation of classical or nonclassical signaling pathways in Sertoli cells within testis explants also resulted in the differential activation of the Zbtb16 and c-Kit genes in adjacent spermatogonia germ cells. Delivery of an inhibitor of either pathway to Sertoli cells of mouse testes disrupted the blood-testis barrier that is essential for spermatogenesis. Furthermore, an inhibitor of nonclassical testosterone signaling blocked meiosis in pubertal mice and caused the loss of meiotic and postmeiotic germ cells in adult mouse testes. An inhibitor of the classical pathway caused the premature release of immature germ cells. Collectively, these observations indicate that classical and nonclassical testosterone signaling regulate overlapping and distinct functions that are required for the maintenance of spermatogenesis and male fertility.

  6. Fgf9 inhibition of meiotic differentiation in spermatogonia is mediated by Erk-dependent activation of Nodal-Smad2/3 signaling and is antagonized by Kit Ligand.

    PubMed

    Tassinari, V; Campolo, F; Cesarini, V; Todaro, F; Dolci, S; Rossi, P

    2015-03-12

    Both fibroblast growth factor 9 (Fgf9) and Kit Ligand (Kl) signal through tyrosine kinase receptors, yet they exert opposite effects on meiotic differentiation in postnatal spermatogonia, Fgf9 acting as a meiosis-inhibiting substance and Kl acting as a promoter of the differentiation process. To understand the molecular mechanisms that might underlie this difference, we tried to dissect the intracellular signaling elicited by these two growth factors. We found that both Fgf9 and Kl stimulate Erk1/2 activation in Kit+ (differentiating) spermatogonia, even though with different time courses, whereas Kl, but not Fgf9, elicits activation of the Pi3k-Akt pathway. Sustained Erk1/2 activity promoted by Fgf9 is required for induction of the autocrine Cripto-Nodal-Smad2/3 signaling loop in these cells. Nodal signaling, in turn, is essential to mediate Fgf9 suppression of the meiotic program, including inhibition of Stra8 and Scp3 expression and induction of the meiotic gatekeeper Nanos2. On the contrary, sustained activation of the Pi3k-Akt pathway is required for the induction of Stra8 expression elicited by Kl and retinoic acid. Moreover, we found that Kl treatment impairs Nodal mRNA expression and Fgf9-mediated Nanos2 induction, reinforcing the antagonistic effect of these two growth factors on the meiotic fate of male germ cells.

  7. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.

    PubMed

    Wilson, P O; Barber, P C; Hamid, Q A; Power, B F; Dhillon, A P; Rode, J; Day, I N; Thompson, R J; Polak, J M

    1988-02-01

    In order to assess the potential of protein gene product (PGP) 9.5 as a marker of the nervous and neuroendocrine systems, we examined its immunolocation in human, rat and guinea-pig tissues, using a rabbit polyclonal antiserum and two new mouse monoclonal antisera, I3C4 and 3IA3. Our results demonstrate immunoreactive PGP 9.5 in neurons and nerve fibres at all levels of the central and peripheral nervous system, in many neuroendocrine cells, in part of the renal tubule, in spermatogonia and Leydig cells of the testis, and in ova and in some cells of the pregnant and non-pregnant corpus luteum. In routinely processed tissues, standard immunohistochemical techniques using the polyclonal antibody demonstrated peripheral nerve fibres of all sizes with striking clarity.

  8. Aym1, a mouse meiotic gene identified by virtue of its ability to activate early meiotic genes in the yeast Saccharomyces cerevisiae.

    PubMed

    Malcov, Mira; Cesarkas, Karen; Stelzer, Gil; Shalom, Sarah; Dicken, Yosef; Naor, Yaniv; Goldstein, Ronald S; Sagee, Shira; Kassir, Yona; Don, Jeremy

    2004-12-01

    Our understanding of the molecular mechanisms that operate during differentiation of mitotically dividing spermatogonia cells into spermatocytes lags way behind what is known about other differentiating systems. Given the evolutionary conservation of the meiotic process, we screened for mouse proteins that could specifically activate early meiotic promoters in Saccharomyces cerevisiae yeast cells, when fused to the Gal4 activation domain (Gal4AD). Our screen yielded the Aym1 gene that encodes a short peptide of 45 amino acids. We show that a Gal4AD-AYM1 fusion protein activates expression of reporter genes through the promoters of the early meiosis-specific genes IME2 and HOP1, and that this activation is dependent on the DNA-binding protein Ume6. Aym1 is transcribed predominantly in mouse primary spermatocytes and in gonads of female embryos undergoing the corresponding meiotic divisions. Aym1 immunolocalized to nuclei of primary spermatocytes and oocytes and to specific type A spermatogonia cells, suggesting it might play a role in the processes leading to meiotic competence. The potential functional relationship between AYM1 and yeast proteins that regulate expression of early meiotic genes is discussed.

  9. Identification of 5-bromo-2'-deoxyuridine-labeled cells during mouse spermatogenesis by heat-induced antigen retrieval in lectin staining and immunohistochemistry.

    PubMed

    Wakayama, Tomohiko; Nakata, Hiroki; Kumchantuek, Tewarat; Gewaily, Mahmoud Saad; Iseki, Shoichi

    2015-03-01

    DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2'-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.

  10. Differential expression of sex-linked and autosomal germ-cell-specific genes during spermatogenesis in the mouse.

    PubMed

    Wang, P Jeremy; Page, David C; McCarrey, John R

    2005-10-01

    We have examined expression during spermatogenesis in the mouse of three Y-linked genes, 11 X-linked genes and 22 autosomal genes, all previously shown to be germ-cell-specific and expressed in premeiotic spermatogonia, plus another 21 germ-cell-specific autosomal genes that initiate expression in meiotic spermatocytes. Our data demonstrate that, like sex-linked housekeeping genes, germ-cell-specific sex-linked genes are subject to meiotic sex-chromosome inactivation (MSCI). Although all the sex-linked genes we investigated underwent MSCI, 14 of the 22 autosomal genes expressed in spermatogonia showed no decrease in expression in meiotic spermatocytes. This along with our observation that an additional 21 germ-cell-specific autosomal genes initiate or significantly up-regulate expression in spermatocytes confirms that MSCI is indeed a sex-chromosome-specific effect. Our results further demonstrate that the chromosome-wide repression imposed by MSCI is limited to meiotic spermatocytes and that postmeiotic expression of sex-linked genes is variable. Thus, 13 of the 14 sex-linked genes we examined showed some degree of postmeiotic reactivation. The extent of postmeiotic reactivation of germ-cell-specific X-linked genes did not correlate with proximity to the X inactivation center or the Xist gene locus. The implications of these findings are discussed with respect to differential gene regulation and the function of MSCI during spermatogenesis, including epigenetic programming of the future paternal genome during spermatogenesis.

  11. Quantitative T2 combined with texture analysis of nuclear magnetic resonance images identify different degrees of muscle involvement in three mouse models of muscle dystrophy: mdx, Largemyd and mdx/Largemyd.

    PubMed

    Martins-Bach, Aurea B; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C M; Almeida, Camila F; Caldeira, Waldir; Ribeiro, Alberto F; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G; Vainzof, Mariz

    2015-01-01

    Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant-T2-measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.

  12. Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx, Largemyd and mdx/Largemyd

    PubMed Central

    Martins-Bach, Aurea B.; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C. M.; Almeida, Camila F.; Caldeira, Waldir; Ribeiro, Alberto F.; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G.; Vainzof, Mariz

    2015-01-01

    Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research

  13. Mouse models for graft arteriosclerosis.

    PubMed

    Qin, Lingfeng; Yu, Luyang; Min, Wang

    2013-05-14

    Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-γ in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-γ followed by administration of mouse IFN-γ (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional

  14. Expression of cubilin in mouse testes and Leydig cells.

    PubMed

    Oh, Y S; Seo, J T; Ahn, H S; Gye, M C

    2016-04-01

    Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes.

  15. In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

    PubMed

    Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

    2016-12-06

    The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation.

  16. The Mouse That Soared

    NASA Astrophysics Data System (ADS)

    2004-09-01

    diameter of about 12 miles. Their formation is associated with a Type II supernova, the collapse and subsequent explosion of a massive star. The origin of a pulsar's high velocity is not known, but many astrophysicists suspect that it is directly related to the explosive circumstances involved in the birth of the pulsar. The rapid rotation and strong magnetic field of a pulsar can generate a wind of high-energy matter and antimatter particles that rush out at near the speed of light. These pulsar winds create large, magnetized bubbles of high-energy particles called pulsar wind nebulae. The X-ray and radio data on the Mouse have enabled Gaensler and his colleagues to constrain the properties of the ambient gas, to estimate the velocity of the pulsar, and to analyze the structure of the various shock waves created by the pulsar, the flow of particles away from the pulsar, and the magnetic field in the nebula. Zoom into Chandra's Image of the Mouse Zoom into Chandra's Image of the Mouse Other members of the research team were Eric van der Swaluw (FOM Institute of Physics, The Netherlands), Fernando Camilo (Columbia Univ., New York), Vicky Kaspi (McGill Univ., Montreal), Frederick K. Baganoff (MIT, Cambridge, Mass.), Farhad Yusef-Zadeh (Northwestern), and Richard Manchester (Australia Telescope National Facility). The pulsar in the Mouse was originally detected by Camilo et al. in 2002 using Australia's Parkes radio telescope. Chandra observed the Mouse on October 23 and 24, 2002. NASA's Marshall Space Flight Center, Huntsville, Ala., manages the Chandra program for NASA's Office of Space Science, Washington. Northrop Grumman of Redondo Beach, Calif., formerly TRW, Inc., was the prime development contractor for the observatory. The Smithsonian Astrophysical Observatory controls science and flight operations from the Chandra X-ray Center in Cambridge, Mass. Additional information and images are available at: http://chandra.harvard.edu and http://chandra.nasa.gov

  17. Retinoic acid availability drives the asynchronous initiation of spermatogonial differentiation in the mouse.

    PubMed

    Snyder, Elizabeth M; Small, Christopher; Griswold, Michael D

    2010-11-01

    Throughout the reproductive lifespan of most male mammals, sperm production is constant because of the regulated differentiation of spermatogonia. Retinoic acid (RA) and a downstream target, Stra8, are required for complete spermatogenesis. To examine the role of RA in initiating spermatogonial differentiation, a transgenic mouse model expressing beta-galactosidase under the control of an RA response element was used. Cells in the neonatal testis undergoing active RA signaling were visualized by beta-galactosidase activity, the relationship between RA and differentiation determined, and the role of RA-degrading enzymes in regulating RA demonstrated. Beta-galactosidase activity was found to be predominantly associated with differentiating, premeiotic germ cells and to be distributed nonuniformly throughout the seminiferous tubules. Additionally, beta-galactosidase activity in premeiotic germ cells colocalized with STRA8 protein and was induced in germ cells with exogenous RA treatment. The RA-degrading enzyme, CYP26B1, was found to have germ cell localization and nonuniform distribution between tubules via immunohistochemistry. Treatment with a CYP26 enzyme inhibitor resulted in an increased number of germ cells with both beta-galactosidase activity and STRA8 protein and an increase in the expression of genes associated with differentiation and reduced expression of a gene associated with undifferentiated germ cells. These results show the action of RA in a subset of spermatogonia leads to nonuniform initiation of differentiation throughout the neonatal testis, potentially mediated through the action of CYP26 enzymes. Thus, the presence of RA is a likely driving factor in the initiation of spermatogonial differentiation and may result in asynchronous spermatogenesis.

  18. Transcription of circular and noncircular forms of Sry in mouse testes.

    PubMed

    Zwingman, T; Fujimoto, H; Lai, L W; Boyer, T; Ao, A; Stalvey, J R; Blecher, S R; Erickson, R P

    1994-04-01

    Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells--RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3' region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes.

  19. The International Mouse Phenotyping Consortium: past and future perspectives on mouse phenotyping

    PubMed Central

    Moore, Mark W.

    2013-01-01

    Determining the function of all mammalian genes remains a major challenge for the biomedical science community in the 21st century. The goal of the International Mouse Phenotyping Consortium (IMPC) over the next 10 years is to undertake broad-based phenotyping of 20,000 mouse genes, providing an unprecedented insight into mammalian gene function. This short article explores the drivers for large-scale mouse phenotyping and provides an overview of the aims and processes involved in IMPC mouse production and phenotyping. PMID:22940749

  20. Bimodal concentration-response of nicotine involves the nicotinic acetylcholine receptor, transient receptor potential vanilloid type 1, and transient receptor potential ankyrin 1 channels in mouse trachea and sensory neurons.

    PubMed

    Kichko, Tatjana I; Lennerz, Jochen; Eberhardt, Mirjam; Babes, Ramona M; Neuhuber, Winfried; Kobal, Gerd; Reeh, Peter W

    2013-11-01

    High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µM (-)-nicotine, a maximum at 100 µM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, - five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

  1. RBE and genetic susceptibility of mouse and rat spermatogonial stem cells to protons, heavy charged particles and 1.5 MeV neutrons

    NASA Astrophysics Data System (ADS)

    Vaglenov, A.; Fedorenko, B.; Kaltenboeck, B.

    The main purpose of the present study is to provide data on RBE and genetic susceptibility in the mouse and the rat when exposed to protons, HZE particles and neutrons. Genetic damage from exposure to 50 MeV and 9 GeV protons, 4 GeV/nucleon helium ions, 4 GeV/nucleon carbon ions and 1.5 MeV neutrons was studied in adult (CBA × C57Bl/6J) F1 mice. Damage from 9 GeV protons and 4 GeV helium ions was studied in adult Wistar rats. The incidence of reciprocal translocations (RT) induced in the spermatogonial stem cells of each species was recorded. RBE values were derived by comparing linear regression coefficients from dose-responses within the same dose-range for each of the radiation types tested and 60Co γ-rays or by means of a direct nonparametric method. RT yields measured after mouse and rat spermatogonial irradiation with protons, heavy charged particles and neutrons fit the linear model of the dose-response relationship. Relative to 60Co γ-rays, RBE values are as follows for mouse spermatogonia: 0.9 for 50 MeV protons; 1.3 for 9 GeV protons; 0.7 for 4 GeV helium ions; and 1.3 for 4 GeV carbon ions. For rat spermatogonia, values were: 1.7 for 9 GeV protons and 1.3 for helium ions. Compared to mice irradiated using the same experimental design, rats were more susceptible to high-LET radiations, with susceptibility assessed by genetic damage to their spermatogonial stem cells. The RBE of 1.5 MeV neutron is about 6.6.

  2. Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling1

    PubMed Central

    Toocheck, Corey; Clister, Terri; Shupe, John; Crum, Chelsea; Ravindranathan, Preethi; Lee, Tae-Kyung; Ahn, Jung-Mo; Raj, Ganesh V.; Sukhwani, Meena; Orwig, Kyle E.; Walker, William H.

    2015-01-01

    Testosterone acts though the androgen receptor in Sertoli cells to support germ cell development (spermatogenesis) and male fertility, but the molecular and cellular mechanisms by which testosterone acts are not well understood. Previously, we found that in addition to acting through androgen receptor to directly regulate gene expression (classical testosterone signaling pathway), testosterone acts through a nonclassical pathway via the androgen receptor to rapidly activate kinases that are known to regulate spermatogenesis. In this study, we provide the first evidence that nonclassical testosterone signaling occurs in vivo as the MAP kinase cascade is rapidly activated in Sertoli cells within the testis by increasing testosterone levels in the rat. We find that either classical or nonclassical signaling regulates testosterone-mediated Rhox5 gene expression in Sertoli cells within testis explants. The selective activation of classical or nonclassical signaling pathways in Sertoli cells within testis explants also resulted in the differential activation of the Zbtb16 and c-Kit genes in adjacent spermatogonia germ cells. Delivery of an inhibitor of either pathway to Sertoli cells of mouse testes disrupted the blood-testis barrier that is essential for spermatogenesis. Furthermore, an inhibitor of nonclassical testosterone signaling blocked meiosis in pubertal mice and caused the loss of meiotic and postmeiotic germ cells in adult mouse testes. An inhibitor of the classical pathway caused the premature release of immature germ cells. Collectively, these observations indicate that classical and nonclassical testosterone signaling regulate overlapping and distinct functions that are required for the maintenance of spermatogenesis and male fertility. PMID:26607719

  3. Mouse models of myasthenia gravis.

    PubMed

    Ban, Joanne; Phillips, William D

    2015-01-01

    Myasthenia gravis is a muscle weakness disease characterized by autoantibodies that target components of the neuromuscular junction, impairing synaptic transmission. The most common form of myasthenia gravis involves antibodies that bind the nicotinic acetylcholine receptors in the postsynaptic membrane. Many of the remaining cases are due to antibodies against muscle specific tyrosine kinase (MuSK). Recently, autoantibodies against LRP4 (another component of the MuSK signaling complex in the postsynaptic membrane) were identified as the likely cause of myasthenia gravis in some patients. Fatiguing weakness is the common symptom in all forms of myasthenia gravis, but muscles of the body are differentially affected, for reasons that are not fully understood. Much of what we have learnt about the immunological and neurobiological aspects of the pathogenesis derives from mouse models. The most widely used mouse models involve either passive transfer of autoantibodies, or active immunization of the mouse with acetylcholine receptors or MuSK protein. These models can provide a robust replication of many of the features of the human disease. Depending upon the protocol, acute fatiguing weakness develops 2 - 14 days after the start of autoantibody injections (passive transfer) or might require repeated immunizations over several weeks (active models). Here we review mouse models of myasthenia gravis, including what they have contributed to current understanding of the pathogenic mechanisms and their current application to the testing of therapeutics.

  4. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage

    PubMed Central

    Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1Ser137 and pPlk1Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1Ser137 was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1Ser137 and pPlk1Thr210, as well as the subcellular distribution of pPlk1Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1Ser137 is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  5. On Involvement.

    ERIC Educational Resources Information Center

    Greene, Michael B.

    Involvement Ratings In Settings (IRIS), a multi-dimensional non-verbal scale of involvement adaptable to a time-sampling method of data collection, was constructed with the aid of the videotapes of second-grade Follow Through classrooms made by CCEP. Scales were defined through observations of involved and alienated behavior, and the IRIS was…

  6. On Involvement.

    ERIC Educational Resources Information Center

    Greene, Michael B.

    Involvement Ratings In Settings (IRIS), a multi-dimensional non-verbal scale of involvement adaptable to a time-sampling method of data collection, was constructed with the aid of the videotapes of second-grade Follow Through classrooms made by CCEP. Scales were defined through observations of involved and alienated behavior, and the IRIS was…

  7. Mouse Curve Biometrics

    SciTech Connect

    Schulz, Douglas A.

    2007-10-08

    A biometric system suitable for validating user identity using only mouse movements and no specialized equipment is presented. Mouse curves (mouse movements with little or no pause between them) are individually classied and used to develop classication histograms, which are representative of an individual's typical mouse use. These classication histograms can then be compared to validate identity. This classication approach is suitable for providing continuous identity validation during an entire user session.

  8. Building a Brainier Mouse.

    ERIC Educational Resources Information Center

    Tsien, Joe Z.

    2000-01-01

    Describes a genetic engineering project to build an intelligent mouse. Cites understanding the molecular basis of learning and memory as a very important step. Concludes that while science will never create a genius mouse that plays the stock market, it can turn a mouse into a quick learner with a better memory. (YDS)

  9. Building a Brainier Mouse.

    ERIC Educational Resources Information Center

    Tsien, Joe Z.

    2000-01-01

    Describes a genetic engineering project to build an intelligent mouse. Cites understanding the molecular basis of learning and memory as a very important step. Concludes that while science will never create a genius mouse that plays the stock market, it can turn a mouse into a quick learner with a better memory. (YDS)

  10. Family Involvement.

    ERIC Educational Resources Information Center

    Liontos, Lynn Balster

    1992-01-01

    Family involvement in schools will work only when perceived as an enlarged concept focusing on all children, including those from at-risk families. Each publication reviewed here is specifically concerned with family involvement strategies concerned with all children or targeted at primarily high risk students. Susan McAllister Swap looks at three…

  11. A starfish homolog of mouse T-brain-1 is expressed in the archenteron of Asterina pectinifera embryos: possible involvement of two T-box genes in starfish gastrulation.

    PubMed

    Shoguchi, E; Satoh, N; Maruyama, Y K

    2000-02-01

    A cDNA clone for a starfish T-box gene (Ap-Tbr) was isolated and characterized. Molecular phylogenetic analysis showed that the Ap-Tbr gene was a member of the T-brain subfamily, which includes mouse T-brain-1 and Xenopus Eomesodermin. Ap-Tbr was expressed as early as in late blastulae, and the transcript was evident in a disc-like region at the vegetal end or the vegetal plate. In early gastrulae, the gene was expressed in the cells of the invaginated archenteron, from which the majority of mesodermal cells as well as some endodermal cells are derived. The Ap-Tbr expression disappeared by the end of gastrulation, and was not detected in early bipinnaria larvae. This expression pattern of Ap-Tbr suggests its role in an early step of archenteron invagination, associated with mesoderm and endoderm formation. Furthermore, double staining of late blastulae and early gastrulae with a probe specific for Ap-Tbr and one for ApBra (the starfish Brachyury gene) demonstrated that the regions of Ap-Tbr and ApBra expression at the vegetal/posterior end of the embryo did not overlap, the region with Ap-Tbr expression being encircled by a ring-shaped region of ApBra expression. A gap without expression of the two genes was located between them, and the gap was seen around the blastoporal lip in the early gastrula. These observations suggest implication of the two T-box genes, with different roles, in starfish gastrulation.

  12. Sulforaphane suppresses vascular adhesion molecule-1 expression in TNF-α-stimulated mouse vascular smooth muscle cells: involvement of the MAPK, NF-κB and AP-1 signaling pathways.

    PubMed

    Kim, Ji-Yun; Park, Hye-Jin; Um, Sung Hee; Sohn, Eun-Hwa; Kim, Byung-Oh; Moon, Eun-Yi; Rhee, Dong-Kwon; Pyo, Suhkneung

    2012-01-01

    Atherosclerosis is a long-term inflammatory disease of the arterial wall. Increased expression of the cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) is associated with increased proliferation of vascular smooth muscle cells (VSMCs), leading to increased neointima or atherosclerotic lesion formation. Therefore, the functional inhibition of adhesion molecules could be a critical therapeutic target of inflammatory disease. In the present study, we investigate the effect of sulforaphane on the expression of VCAM-1 induced by TNF-α in cultured mouse vascular smooth muscle cell lines. Pretreatment of VSMCs for 2h with sulforaphane (1-5μg/ml) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and protein expression of VCAM-1. Sulforaphane also suppressed TNF-α-induced production of intracellular reactive oxygen species (ROS) and activation of p38, ERK and JNK. Furthermore, sulforaphane inhibited NK-κB and AP-1 activation induced by TNF-α. Sulforaphane inhibited TNF-α-induced ΙκΒ kinase activation, subsequent degradation of ΙκΒα and nuclear translocation of p65 NF-κB and decreased c-Jun and c-Fos protein level. This study suggests that sulforaphane inhibits the adhesive capacity of VSMC and downregulates the TNF-α-mediated induction of VCAM-1 in VSMC by inhibiting the MAPK, NF-κB and AP-1 signaling pathways and intracellular ROS production. Thus, sulforaphane may have beneficial effects to suppress inflammation within the atherosclerotic lesion. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. iNOS as a Driver of Inflammation and Apoptosis in Mouse Skeletal Muscle after Burn Injury: Possible Involvement of Sirt1 S-Nitrosylation-Mediated Acetylation of p65 NF-κB and p53

    PubMed Central

    Yasukawa, Takashi; Ishimaru, Kazuhiro; Yasuhara, Shingo; Yu, Yong-Ming; Martyn, J. A. Jeevendra; Tompkins, Ronald. G.; Shimokado, Kentaro; Kaneki, Masao

    2017-01-01

    Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury. PMID:28099528

  14. Suppression of the morphine-induced rewarding effect and G-protein activation in the lower midbrain following nerve injury in the mouse: involvement of G-protein-coupled receptor kinase 2.

    PubMed

    Ozaki, S; Narita, M; Narita, M; Iino, M; Miyoshi, K; Suzuki, T

    2003-01-01

    The present study was designed to investigate whether a state of neuropathic pain induced by sciatic nerve ligation could alter the rewarding effect, antinociception, and G-protein activation induced by a prototype of mu-opioid receptor agonist morphine in the mouse. The sciatic nerve ligation caused a long-lasting and profound thermal hyperalgesia. Under this neuropathic pain-like state, an i.c.v. morphine-induced place preference was observed in sham-operated mice but not in sciatic nerve-ligated mice. However, no differences in the antinociceptive effect of i.c.v.-administered morphine were noted between the groups. The increases in the binding of guanosine-5'-o-(3-[(35)S]thio)triphosphate induced by morphine in lower midbrain membranes including the ventral tegmental area, which contributes to the expression of the rewarding effect of opioid, were significantly attenuated in sciatic nerve-ligated mice. On the other hand, there were no differences in the stimulation of guanosine-5'-o-(3-[(35)S]thio)triphosphate binding to pons/medulla membranes, which plays an important role in the antinociception of mu-opioid receptor agonists, between the groups. In addition, no changes in levels of guanosine-5'-o-(3-[(35)S]thio)triphosphate binding by either the selective delta- or kappa-opioid receptor agonists were noted in membrane of the lower midbrain and limbic forebrain membranes obtained from sciatic nerve-ligated mice. Reverse transcription-polymerase chain reaction analysis showed that sciatic nerve ligation did not alter the mRNA product of mu-opioid receptors in the lower midbrain, indicating that a decrease in some mu-opioid receptor functions may result from the uncoupling of mu-opioid receptors from G-proteins. We found a significant increase in protein levels of G-protein-coupled receptor kinase 2, which causes receptor phosphorylation in membranes of the lower midbrain but not in the pons/medulla, obtained from mice with nerve injury, whereas there were no

  15. Radiation-induced deletions in the 5' end region of Notch1 lead to the formation of truncated proteins and are involved in the development of mouse thymic lymphomas.

    PubMed

    Tsuji, Hideo; Ishii-Ohba, Hiroko; Ukai, Hideki; Katsube, Takanori; Ogiu, Toshiaki

    2003-07-01

    Notch1 protein is a transmembrane receptor that directs various cell fate decisions. Active forms of Notch1 consisting of a transmembrane domain and an intracellular domain (Notch1TM) or only an intracellular domain (Notch1IC) function as oncoproteins. To elucidate the effect of Notch1 abnormalities in radiation-induced lymphomagenesis, we determined the structure of the Notch1 gene and examined the frequency and the sites of Notch1 rearrangements in radiation-induced mouse thymic lymphomas. The Notch1 gene consists of 37 exons, including three exons upstream of the previously reported exon 1. The transcript starting from exon 1 was the major transcript whereas the transcripts read upstream from exon 1a, in which amino acid sequences in the N-terminal region were changed, were minor. More than 50% of radiation-induced thymic lymphomas exhibited Notch1 rearrangements, suggesting that Notch1 acts as a major oncogene in radiation-induced lymphomagenesis. We identified three rearranged sites: novel sites in the 5' end region encompassing exons 1 and 2, the previously identified juxtamembrane extracellular region, and the 3' end region. The 5' deletion and the insertion of murine leukemia virus in the juxtamembrane region led to the production of abnormal transcripts starting from cryptic transcription start sites located halfway through the Notch1 gene and resulted in transcripts lacking most of the extracellular domain. As a result of these rearrangements, truncated Notch1 polypeptides resembling Notch1TM or Notch1IC were formed. In contrast, the 3' deletion led to the production of a C-terminal PEST motif-deleted transcript. The downstream target gene Hes1 was transcribed in a lymphoma with insertion of murine leukemia virus, but not in a lymphoma with a 5' deletion. These results indicate that in addition to Hes1 expression, other Notch1 pathway(s) have a role in thymic lymphomagenesis and suggest the presence of a novel mechanism for oncogenic activation of Notch1

  16. Mouse Models of Human Phenylketonuria

    PubMed Central

    Shedlovsky, A.; McDonald, J. D.; Symula, D.; Dove, W. F.

    1993-01-01

    Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine. Although this inborn error of metabolism was among the first in humans to be understood biochemically and genetically, little is known of the mechanism(s) involved in the pathology of PKU. We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein. Two of these have reduced PAH mRNA and display characteristics of untreated human PKU patients. A low PHE diet partially reverses these abnormalities. Our success in using high frequency random germline point mutagenesis to obtain appropriate disease models illustrates how such mutagenesis can complement the emergent power of targeted mutagenesis in the mouse. The mutants now can be used as models in studying both maternal PKU and somatic gene therapy. PMID:8375656

  17. Radiosensitivity of testicular cells in the fetal mouse

    SciTech Connect

    Vergouwen, R.P.F.A.; Roepers-Gajadien, H.L.; Rooij, D.G. de; Huiskamp, R.; Bas, R.J.; Davids, J.A.G.

    1995-01-01

    The effects of prenatal X irradiation on postnatal development of the CBA/P mouse testis was studied. At days 14, 15 and 18 post coitus pregnant female mice were exposed to single doses of X rays ranging from 0.25-1.5 Gy. Higher doses resulted in extensive loss of fetal mice. In the male offspring, at days 3 and 31 post partum, the numbers of gonocytes, type A spermatogonia and Sertoli cells per testis were determined using the disector method. Furthermore, after irradiation at day 15 post coitus, the numbers of Leydig cells, mesenchymal cells, macrophages, myoid cells, lymphatic endothelial cells, endothelial cells and perivascular cells per testis were also determined at days 3 and 31 post partum. At day 3 post partum, the number of germ cells was decreased after irradiation at days 14 and 15 post coitus. A D{sub o} value of 0.7 Gy was determined for the radiosensitivity of the gonocytes at day 14 post coitus. A D{sub o} value of 0.8 Gy was determined for the gonocytes at day 15 post coitus which, however, seems to be less accurate. No accurate D{sub o} value could be determined for the gonocytes at day 18 post coitus. At day 31 post partum, the repopulation of the seminiferous epithelium as well as testis weights and tubular diameters were more affected by irradiation with increasing age of the mice at the time of irradiation. The percentage of tubular cross sections showing spermatids decreased with increasing dose after irradiation at days 15 and 18 post coitus, but not after irradiation at day 14 post coitus. Furthermore, in tubular cross sections showing spermatids, exposure of testes to 1.25 and 1.5 Gy at day 18 post coitus resulted in significantly lower numbers of spermatids per cross section when compared to those testes exposed to the same doses at day 15 post coitus. 30 refs., 7 figs., 1 tab.

  18. The Knockout Mouse Project

    PubMed Central

    Austin, Christopher P; Battey, James F; Bradley, Allan; Bucan, Maja; Capecchi, Mario; Collins, Francis S; Dove, William F; Duyk, Geoffrey; Dymecki, Susan; Eppig, Janan T; Grieder, Franziska B; Heintz, Nathaniel; Hicks, Geoff; Insel, Thomas R; Joyner, Alexandra; Koller, Beverly H; Lloyd, K C Kent; Magnuson, Terry; Moore, Mark W; Nagy, Andras; Pollock, Jonathan D; Roses, Allen D; Sands, Arthur T; Seed, Brian; Skarnes, William C; Snoddy, Jay; Soriano, Philippe; Stewart, David J; Stewart, Francis; Stillman, Bruce; Varmus, Harold; Varticovski, Lyuba; Verma, Inder M; Vogt, Thomas F; von Melchner, Harald; Witkowski, Jan; Woychik, Richard P; Wurst, Wolfgang; Yancopoulos, George D; Young, Stephen G; Zambrowicz, Brian

    2009-01-01

    Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain. PMID:15340423

  19. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    PubMed

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  20. Toll-Like Receptor 4-Myeloid Differentiation Primary Response Gene 88 Pathway Is Involved in the Inflammatory Development of Polymyositis by Mediating Interferon-γ and Interleukin-17A in Humans and Experimental Autoimmune Myositis Mouse Model.

    PubMed

    Zhang, Hongya; He, Fangyuan; Shi, Ming; Wang, Wenxiu; Tian, Xiaojia; Kang, Juan; Han, Wenjuan; Wu, Rui; Zhou, Linfu; Hu, Mengmeng; Li, Xiaobo; Mi, Fang; Zhao, Gang; Jia, Hongge

    2017-01-01

    Toll-like receptor 4 (TLR4) is one of the key players in the development of many autoimmune diseases. To determine the possible role of TLR4 in polymyositis (PM) development, we collected muscle samples from PM patients and mice subjected to an experimental autoimmune myositis (EAM) model. We measured TLR4-MyD88 pathway-related factors, interferon-γ (IFN-γ), and interleukin-17A (IL-17A) in EAM mice and PM patients. Then, we observed the changes of above factors and the inflammatory development of EAM mice with TLR4 antagonist TAK-242, IFN-γ, or IL-17A antibody treatment. The expression of TLR4, MyD88, and NF-κB was significantly upregulated in the muscle tissues both in 22 patients with PM and in the EAM model. As expected, increased levels of various cytokines, such as IL-1β, IL-6, IL-10, IL-12, tumor necrosis factor-α, TGF-β, IFN-γ, and IL-17A, were evident in the serum of EAM mice. Moreover, mRNA expression levels of IFN-γ and IL-17A were significantly increased in both PM patients and EAM mice. Consistently, the levels of these factors were positively correlated with the degree of muscle inflammation in EAM mice. However, when EAM mice were treated with TLR4 antagonist TAK-242, the expression of IFN-γ and IL-17A was decreased. When the cytokines were neutralized by anti-IFN-γ or anti-IL-17A antibody, the inflammatory development of EAM exacerbated or mitigated. The present study provided the important evidence that the TLR4-MyD88 pathway may be involved in the immune mechanisms of PM by mediating IFN-γ and IL-17A.

  1. Synaptonemal Complex Components Persist at Centromeres and Are Required for Homologous Centromere Pairing in Mouse Spermatocytes

    PubMed Central

    Kouznetsova, Anna; Scherthan, Harry; Höög, Christer; Dawson, Dean S.; Pezza, Roberto J.

    2012-01-01

    Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and

  2. Optical mouse acting as biospeckle sensor

    NASA Astrophysics Data System (ADS)

    da Silva, Michel Melo; Nozela, Jose Roberto de Almeida; Chaves, Marcio Jose; Alves Braga, Roberto; Rabal, Hector Jorge

    2011-04-01

    In this work we propose some experiments with the use of optical computer mouse, associated to low cost lasers that can be used to perform several measurements with applications in industry and in human health monitoring. The mouse was used to grab the movements produced by speckle pattern changes and to get information through the adaptation of its structure. We measured displacements in wood samples under strain, variations of the diameter of an artery due to heart beat and, through a hardware simulation, the movement of an eye, an experiment that could be of low cost help for communication to severely handicapped motor patients. Those measurements were done in spite of the fact that the CCD sensor of the mice is monolithically included into an integrated circuit so that the raw image cannot be accessed. If, as was the case with primitive optical mouse, that signal could be accessed, the quality and usefulness of the measurements could be significantly increased. As it was not possible, a webcam sensor was used for measuring the drying of paint, a standard phenomenon for testing biospeckle techniques, in order to prove the usefulness of the mouse design. The results showed that the use of the mouse associated to a laser pointer could be the way to get metrological information from many phenomena involving the whole field spatial displacement, as well as the use of the mouse as in its prime version allowed to get images of the speckle patterns and to analyze them.

  3. The MOUSE Squad

    ERIC Educational Resources Information Center

    Borja, Rhea R.

    2004-01-01

    This article presents a New York city after-school program started by MOUSE (Making Opportunities for Upgrading Schools and Education), a national nonprofit group that teaches students how to fix computers, and equips them with the communication and problem-solving skills to help them in the working world. The MOUSE program is part of a trend…

  4. Mouse homologues of human hereditary disease.

    PubMed Central

    Searle, A G; Edwards, J H; Hall, J G

    1994-01-01

    Details are given of 214 loci known to be associated with human hereditary disease, which have been mapped on both human and mouse chromosomes. Forty two of these have pathological variants in both species; in general the mouse variants are similar in their effects to the corresponding human ones, but exceptions include the Dmd/DMD and Hprt/HPRT mutations which cause little, if any, harm in mice. Possible reasons for phenotypic differences are discussed. In most pathological variants the gene product seems to be absent or greatly reduced in both species. The extensive data on conserved segments between human and mouse chromosomes are used to predict locations in the mouse of over 50 loci of medical interest which are mapped so far only on human chromosomes. In about 80% of these a fairly confident prediction can be made. Some likely homologies between mapped mouse loci and unmapped human ones are also given. Sixty six human and mouse proto-oncogene and growth factor gene homologies are also listed; those of confirmed location are all in known conserved segments. A survey of 18 mapped human disease loci and chromosome regions in which the manifestation or severity of pathological effects is thought to be the result of genomic imprinting shows that most of the homologous regions in the mouse are also associated with imprinting, especially those with homologues on human chromosomes 11p and 15q. Useful methods of accelerating the production of mouse models of human hereditary disease include (1) use of a supermutagen, such as ethylnitrosourea (ENU), (2) targeted mutagenesis involving ES cells, and (3) use of gene transfer techniques, with production of 'knockout mutations'. PMID:8151633

  5. Mouse genome database 2016

    PubMed Central

    Bult, Carol J.; Eppig, Janan T.; Blake, Judith A.; Kadin, James A.; Richardson, Joel E.

    2016-01-01

    The Mouse Genome Database (MGD; http://www.informatics.jax.org) is the primary community model organism database for the laboratory mouse and serves as the source for key biological reference data related to mouse genes, gene functions, phenotypes and disease models with a strong emphasis on the relationship of these data to human biology and disease. As the cost of genome-scale sequencing continues to decrease and new technologies for genome editing become widely adopted, the laboratory mouse is more important than ever as a model system for understanding the biological significance of human genetic variation and for advancing the basic research needed to support the emergence of genome-guided precision medicine. Recent enhancements to MGD include new graphical summaries of biological annotations for mouse genes, support for mobile access to the database, tools to support the annotation and analysis of sets of genes, and expanded support for comparative biology through the expansion of homology data. PMID:26578600

  6. Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure.

    PubMed

    Favor, Jack; Shelby, Michael D

    2005-02-07

    An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.

  7. Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis

    SciTech Connect

    Petit, J.M.; Ratinaud, M.H.; Cordelli, E.; Spano, M.; Julien, R.

    1995-04-01

    Flow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10-N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S-phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis. 45 refs., 5 figs., 2 tabs.

  8. Distribution of the sex chromosome during mouse spermatogenesis in testis tissue sections

    PubMed Central

    OTAKA, Kosuke; HIRADATE, Yuuki; KOBAYASHI, Norio; SHIRAKATA, Yoshiki; TANEMURA, Kentaro

    2015-01-01

    During mammalian spermatogenesis, spermatogenic cells undergo mitotic division and are subsequently divided into haploid spermatids by meiotic division, but the dynamics of sex chromosomes during spermatogenesis are unclear in vivo. To gain insight into the distribution of sex chromosomes in the testis, we examined the localization of sex chromosomes before and after meiosis in mouse testis sections. Here, we developed a method of fluorescence in situ hybridization (FISH) using specific probes for the X and Y chromosomes to obtain their positional information in histological testis sections. FISH analysis revealed the sex chromosomal position during spermatogenesis in each stage of seminiferous epithelia and in each spermatogenic cell. In the spermatogonia and leptotene spermatocytes, sex chromosomes were distantly positioned in the cell. In the zygotene and pachytene spermatocytes at prophase I, X and Y chromosomes had a random distribution. After meiosis, the X and Y spermatids were random in every seminiferous epithelium. We also detected aneuploidy of sex chromosomes in spermatogenic cells using our developed FISH analysis. Our results provide further insight into the distribution of sex chromosomes during spermatogenesis, which could help to elucidate a specific difference between X and Y spermatids and sex chromosome-specific behavior. PMID:26073979

  9. DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment.

    PubMed

    Zhang, Teng; Oatley, Jon; Bardwell, Vivian J; Zarkower, David

    2016-09-01

    Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion.

  10. DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment

    PubMed Central

    Zhang, Teng; Oatley, Jon; Bardwell, Vivian J.; Zarkower, David

    2016-01-01

    Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion. PMID:27583450

  11. Growth and differentiation of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons

    SciTech Connect

    van den Aardweg, G.J.M.J.; de Ruiter-Bootsma, A.L.; Kramer, M.F.; Davids, J.A.G.

    1983-06-01

    The longitudinal outgrowth of spermatogenetic colonies arising from stem cells that survived neutron doses of 150, 300, and 350 rad was studied up to 30 weeks in histological sections of CBA mouse testes. Two methods were used: (1) the repopulation index (RI) as a measure of the length of total colonies per testis and (2) measurement of the individual length of all colonies in serially sectioned testes 4 and 15 weeks after 300 rad and 15 weeks after 350 rad. The mean initial growth of the colonies is linear up to 8, 15, and 20 weeks after 150, 300, and 350 rad, respectively. Counting of colonies after 300 rad showed that all surviving stem cells had started to form a colony within 4 weeks after irradiation. The development of spermatogenetic cells to mature spermatozoa was studied after 100, 150, 300, and 350 rad in sections of repopulating tubules used for RI determination as well as in serial sections of individual colonies. Although after 300 and 350 rad spermatogenetic cell types beyond the stage of young spermatocytes reappeared 1 week late, we found no great disturbances in the regular reappearance of the successive spermatogenetic cell types after irradiation. Our data suggest that this retardation in the reappearance of further developed cells is caused by a delay in the production of developed cells in spermatogonia in an increasing fraction of the colonies after higher neutron doses. Even in fully developed colonies the production of differentiating spermatogenetic cell types was subnormal after 300 and 350 rad.

  12. Which Governmental Agencies are Involved in Rat and Mouse Control?

    EPA Pesticide Factsheets

    EPA works with the Centers for Disease Control and Prevention (CDC) and various other state and local agencies and institutions to provide to the public information and tools for controlling rodents and the risks they may pose.

  13. Transvection effects involving DNA methylation during meiosis in the mouse

    PubMed Central

    Rassoulzadegan, Minoo; Magliano, Marc; Cuzin, François

    2002-01-01

    High efficiencies of recombination between LoxP elements were initially recorded when the Cre recombinase was expressed in meiotic spermatocytes. However, it was unexpectedly found that LoxP recombination fell to very low values at the second generation of mice expressing Cre during meiosis. The inability of the LoxP elements to serve as recombination substrates was correlated with cytosine methylation, initially in LoxP and transgene sequences, but later extending for distances of at least several kilobases into chromosomal sequences. It also affected the allelic locus, implying a transfer of structural information between alleles similar to the transvection phenomenon described in Drosophila. Once initiated following Cre–LoxP interaction, neither cis-extension nor transvection of the methylated state required the continuous expression of Cre, as they occurred both in germinal and somatic cells and in the fraction of the offspring that had not inherited the Sycp1-Cre transgene. Therefore, these processes depend on a physiological mechanism of establishment and extension of an epigenetic state, for which they provide an experimental model. PMID:11823436

  14. Effects of male germ-cell stage on the frequency, nature, and spectrum of induced specific-locus mutations in the mouse.

    PubMed

    Russell, Liane B

    2004-09-01

    By means of the mouse specific-locus test (SLT) with visible markers, which is capable of detecting intragenic mutations as well as larger lesions, about 20 mutagens have been studied comparatively across arrays of male germ-cell stages. In addition, a very large historical control, accumulated over decades, provides data on spontaneous mutations in males. Each mutagen has a characteristic germ-cell-stage sensitivity pattern. Although most chemicals yield their maximum numbers of mutations following exposure of spermatozoa and late spermatids, mutagens have now been identified that peak in each of the major stages of spermatogenesis and spermiogenesis, including those in which effects on recombination can also be induced. Stem-cell spermatogonia have yielded positive results with only five of 15 mutagenic chemicals. In postspermatogonial stages, all chemicals, as well as radiations, induce primarily large lesions (LL). By contrast, in spermatogonia (either stem-cell or differentiating) all chemicals except one (bleomycin) produce very few such lesions. The spectrum of relative mutation frequencies at the seven loci of the SLT is characteristic for treated germ-cell stage and mutagen. Treatments that induce primarily LL are characterized by a great preponderance of s (Ednrb)-locus mutations (possibly due to a paucity of haplo-insufficient genes in the surrounding region); and those that induce very few, if any, LL by a great preponderance of p-locus mutations. Spontaneous locus-spectra differ from both types of treatment-induced spectra; moreover, there are two distinct types of spontaneous spectra, depending on whether mutations occurred in mitotic cells or during the perigametic interval.

  15. [Echocardiography in mouse].

    PubMed

    Fayssoil, A

    2008-06-01

    Assessing cardiac phenotype requires invasive or noninvasive techniques in mouse. Echocardiography is a noninvasive technique for evaluating cardiac function. The purpose of this paper is to underline echocardiography modalities and new tools Doppler applications like tissue Doppler imaging.

  16. Mouse Cleaning Apparatus and Method

    NASA Technical Reports Server (NTRS)

    Williams, Glenn L. (Inventor)

    2005-01-01

    The method of using the mouse pad cleaning apparatus is disclosed and claimed. The method comprises the steps of uncovering the mouse cleaning surface, applying the mouse and ball of the mouse to the cleaning surface, moving the mouse in a rotational pattern on the mouse cleaning surface, removing the mouse form the mouse cleaning surface, washing the cleaning surface, and covering the mouse cleaning surface. A mouse pad cleaning apparatus comprising a plurality of substrates, each said substrate having adhesive thereon, said plurality of substrates residing in and affixed to a receptacle. A single substrate having adhesive, which may be washable or non-washable, thereon may be employed. The washable adhesive may be an organopolysiloxane or gelatinous elastomer.

  17. Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue.

    PubMed

    Yildiz, Cengiz; Mullen, Brendan; Jarvi, Keith; McKerlie, Colin; Lo, Kirk C

    2013-08-01

    Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank's balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen-thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen-thawed-graft groups (p<0.05). Fresh grafts and frozen-thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p>0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p<0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p<0.05). The typical damage observed in the frozen-thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings

  18. Distribution of lectin-bindings in the testis of the lesser mouse deer, Tragulus javanicus.

    PubMed

    Agungpriyono, S; Kurohmaru, M; Kimura, J; Wahid, A H; Sasaki, M; Kitamura, N; Yamada, J; Fukuta, K; Zuki, A B

    2009-06-01

    The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d-galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N-acetyl-d-galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N-acetyl-D-glucosamine and sialic acid (wheat germ agglutinin WGA, s-WGA), D-mannose and d-glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), L-fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA-E) sugar residues. In Golgi-, cap-, and acrosome-phase spermatids, lectin-bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s-WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA-E). s-WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA-E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA-E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA-E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin-bindings noted in the testis of lesser mouse deer included the limited distribution of s-WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.

  19. Mouse Auditory Brainstem Response Testing

    PubMed Central

    Akil, Omar; Oursler, A. E.; Fan, Kevin; Lustig, Lawrence R.

    2016-01-01

    The auditory brainstem response (ABR) test provides information about the inner ear (cochlea) and the central pathways for hearing. The ABR reflects the electrical responses of both the cochlear ganglion neurons and the nuclei of the central auditory pathway to sound stimulation (Zhou et al., 2006; Burkard et al., 2007). The ABR contains 5 identifiable wave forms, labeled as I-V. Wave I represents the summated response from the spiral ganglion and auditory nerve while waves II-V represent responses from the ascending auditory pathway. The ABR is recorded via electrodes placed on the scalp of an anesthetized animal. ABR thresholds refer to the lowest sound pressure level (SPL) that can generate identifiable electrical response waves. This protocol describes the process of measuring the ABR of small rodents (mouse, rat, guinea pig, etc.), including anesthetizing the mouse, placing the electrodes on the scalp, recording click and tone burst stimuli and reading the obtained waveforms for ABR threshold values. As technology continues to evolve, ABR will likely provide more qualitative and quantitative information regarding the function of the auditory nerve and brainstem pathways involved in hearing.

  20. Head Transplantation in Mouse Model.

    PubMed

    Ren, Xiao-Ping; Ye, Yi-Jie; Li, Peng-Wei; Shen, Zi-Long; Han, Ke-Cheng; Song, Yang

    2015-08-01

    The mouse model of allo-head and body reconstruction (AHBR) has recently been established to further the clinical development of this strategy for patients who are suffering from mortal bodily trauma or disease, yet whose mind remains healthy. Animal model studies are indispensable for developing such novel surgical practices. The goal of this work was to establish head transplant mouse model, then the next step through the feasible biological model to investigate immune rejection and brain function in next step, thereby promoting the goal of translation of AHBR to the clinic in the future. Our approach involves retaining adequate blood perfusion in the transplanted head throughout the surgical procedure by establishing donor-to-recipient cross-circulation by cannulating and anastomosing the carotid artery on one side of the body and the jugular vein on the other side. Neurological function was preserved by this strategy as indicated by electroencephalogram and intact cranial nerve reflexes. The results of this study support the feasibility of this method for avoiding brain ischemia during transplantation, thereby allowing for the possibility of long-term studies of head transplantation. © 2015 John Wiley & Sons Ltd.

  1. Mouse bladder wall injection.

    PubMed

    Fu, Chi-Ling; Apelo, Charity A; Torres, Baldemar; Thai, Kim H; Hsieh, Michael H

    2011-07-12

    Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

  2. Lack of effect of dibromochloropropane on the mouse testis

    SciTech Connect

    Oakberg, E.F.; Cummings, C.C.

    1984-01-01

    A single intraperitoneal injection of male mice with 110 mg/kg of 1,2-dibromo-3-chloropropane (DBCP) showed no effect on spermatogonial stem cells or on differentiating spermatogonia as measured by cell counts made 3, 5, and 8 days after injection.

  3. Apoptotic Signaling in Mouse Odontogenesis

    PubMed Central

    Svandova, Eva; Tucker, Abigail S.

    2012-01-01

    Abstract Apoptosis is an important morphogenetic event in embryogenesis as well as during postnatal life. In the last 2 decades, apoptosis in tooth development (odontogenesis) has been investigated with gradually increasing focus on the mechanisms and signaling pathways involved. The molecular machinery responsible for apoptosis exhibits a high degree of conservation but also organ and tissue specific patterns. This review aims to discuss recent knowledge about apoptotic signaling networks during odontogenesis, concentrating on the mouse, which is often used as a model organism for human dentistry. Apoptosis accompanies the entire development of the tooth and corresponding remodeling of the surrounding bony tissue. It is most evident in its role in the elimination of signaling centers within developing teeth, removal of vestigal tooth germs, and in odontoblast and ameloblast organization during tooth mineralization. Dental apoptosis is caspase dependent and proceeds via mitochondrial mediated cell death with possible amplification by Fas-FasL signaling modulated by Bcl-2 family members. PMID:22204278

  4. Apoptotic signaling in mouse odontogenesis.

    PubMed

    Matalova, Eva; Svandova, Eva; Tucker, Abigail S

    2012-01-01

    Apoptosis is an important morphogenetic event in embryogenesis as well as during postnatal life. In the last 2 decades, apoptosis in tooth development (odontogenesis) has been investigated with gradually increasing focus on the mechanisms and signaling pathways involved. The molecular machinery responsible for apoptosis exhibits a high degree of conservation but also organ and tissue specific patterns. This review aims to discuss recent knowledge about apoptotic signaling networks during odontogenesis, concentrating on the mouse, which is often used as a model organism for human dentistry. Apoptosis accompanies the entire development of the tooth and corresponding remodeling of the surrounding bony tissue. It is most evident in its role in the elimination of signaling centers within developing teeth, removal of vestigal tooth germs, and in odontoblast and ameloblast organization during tooth mineralization. Dental apoptosis is caspase dependent and proceeds via mitochondrial mediated cell death with possible amplification by Fas-FasL signaling modulated by Bcl-2 family members.

  5. Virtual simulation of mouse anatomy and procedural techniques.

    PubMed

    Stredney, Don; Hittle, Bradley; Chen, Chun-Ming; Kerwin, Thomas; Bratasz, Anna; Manivannan, Niranchana; Powell, Kimerly

    2012-01-01

    Translational science requires the use of mouse models for the characterization of disease and evaluation of treatment therapies. However, often there is a lack of comprehensive training for scientists in the systemic and regional anatomy of the mouse that limits their ability to perform studies involving complex interventional procedures. We present our methodologies for the development, evaluation, and dissemination of an interactive 3D mouse atlas that includes designs for presenting emulation of procedural technique. We present the novel integration of super-resolution imaging techniques, depth-of-field interactive volume rendering of large data, and the seamless delivery of remote visualization and interaction to thin clients.

  6. An encyclopedia of mouse DNA elements (Mouse ENCODE)

    PubMed Central

    2012-01-01

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome. PMID:22889292

  7. Researchers Create Artificial Mouse 'Embryo'

    MedlinePlus

    ... news/fullstory_163881.html Researchers Create Artificial Mouse 'Embryo' Experiment used two types of gene-modified stem ... they've created a kind of artificial mouse embryo using stem cells, which can be coaxed to ...

  8. The Mouse SAGE Site: database of public mouse SAGE libraries.

    PubMed

    Divina, Petr; Forejt, Jirí

    2004-01-01

    The Mouse SAGE Site is a web-based database of all available public libraries generated by the Serial Analysis of Gene Expression (SAGE) from various mouse tissues and cell lines. The database contains mouse SAGE libraries organized in a uniform way and provides web-based tools for browsing, comparing and searching SAGE data with reliable tag-to-gene identification. A modified approach based on the SAGEmap database is used for reliable tag identification. The Mouse SAGE Site is maintained on an ongoing basis at the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic and is accessible at the internet address http://mouse.biomed.cas.cz/sage/.

  9. Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

    PubMed Central

    Vance, V B; Thompson, E A; Bowman, L H

    1985-01-01

    Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences. Images PMID:2997749

  10. Mouse models of intracranial aneurysm.

    PubMed

    Wang, Yutang; Emeto, Theophilus I; Lee, James; Marshman, Laurence; Moran, Corey; Seto, Sai-wang; Golledge, Jonathan

    2015-05-01

    Subarachnoid hemorrhage secondary to rupture of an intracranial aneurysm is a highly lethal medical condition. Current management strategies for unruptured intracranial aneurysms involve radiological surveillance and neurosurgical or endovascular interventions. There is no pharmacological treatment available to decrease the risk of aneurysm rupture and subsequent subarachnoid hemorrhage. There is growing interest in the pathogenesis of intracranial aneurysm focused on the development of drug therapies to decrease the incidence of aneurysm rupture. The study of rodent models of intracranial aneurysms has the potential to improve our understanding of intracranial aneurysm development and progression. This review summarizes current mouse models of intact and ruptured intracranial aneurysms and discusses the relevance of these models to human intracranial aneurysms. The article also reviews the importance of these models in investigating the molecular mechanisms involved in the disease. Finally, potential pharmaceutical targets for intracranial aneurysm suggested by previous studies are discussed. Examples of potential drug targets include matrix metalloproteinases, stromal cell-derived factor-1, tumor necrosis factor-α, the renin-angiotensin system and the β-estrogen receptor. An agreed clear, precise and reproducible definition of what constitutes an aneurysm in the models would assist in their use to better understand the pathology of intracranial aneurysm and applying findings to patients.

  11. Signaling pathways in mouse and human trophoblast differentiation: a comparative review

    PubMed Central

    Soncin, Francesca; Natale, David; Parast, Mana M.

    2014-01-01

    The mouse is often used as a model for understanding human placentation and offers multiple advantages, including the ability to manipulate gene expression in specific compartments and to derive trophoblast stem cells, which can be maintained or differentiated in vitro. Nevertheless, there are numerous differences between the mouse and human placentas, only the least of which are structural. This review aims to compare mouse and human placentation, with a focus on signaling pathways involved in trophoblast lineage-specific differentiation. PMID:25430479

  12. The nude mouse model for the study of human skin disorders.

    PubMed

    Gilhar, A; Etzioni, A

    1994-01-01

    Normal human skin grafted onto the nude mouse can be maintained without any signs of rejection throughout the life-span of the animal. Indeed, the nude mouse model is a powerful tool for understanding the pathological process of the skin. Until now many skin diseases such as psoriasis, cutaneous lupus, pemphigus and vitiligo have been looked at using the nude mouse model, which has helped to clarify the role of the various factors involved.

  13. Mouse Phenome Database

    PubMed Central

    Grubb, Stephen C.; Bult, Carol J.; Bogue, Molly A.

    2014-01-01

    The Mouse Phenome Database (MPD; phenome.jax.org) was launched in 2001 as the data coordination center for the international Mouse Phenome Project. MPD integrates quantitative phenotype, gene expression and genotype data into a common annotated framework to facilitate query and analysis. MPD contains >3500 phenotype measurements or traits relevant to human health, including cancer, aging, cardiovascular disorders, obesity, infectious disease susceptibility, blood disorders, neurosensory disorders, drug addiction and toxicity. Since our 2012 NAR report, we have added >70 new data sets, including data from Collaborative Cross lines and Diversity Outbred mice. During this time we have completely revamped our homepage, improved search and navigational aspects of the MPD application, developed several web-enabled data analysis and visualization tools, annotated phenotype data to public ontologies, developed an ontology browser and released new single nucleotide polymorphism query functionality with much higher density coverage than before. Here, we summarize recent data acquisitions and describe our latest improvements. PMID:24243846

  14. Neuroanatomy and Neurochemistry of Mouse Cornea.

    PubMed

    He, Jiucheng; Bazan, Haydee E P

    2016-02-01

    To investigate the entire nerve architecture and content of the two main sensory neuropeptides in mouse cornea to determine if it is a good model with similarities to human corneal innervation. Mice aged 1 to 24 weeks were used. The corneas were stained with neuronal-class βIII-tubulin, calcitonin gene-related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. To test the origin of CGRP and SP, trigeminal ganglia (TG) were processed for immunofluorescence. Relative corneal nerve fiber densities or neuron numbers were assessed by computer-assisted analysis. Between 1 and 3 weeks after birth, mouse cornea was mainly composed of a stromal nerve network. At 4 weeks, a whorl-like structure (or vortex) appeared that gradually became more defined. By 8 weeks, anatomy of corneal nerves had reached maturity. Epithelial bundles converged into the central area to form the vortex. The number and pattern of whorl-like structures were different. Subbasal nerve density and nerve terminals were greater in the center than the periphery. Nerve fibers and terminals that were CGRP-positive were more abundant than SP-positive nerves and terminals. In trigeminal ganglia, the number of CGRP-positive neurons significantly outnumbered those positive for SP. This is the first study to show a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the mouse cornea. This finding shows mouse corneal innervation has many similarities to human cornea and makes the mouse an appropriate model to study pathologies involving corneal nerves.

  15. Neuroanatomy and Neurochemistry of Mouse Cornea

    PubMed Central

    He, Jiucheng; Bazan, Haydee E. P.

    2016-01-01

    Purpose To investigate the entire nerve architecture and content of the two main sensory neuropeptides in mouse cornea to determine if it is a good model with similarities to human corneal innervation. Methods Mice aged 1 to 24 weeks were used. The corneas were stained with neuronal-class βIII-tubulin, calcitonin gene–related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. To test the origin of CGRP and SP, trigeminal ganglia (TG) were processed for immunofluorescence. Relative corneal nerve fiber densities or neuron numbers were assessed by computer-assisted analysis. Results Between 1 and 3 weeks after birth, mouse cornea was mainly composed of a stromal nerve network. At 4 weeks, a whorl-like structure (or vortex) appeared that gradually became more defined. By 8 weeks, anatomy of corneal nerves had reached maturity. Epithelial bundles converged into the central area to form the vortex. The number and pattern of whorl-like structures were different. Subbasal nerve density and nerve terminals were greater in the center than the periphery. Nerve fibers and terminals that were CGRP-positive were more abundant than SP-positive nerves and terminals. In trigeminal ganglia, the number of CGRP-positive neurons significantly outnumbered those positive for SP. Conclusions This is the first study to show a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the mouse cornea. This finding shows mouse corneal innervation has many similarities to human cornea and makes the mouse an appropriate model to study pathologies involving corneal nerves. PMID:26906155

  16. Development and testing of a mouse simulated space flight model

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, Gerald

    1987-01-01

    The development and testing of a mouse model for simulating some aspects of weightlessness that occurs during space flight, and the carrying out of immunological experiments on animals undergoing space flight is examined. The mouse model developed was an antiorthostatic, hypokinetic, hypodynamic suspension model similar to one used with rats. The study was divided into two parts. The first involved determination of which immunological parameters should be observed on animals flown during space flight or studied in the suspension model. The second involved suspending mice and determining which of those immunological parameters were altered by the suspension. Rats that were actually flown in Space Shuttle SL-3 were used to test the hypotheses.

  17. RIKEN mouse genome encyclopedia.

    PubMed

    Hayashizaki, Yoshihide

    2003-01-01

    We have been working to establish the comprehensive mouse full-length cDNA collection and sequence database to cover as many genes as we can, named Riken mouse genome encyclopedia. Recently we are constructing higher-level annotation (Functional ANnoTation Of Mouse cDNA; FANTOM) not only with homology search based annotation but also with expression data profile, mapping information and protein-protein database. More than 1,000,000 clones prepared from 163 tissues were end-sequenced to classify into 159,789 clusters and 60,770 representative clones were fully sequenced. As a conclusion, the 60,770 sequences contained 33,409 unique. The next generation of life science is clearly based on all of the genome information and resources. Based on our cDNA clones we developed the additional system to explore gene function. We developed cDNA microarray system to print all of these cDNA clones, protein-protein interaction screening system, protein-DNA interaction screening system and so on. The integrated database of all the information is very useful not only for analysis of gene transcriptional network and for the connection of gene to phenotype to facilitate positional candidate approach. In this talk, the prospect of the application of these genome resourced should be discussed. More information is available at the web page: http://genome.gsc.riken.go.jp/.

  18. Mouse Models of Aneuploidy

    PubMed Central

    Sheppard, Olivia; Wiseman, Frances K.; Ruparelia, Aarti; Tybulewicz, Victor L. J.; Fisher, Elizabeth M. C.

    2012-01-01

    Abnormalities of chromosome copy number are called aneuploidies and make up a large health load on the human population. Many aneuploidies are lethal because the resulting abnormal gene dosage is highly deleterious. Nevertheless, some whole chromosome aneuploidies can lead to live births. Alterations in the copy number of sections of chromosomes, which are also known as segmental aneuploidies, are also associated with deleterious effects. Here we examine how aneuploidy of whole chromosomes and segmental aneuploidy of chromosomal regions are modeled in the mouse. These models provide a whole animal system in which we aim to investigate the complex phenotype-genotype interactions that arise from alteration in the copy number of genes. Although our understanding of this subject is still in its infancy, already research in mouse models is highlighting possible therapies that might help alleviate the cognitive effects associated with changes in gene number. Thus, creating and studying mouse models of aneuploidy and copy number variation is important for understanding what it is to be human, in both the normal and genomically altered states. PMID:22262951

  19. Mouse models in oncoimmunology.

    PubMed

    Zitvogel, Laurence; Pitt, Jonathan M; Daillère, Romain; Smyth, Mark J; Kroemer, Guido

    2016-12-01

    Fundamental cancer research and the development of efficacious antineoplastic treatments both rely on experimental systems in which the relationship between malignant cells and immune cells can be studied. Mouse models of transplantable, carcinogen-induced or genetically engineered malignancies - each with their specific advantages and difficulties - have laid the foundations of oncoimmunology. These models have guided the immunosurveillance theory that postulates that evasion from immune control is an essential feature of cancer, the concept that the long-term effects of conventional cancer treatments mostly rely on the reinstatement of anticancer immune responses and the preclinical development of immunotherapies, including currently approved immune checkpoint blockers. Specific aspects of pharmacological development, as well as attempts to personalize cancer treatments using patient-derived xenografts, require the development of mouse models in which murine genes and cells are replaced with their human equivalents. Such 'humanized' mouse models are being progressively refined to characterize the leukocyte subpopulations that belong to the innate and acquired arms of the immune system as they infiltrate human cancers that are subjected to experimental therapies. We surmise that the ever-advancing refinement of murine preclinical models will accelerate the pace of therapeutic optimization in patients.

  20. A Comparative Encyclopedia of DNA Elements in the Mouse Genome

    PubMed Central

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D.; Shen, Yin; Pervouchine, Dmitri D.; Djebali, Sarah; Thurman, Bob; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K.; Williams, Brian A.; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M. A.; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T.; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D.; Bansal, Mukul S.; Keller, Cheryl A.; Morrissey, Christapher S.; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S.; Cayting, Philip; Kawli, Trupti; Boyle, Alan P.; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S.; Cline, Melissa S.; Erickson, Drew T.; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A.; Rosenbloom, Kate R.; de Sousa, Beatriz Lacerda; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W. James; Santos, Miguel Ramalho; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J.; Wilken, Matthew S.; Reh, Thomas A.; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P.; Neph, Shane; Humbert, Richard; Hansen, R. Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E.; Orkin, Stuart H.; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J.; Blobel, Gerd A.; Good, Peter J.; Lowdon, Rebecca F.; Adams, Leslie B.; Zhou, Xiao-Qiao; Pazin, Michael J.; Feingold, Elise A.; Wold, Barbara; Taylor, James; Kellis, Manolis; Mortazavi, Ali; Weissman, Sherman M.; Stamatoyannopoulos, John; Snyder, Michael P.; Guigo, Roderic; Gingeras, Thomas R.; Gilbert, David M.; Hardison, Ross C.; Beer, Michael A.; Ren, Bing

    2014-01-01

    Summary As the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases. PMID:25409824

  1. A comparative encyclopedia of DNA elements in the mouse genome.

    PubMed

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D; Shen, Yin; Pervouchine, Dmitri D; Djebali, Sarah; Thurman, Robert E; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K; Williams, Brian A; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M A; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D; Bansal, Mukul S; Kellis, Manolis; Keller, Cheryl A; Morrissey, Christapher S; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S; Cayting, Philip; Kawli, Trupti; Boyle, Alan P; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S; Cline, Melissa S; Erickson, Drew T; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A; Rosenbloom, Kate R; Lacerda de Sousa, Beatriz; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W James; Ramalho Santos, Miguel; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J; Wilken, Matthew S; Reh, Thomas A; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P; Neph, Shane; Humbert, Richard; Hansen, R Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E; Orkin, Stuart H; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J; Blobel, Gerd A; Cao, Xiaoyi; Zhong, Sheng; Wang, Ting; Good, Peter J; Lowdon, Rebecca F; Adams, Leslie B; Zhou, Xiao-Qiao; Pazin, Michael J; Feingold, Elise A; Wold, Barbara; Taylor, James; Mortazavi, Ali; Weissman, Sherman M; Stamatoyannopoulos, John A; Snyder, Michael P; Guigo, Roderic; Gingeras, Thomas R; Gilbert, David M; Hardison, Ross C; Beer, Michael A; Ren, Bing

    2014-11-20

    The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

  2. Micrognathia in mouse models of ciliopathies.

    PubMed

    Adel Al-Lami, Hadeel; Barrell, William B; Liu, Karen J

    2016-12-15

    Defects in the development of the mandible can lead to micrognathia, or small jaw, which manifests in ciliopathic conditions, such as orofaciodigital syndrome, Meckel-Gruber syndrome, and Bardet-Biedl syndrome. Although micrognathia occurs frequently in human and mouse ciliopathies, it has been difficult to pinpoint the underlying cellular causes. In this mini-review, we shed light on the tissue-specific contributions to ciliary dysfunction in the development of the mandible. First, we outline the steps involved in setting up the jaw primordium and subsequent steps in the outgrowth of the mandibular skeleton. We then determine the critical tissue interactions using mice carrying a conditional mutation in the cilia gene Ofd1 Our studies highlight the usefulness of the Ofd1 mouse model and illustrate long-term possibilities for understanding the cellular and biochemical events underlying micrognathia. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  3. Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production

    PubMed Central

    Limaye, Advait; Hall, Bradford; Kulkarni, Ashok B

    2009-01-01

    The establishment of mouse embryonic stem (ES) cell liness has allowed for the generation of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. PMID:19731225

  4. Genome-Scale Assessment of Age-Related DNA Methylation Changes in Mouse Spermatozoa

    PubMed Central

    Kobayashi, Norio; Okae, Hiroaki; Hiura, Hitoshi; Chiba, Hatsune; Shirakata, Yoshiki; Hara, Kenshiro; Tanemura, Kentaro; Arima, Takahiro

    2016-01-01

    DNA methylation plays important roles in the production and functioning of spermatozoa. Recent studies have suggested that DNA methylation patterns in spermatozoa can change with age, but the regions susceptible to age-related methylation changes remain to be fully elucidated. In this study, we conducted genome-scale DNA methylation profiling of spermatozoa obtained from C57BL/6N mice at 8 weeks (8w), 18 weeks (18w) and 17 months of age (17m). There was no substantial difference in the global DNA methylation patterns between 18w and 17m samples except for a slight increase of methylation levels in long interspersed nuclear elements in the 17m samples. We found that maternally methylated imprinting control regions (mICRs) and spermatogenesis-related gene promoters had 5–10% higher methylation levels in 8w samples than in 18w or 17m samples. Analysis of individual sequence reads suggested that these regions were fully methylated (80–100%) in a subset of 8w spermatozoa. These regions are also known to be highly methylated in a subset of postnatal spermatogonia, which might be the source of the increased DNA methylation in 8w spermatozoa. Another possible source was contamination by somatic cells. Although we carefully purified the spermatozoa, it was difficult to completely exclude the possibility of somatic cell contamination. Further studies are needed to clarify the source of the small increase in DNA methylation in the 8w samples. Overall, our findings suggest that DNA methylation patterns in mouse spermatozoa are relatively stable throughout reproductive life. PMID:27880848

  5. Mouse genetics: catalogue and scissors.

    PubMed

    Sung, Young Hoon; Baek, In-Jeoung; Seong, Je Kyung; Kim, Jin Soo; Lee, Han-Woong

    2012-12-01

    Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-Like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics.

  6. A new mouse model for infantile neuroaxonal dystrophy, inad mouse, maps to mouse chromosome 1.

    PubMed

    Matsushima, Yoshibumi; Kikuchi, Tateki; Kikuchi, Hisae; Ichihara, Nobutsune; Ishikawa, Akira; Ishijima, Yasushi; Tachibana, Masayoshi

    2005-02-01

    Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive hereditary neurodegenerative disease of humans. So far, no responsible gene has been cloned or mapped to any chromosome. For chromosome mapping and positional cloning of the responsible gene, establishment of an animal model would be useful. Here we describe a new mouse model for INAD, named inad mouse. In this mouse, the phenotype is inherited in an autosomal recessive manner, symptoms occur in the infantile period, and the mouse dies before sexual maturity. Axonal dystrophic change appearing as spheroid bodies in central and peripheral nervous system was observed. These features more closely resembled human INAD than did those of the gad mouse, the traditional mouse model for INAD. Linkage analysis linked the inad gene to mouse Chromosome 1, with the highest LOD score (=128.6) at the D1Mit45 marker, and haplotype study localized the inad gene to a 7.5-Mb region between D1Mit84 and D1Mit25. In this linkage area some 60 genes exist: Mutation of one of these 60 genes is likely responsible for the inad mouse phenotype. Our preliminary mutation analysis in 15 genes examining the nucleotide sequence of exons of these genes did not find any sequence difference between inad mouse and C57BL/6 mouse.

  7. EPAS1 Is Required for Spermatogenesis in the Postnatal Mouse Testis1

    PubMed Central

    Gruber, Michaela; Mathew, Lijoy K.; Runge, Anja C.; Garcia, Joseph A.; Simon, M. Celeste

    2010-01-01

    Spermatogenesis, a process involving the differentiation of spermatogonial stem cells into mature spermatozoa, takes place throughout masculine life. A complex system in the testis, including endocrine signaling, physical interactions between germ and somatic cells, spermatocyte meiosis, and timely release of spermatozoa, controls this cycle. We demonstrate herein that decreased O2 levels and Epas1 activation are critical components of spermatogenesis. Postnatal Epas1 ablation leads to male infertility, with reduced testis size and weight. While immature spermatogonia and spermatocytes are present in Epas1Delta/Delta testes, spermatid and spermatozoan numbers are dramatically reduced. This is not due to germ cell-intrinsic defects. Rather, EpasDelta/Delta Sertoli cells exhibit decreased ability to form tight junctions, thereby disrupting the blood-testis barrier necessary for proper spermatogenesis. Reduced numbers of tight junction complexes are due to decreased expression of multiple genes encoding tight junction proteins, including TJP1 (ZO1), TJP2 (ZO2), and occludin. Furthermore, Epas1Delta/Delta testes exhibit disrupted basement membranes surrounding the seminiferous tubules, causing the premature release of incompletely differentiated germ cells. We conclude that low O2 levels in the male gonad regulate germ cell homeostasis in this organ via EPAS1. PMID:20181618

  8. Human anti-mouse antibodies.

    PubMed

    Klee, G G

    2000-06-01

    Human anti-mouse antibodies (HAMA) are human immunoglobulins with specificity for mouse immunoglobulins. This topic currently is of interest because of the increased use of monoclonal mouse antibodies as diagnostic reagents both for in vitro laboratory measurements and for in vivo imaging studies. Monoclonal mouse antibodies also are being used therapeutically. This short article reviews the production of HAMA in patients receiving monoclonal antibodies and illustrates the potential ways that HAMA can interfere with immunoassay measurements. Methods for measuring and neutralizing HAMA also are discussed.

  9. Modeling cutaneous squamous carcinoma development in the mouse.

    PubMed

    Huang, Phillips Y; Balmain, Allan

    2014-09-02

    Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers in Caucasian populations and is associated with a significant risk of morbidity and mortality. The classic mouse model for studying SCC involves two-stage chemical carcinogenesis, which has been instrumental in the evolution of the concept of multistage carcinogenesis, as widely applied to both human and mouse cancers. Much is now known about the sequence of biological and genetic events that occur in this skin carcinogenesis model and the factors that can influence the course of tumor development, such as perturbations in the oncogene/tumor-suppressor signaling pathways involved, the nature of the target cell that acquires the first genetic hit, and the role of inflammation. Increasingly, studies of tumor-initiating cells, malignant progression, and metastasis in mouse skin cancer models will have the potential to inform future approaches to treatment and chemoprevention of human squamous malignancies. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  10. MouseBook: an integrated portal of mouse resources.

    PubMed

    Blake, Andrew; Pickford, Karen; Greenaway, Simon; Thomas, Steve; Pickard, Amanda; Williamson, Christine M; Adams, Niels C; Walling, Alison; Beck, Tim; Fray, Martin; Peters, Jo; Weaver, Tom; Brown, Steve D M; Hancock, John M; Mallon, Ann-Marie

    2010-01-01

    The MouseBook (http://www.mousebook.org) databases and web portal provide access to information about mutant mouse lines held as live or cryopreserved stocks at MRC Harwell. The MouseBook portal integrates curated information from the MRC Harwell stock resource, and other Harwell databases, with information from external data resources to provide value-added information above and beyond what is available through other routes such as International Mouse Stain Resource (IMSR). MouseBook can be searched either using an intuitive Google style free text search or using the Mammalian Phenotype (MP) ontology tree structure. Text searches can be on gene, allele, strain identifier (e.g. MGI ID) or phenotype term and are assisted by automatic recognition of term types and autocompletion of gene and allele names covered by the database. Results are returned in a tabbed format providing categorized results identified from each of the catalogs in MouseBook. Individual result lines from each catalog include information on gene, allele, chromosomal location and phenotype, and provide a simple click-through link to further information as well as ord