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Sample records for murine sertoli cells

  1. The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis.

    PubMed

    Timmons, Paula M; Rigby, Peter W J; Poirier, Françoise

    2002-02-01

    The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages. PMID:11830565

  2. Sertoli cells as biochambers

    NASA Technical Reports Server (NTRS)

    Cameron, Don F. (Inventor); Sanberg, Paul R. (Inventor); Saporta, Samuel (Inventor); Hushen, Joelle J. (Inventor)

    2004-01-01

    According to the present invention, there is provided a biological chamber system having a biochamber defined by outer walls of Sertoli cells. Also provided is a transplantation facilitator including a biochamber. A method of making biochambers by co-culturing facilitator cells and therapeutic cells and then aggregating the facilitator celes is also provided. Also provided is a method of transplanting cells by incorporating transplant cells into a biochamber and transplanting the biochamber containing the transplant cells.

  3. Constitutive WNT/Beta-Catenin Signaling in Murine Sertoli Cells Disrupts Their Differentiation and Ability to Support Spermatogenesis1

    PubMed Central

    Tanwar, Pradeep S.; Kaneko-Tarui, Tomoko; Zhang, LiHua; Rani, Poonam; Taketo, Makoto M.; Teixeira, Jose

    2009-01-01

    Sertoli and germ cell interactions are essential for spermatogenesis and, thus, male fertility. Sertoli cells provide a specialized microenvironment for spermatogonial stem cells to divide, allowing both self-renewal and spermatogenesis. In the present study, we used mice with a conditional activated allele of the beta-catenin gene (Ctnnb1tm1Mmt/+) in Sertoli cells expressing Cre recombinase driven by the anti-Müllerian hormone (AMH; also known as Müllerian-inhibiting substance) type II receptor promoter (Amhr2tm3(cre)Bhr/+) to show that constitutively activated beta-catenin leads to their continuous proliferation and compromised differentiation. Compared to controls, Sertoli cells in mature mutant mice continue to express high levels of both AMH and glial cell-derived neurotrophic factor (GDNF), which normally are expressed only in immature Sertoli cells. We also show evidence that LiCl treatment, which activates endogenous nuclear beta-catenin activity, regulates both AMH and GDNF expression at the transcriptional level. The epididymides were devoid of sperm in the Amhr2tm3(cre)Bhr/+;Ctnnb1tm1Mmt/+ mice at all ages examined. We show that the mutant mice are infertile because of defective differentiation of germ cells and increased apoptosis, both of which are characteristic of GDNF overexpression in Sertoli cells. Constitutive activation of beta-catenin in Amhr2-null mice showed the same histology, suggesting that the phenotype was the result of persistent overexpression of GDNF. These results show that dysregulated wingless-related MMTV integration site/beta-catenin signaling in Sertoli cells inhibits their postnatal differentiation, resulting in increased germ cell apoptosis and infertility. PMID:19794154

  4. The kinase DYRKIA regulates pre-mRNA splicing in spermatogonia and proliferation of spermatogonia and Sertoli cells by phosphorylating a spliceosomal component, SAP155, in postnatal murine testes.

    PubMed

    Eto, Ko; Sonoda, Yoshiyuki; Abe, Shin-Ichi

    2011-09-01

    SAP155 is an essential component of the spliceosome and its phosphorylation is required for splicing catalysis, but little is known concerning its function and regulation during spermatogenesis in postnatal murine testes. We report that inhibition of dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) IA strongly suppressed the mitogen-stimulated SAP155 phosphorylation and constitutive splicing of I?B pre-mRNA as well as the proliferation of spermatogonial and Sertoli cells in cultures of the 6-day post partum testes and a spermatogonial cell line, but not in a Sertoli cell line. Our findings suggest that the active spliceosome, containing SAP155 phosphorylated by DYRKIA, performs pre-mRNA splicing in spermatogonia during testicular development. PMID:21553260

  5. Postnatal development of bovine Sertoli cells.

    PubMed

    Sinowatz, F; Amselgruber, W

    1986-01-01

    The fine structure of bovine Sertoli cells was studied from the 4th to the 40th week post natum in order to correlate the progressive acquisition of normal adult morphology with functional development. The considerable increase in tubular size during the first 20 weeks is due to the proliferation of both presumptive Sertoli and germ cells. Aside from this, the presumptive Sertoli cells are seen to expand radially and lengthen considerably. From then on however, the observed increase in tubular diameter during the later period of postnatal development is solely due to the great increase in the number of germ cells. Presumptive Sertoli cells undergo morphological differentiation to mature Sertoli cells during the first 28 weeks of proliferative development. The maturation process includes distinct changes in cell shape, nucleus and cellular organelles, as well as an increase in and differentiation of Sertoli cell surface specializations. At 24 weeks the development of inter-Sertoli cell junctions has reached a point of differentiation where, in our opinion, a functional blood-testis barrier can be expected. During the first 8 weeks an extensive development of rough endoplasmic reticulum and a well-developed Golgi apparatus can be observed, which suggests a high secretory activity in the presumptive Sertoli cells at this time. We speculate that these secretory activities may play a role in the formation of the basal lamina which is extremely well developed during early postnatal life. The subsequent reduction of the basal lamina correlates well with diminished secretory activity in the Sertoli cells. PMID:3766996

  6. Sertoli cell tumour in an Amur tiger.

    PubMed

    Scudamore, C L; Meredith, A L

    2001-01-01

    The histological and immunohistochemical characteristics of a malignant Sertoli cell tumour in a 17-year-old Amur tiger (Panthera tigris altaica) are described. Histological examination of the primary lesion in the right testis and metastatic lesions throughout the internal organs showed a variable cellular pattern with an admixture of tubular structures divided by fine stroma filled with fusiform to stellate cells, and sheets of polygonal cells with abundant vacuolated cytoplasm. Immunohistochemical techniques demonstrated strong positive staining for neuron-specific enolase and variable positive staining for vimentin in neoplastic cells, supporting a diagnosis of a tumour of Sertoli cell origin. PMID:11428192

  7. Sertoli Cell Differentiation in Pubertal Boars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Meishan boars experience puberty at a younger age than crossbred (BX) boars in association with earlier cessation of Sertoli cell proliferation and smaller post pubertal testicular size. The current study defined changes in expression, assessed by immunohistochemistry, of anti-Mullerian hormone (AMH...

  8. Apoptosis of Sertoli cells after conditional ablation of murine double minute 2 (Mdm2) gene is p53-dependent and results in male sterility.

    PubMed

    Fouchécourt, S; Livera, G; Messiaen, S; Fumel, B; Parent, A-S; Marine, J-C; Monget, P

    2016-03-01

    Beside its well-documented role in carcinogenesis, the function of p53 family has been more recently revealed in development and female reproduction, but it is still poorly documented in male reproduction. We specifically tested this possibility by ablating Mdm2, an E3 ligase that regulates p53 protein stability and transactivation function, specifically in Sertoli cells (SCs) using the AMH-Cre line and created the new SC-Mdm2(-/-) line. Heterozygous SC-Mdm2(-/+) adult males were fertile, but SC-Mdm2(-/-) males were infertile and exhibited: a shorter ano-genital distance, an extra duct along the vas deferens that presents a uterus-like morphology, degenerated testes with no organized seminiferous tubules and a complete loss of differentiated germ cells. In adults, testosterone levels as well as StAR, P450c17 (Cyp17a1) and P450scc (Cyp11a1) mRNA levels decreased significantly, and both plasma LH and FSH levels increased. A detailed investigation of testicular development indicated that the phenotype arose during fetal life, with SC-Mdm2(-/-) testes being much smaller at birth. Interestingly, Leydig cells remained present until adulthood and fetal germ cells abnormally initiated meiosis. Inactivation of Mdm2 in SCs triggered p53 activation and apoptosis as early as 15.5 days post conception with significant increase in apoptotic SCs. Importantly, testis development occurred normally in SC-Mdm2(-/-) lacking p53 mice (SC-Mdm2(-/-)p53(-/-)) and accordingly, these mice were fertile indicating that the aforementioned phenotypes are entirely p53-dependent. These data not only highlight the importance of keeping p53 in check for proper testicular development and male fertility but also certify the critical role of SCs in the maintenance of meiotic repression. PMID:26470726

  9. Nongenomic actions of androgen in Sertoli cells.

    PubMed

    Walker, William H

    2003-01-01

    Nongenomic actions mediated by androgens have now been described in more than 10 cell types. Some of these cells transduce androgen signals using surface receptors that await final characterization, whereas other cells employ the classical AR. Various second messengers can be activated by androgens, including cAMP, IP3, phospholipase C, DAG, and Ca2+. Each of these second messengers is capable of activating multiple kinases. One of the most important kinase networks to be regulated by androgens is the MAP kinase cascade. This series of kinase reactions is capable of altering the activity of many transcription factors with important implications for the regulation of gene expression. Because there is evidence that androgen is capable of regulating CREB-mediated gene expression via the MAP kinase pathway, it is now somewhat misleading to characterize androgen actions in Sertoli cells as nongenomic. Instead, it may be more appropriate to label these activities as independent of AR-DNA interactions, or more simply as nonclassical. The nonclassical regulation of gene expression in Sertoli cells is particularly relevant for providing an answer to the paradox of how testosterone can support spermatogenesis yet regulate few genes via AR-promoter interactions. It is expected that with the increasing use of microarray and related technologies, additional AR-regulated genes will be identified. However, the androgen-induced increases in [Ca2+]i, the activation of Src kinase, and the MAP kinase cascade that have been characterized thus far have the potential to regulate the expression of many more genes than is possible by direct AR-promoter interactions. Thus, it is likely that nonclassical actions of testosterone in Sertoli cells will be found to be a necessary complement to the classical actions that are required to maintain spermatogenesis. PMID:14584725

  10. Defined pattern of Sertoli cell differentiation in pubertal porcine testes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Number of Sertoli cells is a primary determinant of mature testicular size and sperm production. In boars, formation of the blood/testis barrier, which occurs by 4 mo of age in commercial breeds, signals the end of Sertoli cell proliferation. Previous studies established that expression of p27Kip1, ...

  11. Sertoli-Leydig cell tumor

    MedlinePLUS

    ... the female ovaries, usually in younger women. The cancer cells release a male sex hormone. As a result, the woman may develop symptoms such as: A deep voice Enlarged clitoris Facial hair Loss in breast size Stopping of menstrual periods Pain in the lower ...

  12. Porcine sertoli cell proliferation after androgen receptor inactivation.

    PubMed

    Legacki, Erin; Conley, Alan J; Nitta-Oda, Barbara Jean; Berger, Trish

    2015-04-01

    Sertoli cell proliferation in neonatal boars is potentially androgen dependent. Hence, the immediate objective was to evaluate effects of androgen receptor-mediated signaling on the first wave of Sertoli cell proliferation. The experimental design employed littermate pairs of boars with one member assigned to receive a daily oral dose of flutamide, an androgen receptor antagonist, beginning at 1 wk of age and the littermate the canola oil vehicle. Experiment 1 examined the response at 6.5 wk of age after completion of the first wave of Sertoli cell proliferation, and experiment 2 examined the response at 11 wk of age after initiation of the second wave of Sertoli cell proliferation. Experiment 3 was designed to evaluate initial responses at 2, 3, or 4 wk of age. Additional littermates from four of the litters evaluated at 2 wk of age were hemicastrated at 8 days of age. Testis weight increased approximately 50% in the flutamide-treated boars compared with vehicle-treated littermates (P = 0.01) by 6.5 wk of age. Approximately 80% more Sertoli cells/testis were present in flutamide-treated boars at 6.5 wk of age compared with their vehicle-treated littermates (P < 0.01). Animals that were hemicastrated at 8 days of age had more Sertoli cells/testis than their intact littermates at 2 wk of age (P < 0.01), but flutamide inhibited the hemicastration response. Androgen receptor antagonism during postnatal Sertoli cell proliferation increases Sertoli cell numbers, as does hemicastration, but receptor antagonism initially inhibits Sertoli cell proliferation induced by hemicastration. PMID:25715793

  13. Characterization and Functionality of Proliferative Human Sertoli Cells

    PubMed Central

    Chui, Kitty; Trivedi, Alpa; Cheng, C. Yan; Cherbavaz, Diana B.; Dazin, Paul F.; Huynh, Ai Lam Thu; Mitchell, James B.; Rabinovich, Gabriel A.; Noble-Haeusslein, Linda J.; John, Constance M.

    2014-01-01

    It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy. PMID:21054948

  14. Sertoli cells- Immunological sentinels of spermatogenesis

    PubMed Central

    Kaur, Gurvinder; Thompson, Lea Ann; Dufour, Jannette M.

    2014-01-01

    Testicular germ cells, which appear after the establishment of central tolerance, express novel cell surface and intracellular proteins that can be recognized as foreign antigens by the hosts immune system. However, normally these germ cells do not evoke an auto-reactive immune response. The focus of this manuscript is to review the evidence that the Blood-Testis-Barrier (BTB)/Sertoli cell (SC) barrier along with the SCs ability to modulate the immune response is vital for protecting auto-antigenic germ cells. In normal testis, the BTB/SC barrier protects the majority of the auto-antigenic germ cells by limiting access by the immune system and sequestering these new antigens. SCs also modulate testis immune cells (induce regulatory immune cells) by expressing several immunoregulatory factors, thereby creating a local tolerogenic environment optimal for survival of nonsequesetred auto-antigenic germ cells. Collectively, the fortress created by the BTB/SC barrier along with modulation of the immune response is pivotal for completion of spermatogenesis and species survival. PMID:24603046

  15. Testicular histopathology associated with disruption of the Sertoli cell cytoskeleton

    PubMed Central

    Johnson, Kamin J

    2014-01-01

    Testicular histological alterations following Sertoli cell cytoskeleton disruption are numerous. The Sertoli cell cytoskeleton is comprised of intermediate filaments, microtubules, microfilaments and their direct interacting proteins and performs essential functions including structural support of the seminiferous epithelium, apicobasal movement of elongate spermatids, and release of elongate spermatids from the seminiferous epithelium during spermiation. This review summarizes the histological changes occurring after disruption of the Sertoli cell cytoskeleton, including the signature lesion of seminiferous epithelium sloughing. By presenting examples of histological changes after exposure to toxins or toxicants directly affecting the Sertoli cell cytoskeleton or genetic manipulations of this cytoskeleton, the toxicologist observing similar histological changes associated with exposure to novel compounds can use this information to generate hypotheses about a potential mode of action. PMID:26413393

  16. A Rare Cause of Prepubertal Gynecomastia: Sertoli Cell Tumor

    PubMed Central

    Dursun, Fatma; Su Dur, ?eyma Meliha; ?ahin, Ceyhan; K?rm?z?bekmez, Heves; Karabulut, Murat Hakan; Yrk, As?m

    2015-01-01

    Prepubertal gynecomastia due to testis tumors is a very rare condition. Nearly 5% of the patients with testicular mass present with gynecomastia. Sertoli cell tumors are sporadic in 60% of the reported cases, while the remaining is a component of multiple neoplasia syndromes such as Peutz-Jeghers syndrome and Carney complex. We present a 4-year-old boy with gynecomastia due to Sertoli cell tumor with no evidence of Peutz-Jeghers syndrome or Carney complex. PMID:26366315

  17. Retinol uptake and esterification in the rate sertoli cell

    SciTech Connect

    Shingleton, J.L.

    1989-01-01

    The mechanism by which Sertoli cells accumulate retinol from retinol-binding protein (RBP) and the cellular metabolism of the accumulated retinol were investigated here using primary cultures of Sertoli cells isolated from 20 day-old rats. Cells incubated with ({sup 3}H)retinol-RBP accumulated ({sup 3}H)retinol in a time- and temperature dependent manner. The rate of ({sup 3}H) retinol accumulation declined when cellular ({sup 3}H) retinol concentrations reached approximately 0.53 pmol of retinol per {mu}g of cellular DNA, equivalent to the cellular content of cellular retinol-binding protein (CRBP). Excess unlabeled retinol-RBP competed with ({sup 3}H) retinol-RBP for ({sup 3}H) retinol delivery to the cells but free retinol did not. Furthermore, free ({sup 3}H) retinol associated with Sertoli cells in a non-saturable manner. The transport constant for specific retinol accumulation from RBP was 1.9 {mu}M, suggesting that any change in the normal circulating retinol-RBP level would directly affect the rate of retinol accumulation. Competition studies and studies using labeled RBP, cellular energy inhibitors, and lysosomal poisons indicated that the specific retinol accumulation by Sertoli cells occurs by interaction with a cell-surface receptor that internalizes retinol without concomitant internalization of RBP. Extraction and HPLC analysis of the radioactivity associated with Sertoli cells after incubation with ({sup 3}H) retinol-RBP yielded retinol and retinyl esters.

  18. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    SciTech Connect

    Dai, R.X.; Djakiew, D.; Dym, M.

    1987-07-01

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding /sup 125/I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.

  19. Sertoli Cell-Only Syndrome: Behind the Genetic Scenes.

    PubMed

    Stouffs, Katrien; Gheldof, Alexander; Tournaye, Herman; Vandermaelen, Deborah; Bonduelle, Maryse; Lissens, Willy; Seneca, Sara

    2016-01-01

    Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of "idiopathic patients" showed no known infertility related copy number variations. PMID:26925412

  20. Sertoli Cell-Only Syndrome: Behind the Genetic Scenes

    PubMed Central

    Stouffs, Katrien; Gheldof, Alexander; Tournaye, Herman; Vandermaelen, Deborah; Bonduelle, Maryse; Lissens, Willy; Seneca, Sara

    2016-01-01

    Sertoli cell-only syndrome is defined by the complete absence of germ cells in testicular tissues and always results in male infertility. The aetiology often remains unknown. In this paper, we have investigated possible causes of Sertoli cell-only syndrome with a special focus on genetic causes. Our results show that, for a large part of the patients (>23% in an unselected group), the sex chromosomes are involved. The majority of patients had a Klinefelter syndrome, followed by patients with Yq microdeletions. Array comparative genomic hybridization in a selected group of “idiopathic patients” showed no known infertility related copy number variations. PMID:26925412

  1. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  2. Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

    PubMed Central

    Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

    2012-01-01

    Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

  3. Bilateral Sertoli cell adenoma in gonads, associated with serous cystadenoma.

    PubMed

    Fernandez-Vega, I; Santos-Juanes, J; García-Pravia, C

    2014-06-01

    Complete androgen insensitivity syndrome is an extremely infrequent disease. The patients exhibit female phenotype because of insensitivity to the androgen receptor and may develop tumors, especially in their undescended gonads. We report a case of bilateral Sertoli cell adenoma in gonads with unilateral serous cystadenoma, in an elderly phenotypic woman with primary amenorrhea. We also provide radiological and pathological studies. PMID:25119177

  4. The actin filament network associated to Sertoli cell ectoplasmic specializations.

    PubMed

    Cavicchia, Juan Carlos; Fscolo, Mabel; Ibaez, Jorge; Lillig, Christopher; Capani, Francisco

    2011-12-01

    Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle. PMID:22423484

  5. Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling.

    PubMed

    Chen, Su-Ren; Tang, Ji-Xin; Cheng, Jin-Mei; Li, Jian; Jin, Cheng; Li, Xiao-Yu; Deng, Shou-Long; Zhang, Yan; Wang, Xiu-Xia; Liu, Yi-Xun

    2015-11-10

    Sertoli cells, the primary somatic cell in the seminiferous epithelium, provide the spermatogonial stem cell (SSC) microenvironment (niche) through physical support and the expression of paracrine factors. However, the regulatory mechanisms within the SSC niche, which is primarily controlled by Sertoli cells, remain largely unknown. GATA4 is a Sertoli cell marker, involved in genital ridge initiation, sex determination and differentiation during the embryonic stage. Here, we showed that neonatal mice with a targeted disruption of Gata4 in Sertoli cells (Gata4flox/flox; Amh-Cre; hereafter termed Gata4 cKO) displayed a loss of the establishment and maintenance of the SSC pool and apoptosis of both gonocyte-derived differentiating spermatogonia and meiotic spermatocytes. Thus, progressive germ cell depletion and a Sertoli-cell-only syndrome were observed as early as the first wave of murine spermatogenesis. Transplantation of germ cells from postnatal day 5 (P5) Gata4 cKO mice into KitW/W-v recipient seminiferous tubules restored spermatogenesis. In addition, microarray analyses of P5 Gata4 cKO mouse testes showed alterations in chemokine signaling factors, including Cxcl12, Ccl3, Cxcr4 (CXCL12 receptor), Ccr1 (CCL3 receptor), Ccl9, Xcl1 and Ccrl2. Deletion of Gata4 in Sertoli cells markedly attenuated Sertoli cell chemotaxis, which guides SSCs or prospermatogonia to the stem cell niche. Finally, we showed that GATA4 transcriptionally regulated Cxcl12 and Ccl9, and the addition of CXCL12 and CCL9 to an in vitro testis tissue culture system increased the number of PLZF+ undifferentiated spermatogonia within Gata4 cKO testes. Together, these results reveal a novel role for GATA4 in controlling the SSC niche via the transcriptional regulation of chemokine signaling shortly after birth. PMID:26473289

  6. Loss of Gata4 in Sertoli cells impairs the spermatogonial stem cell niche and causes germ cell exhaustion by attenuating chemokine signaling

    PubMed Central

    Chen, Su-Ren; Tang, Ji-Xin; Cheng, Jin-Mei; Li, Jian; Jin, Cheng; Li, Xiao-Yu; Deng, Shou-Long; Zhang, Yan; Wang, Xiu-Xia; Liu, Yi-Xun

    2015-01-01

    Sertoli cells, the primary somatic cell in the seminiferous epithelium, provide the spermatogonial stem cell (SSC) microenvironment (niche) through physical support and the expression of paracrine factors. However, the regulatory mechanisms within the SSC niche, which is primarily controlled by Sertoli cells, remain largely unknown. GATA4 is a Sertoli cell marker, involved in genital ridge initiation, sex determination and differentiation during the embryonic stage. Here, we showed that neonatal mice with a targeted disruption of Gata4 in Sertoli cells (Gata4flox/flox; Amh-Cre; hereafter termed Gata4 cKO) displayed a loss of the establishment and maintenance of the SSC pool and apoptosis of both gonocyte-derived differentiating spermatogonia and meiotic spermatocytes. Thus, progressive germ cell depletion and a Sertoli-cell-only syndrome were observed as early as the first wave of murine spermatogenesis. Transplantation of germ cells from postnatal day 5 (P5) Gata4 cKO mice into KitW/W-v recipient seminiferous tubules restored spermatogenesis. In addition, microarray analyses of P5 Gata4 cKO mouse testes showed alterations in chemokine signaling factors, including Cxcl12, Ccl3, Cxcr4 (CXCL12 receptor), Ccr1 (CCL3 receptor), Ccl9, Xcl1 and Ccrl2. Deletion of Gata4 in Sertoli cells markedly attenuated Sertoli cell chemotaxis, which guides SSCs or prospermatogonia to the stem cell niche. Finally, we showed that GATA4 transcriptionally regulated Cxcl12 and Ccl9, and the addition of CXCL12 and CCL9 to an in vitro testis tissue culture system increased the number of PLZF+ undifferentiated spermatogonia within Gata4 cKO testes. Together, these results reveal a novel role for GATA4 in controlling the SSC niche via the transcriptional regulation of chemokine signaling shortly after birth. PMID:26473289

  7. Clinicopathologic features of ovarian Sertoli-Leydig cell tumors

    PubMed Central

    Zhang, Hai-Yan; Zhu, Jia-Er; Huang, Wen; Zhu, Jin

    2014-01-01

    Background: Ovarian Stertoli-Ledig cell tumor (SLCT) is a rare type of sex cord-stromal tumor of the ovary. The present study was to evaluate clinicalopahologic features and prognosis of patients with Sertoli-Leydig cell tumor treated by surgery and adjuvant chemotherapy during short term follow-up. Methods: A total of sixteen patients with ovarian Sertoli-Leydig cell tumor treated at the Obstetrics and Gynecology Hospital, Shanghai, China, between Jan 2001 and Dec 2011 were reviewed. The clinical data, treatment and prognosis were obtained from medical records. Results: The median age of the patients with ovarian Sertoli-Leydig cell tumor was about 27.5 years old in non-menopausal women, while the median age of menopausal women was about 63 years old. The most common complaint was with hormonal-related symptoms in the form of secondary amenorrhea and infinity, features of virilization, abdominal mass or irregular vaginal bleeding. All of sixteen patients underwent surgical staging and all were found to have stage I disease at the time of diagnosis. Eleven patients with intermediate and two patients with poorly differentiated tumors received adjuvant chemotherapy. There were differences found in operative time, blood loss and postoperative recovery time between laparotomy and laparoscopy. There were no disease-related deaths and all patients were under complete remission at the last follow-up. Conclusions: Ovarian Sertoli-Leydig cell tumors could happen in any period age of women. However, the tumors typically occur in the single side while still at the early stage, a favorable outcome could be achieved by surgery and adjuvant chemotherapy. Laparoscopy has similar surgical effects as laparotomy, but has a number of advantages. PMID:25400781

  8. Impaired Sertoli cell function in males diagnosed with Noonan syndrome.

    PubMed

    Marcus, K A; Sweep, C G J; van der Burgt, I; Noordam, C

    2008-11-01

    In order to study male gonadal function in Noonan syndrome, clinical and laboratory data, including inhibin B, were gathered in nine pubertal males diagnosed with Noonan syndrome. Bilateral testicular maldescent was observed in four, and unilateral cryptorchidism occurred in two. Puberty was delayed in three patients. Luteinising hormone (LH) levels were normal in all patients in our series, while follicle stimulating hormone (FSH) levels were raised in seven. Inhibin B was low in six males and just above the lower limit of normal in two. Importantly, all three men with normal testicular descent displayed signs of Sertoli cell dysfunction, indicating, in contrast to earlier reports, that bilateral cryptorchidism does not seem to be the main contributing factor to impairment of testicular function in Noonan syndrome. These findings suggest different mechanisms of disturbance in male gonadal function, which is frequently associated with Sertoli dysfunction. PMID:19189703

  9. Rictor Regulates Spermatogenesis by Controlling Sertoli Cell Cytoskeletal Organization and Cell Polarity in the Mouse Testis.

    PubMed

    Dong, Heling; Chen, Zhenguo; Wang, Caixia; Xiong, Zhi; Zhao, Wanlu; Jia, Chunhong; Lin, Jun; Lin, Yan; Yuan, Weiping; Zhao, Allan Z; Bai, Xiaochun

    2015-11-01

    Maintenance of cell polarity is essential for Sertoli cell and blood-testis barrier (BTB) function and spermatogenesis; however, the signaling mechanisms that regulate the integrity of the cytoskeleton and polarity of Sertoli cells are not fully understood. Here, we demonstrate that rapamycin-insensitive component of target of rapamycin (TOR) (Rictor), a core component of mechanistic TOR complex 2 (mTORC2), was expressed in the seminiferous epithelium during testicular development, and was down-regulated in a cadmium chloride-induced BTB damage model. We then conditionally deleted the Rictor gene in Sertoli cells and mutant mice exhibited azoospermia and were sterile as early as 3 months old. Further study revealed that Rictor may regulate actin organization via both mTORC2-dependent and mTORC2-independent mechanisms, in which the small GTPase, ras-related C3 botulinum toxin substrate 1, and phosphorylation of the actin filament regulatory protein, Paxillin, are involved, respectively. Loss of Rictor in Sertoli cells perturbed actin dynamics and caused microtubule disarrangement, both of which accumulatively disrupted Sertoli cell polarity and BTB integrity, accompanied by testicular developmental defects, spermiogenic arrest and excessive germ cell loss in mutant mice. Together, these findings establish the importance of Rictor/mTORC2 signaling in Sertoli cell function and spermatogenesis through the maintenance of Sertoli cell cytoskeletal dynamics, BTB integrity, and cell polarity. PMID:26360620

  10. Changes of heavy metal, metallothionein and heat shock proteins in Sertoli cells induced by cadmium exposure.

    PubMed

    Kusakabe, Takahiko; Nakajima, Katsuyuki; Nakazato, Kyoumi; Suzuki, Keiji; Takada, Hisashi; Satoh, Takahiro; Oikawa, Masakazu; Arakawa, Kazuo; Nagamine, Takeaki

    2008-09-01

    In this study, we examined the levels of Cadmium (Cd), iron (Fe) and zinc (Zn), which were considered to be involved in Sertoli cell damage caused by Cd exposure. We also examined metallothionein (MT), heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1) expressions in Sertoli cells induced by Cd exposure. Evaluation by the in-air micro-particle induced X-ray emission (PIXE) method revealed that Cd and Fe distribution was increased in the cytoplasm of Sertoli cells after Cd exposure. By contrast, Zn was decreased in the cytoplasm of Sertoli cells after Cd exposure. It was suggested that the target of Cd toxicity was the cytoplasm of Sertoli cells, Fe was considered to enhance damage to Sertoli cells caused by Cd exposure. The DNA fragmentation rate was determined by ELISA after Cd exposure to Sertoli cells. It remained essentially unchanged with 2.5 microM Cd exposure of Sertoli cells; however, MT, Hsp70 and HO-1 were significantly increased by Cd exposure. As a result, Cd-induced MT was protected Sertoli cells against apoptosis, and Cd-induced HO-1 was involved in protection against oxidative stress. Incidentally, MT, Hsp70 and HO-1 showed similar responses to Cd exposure. PMID:18556172

  11. Sertoli cell development and function in an animal model of testicular dysgenesis syndrome.

    PubMed

    Hutchison, Gary R; Scott, Hayley M; Walker, Marion; McKinnell, Chris; Ferrara, Diana; Mahood, I Kim; Sharpe, Richard M

    2008-02-01

    Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-mllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells. PMID:17928633

  12. Effects of simulated microgravity on mouse Sertoli cells in culture

    NASA Astrophysics Data System (ADS)

    Angela, Masini Maria; Prato, Paola; Linda, Scarabelli; Lanza, Cristina; Palmero, Silvio; Pointis, Georges; Ricci, Franco; Strollo, Felice

    With the advent of space flights questions concerning the effects of microgravity (0xG) on hu-man reproduction physiology have got priority Spermatogenesis is a complex, highly ordered process of cell division and differentiation by which spermatogonial cells give rise to mature spermatozoa. Sertoli cells play a crucial role in the development of germ cells and the regulation of spermatogenesis. In this study the influence of 0xG on Sertoli cells was evaluated. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal (using the 3D reconstruction generated from a stack of confocal images) and SHBG changes by immunohistochemistry, for antioxidant agents by RT-PCR and for culture medium lactate concentrations by wet chemistry. Cells were cultured for 6, 24 and 48 hrs on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1xG) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or RNA-extracted or used for culture medium lactate measurements as needed. At 0xG Sertoli cytoskeleton got disorganized, microtubules fragmented and SHBG undetectable already after 24 hrs, with alterations wors-ening further until 48 hrs; various antioxidant systems (SOD, GST, PARP, MTs) appreciably increased during the first 24 hrs but significantly decreased at 48 hrs. No changes occurred in 1xG samples. At least initially, 0xG seems to perturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0xG slightly decreased only after 24 hrs. Further experiments need to be carried out in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out eventually pending male infertility consequences, which would be a problem nowadays, when life expectancy increases and male fertility might become a social issue often extending into 60 years and over. (experiment funded by ASI, through a grant within the OSMA project).

  13. Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice.

    PubMed

    Hu, Xiaopeng; Tang, Zhenzhou; Li, Yang; Liu, Wensheng; Zhang, Shuang; Wang, Bingyan; Tian, Yingpu; Zhao, Yinan; Ran, Hao; Liu, Wenjie; Feng, Gen-Sheng; Shuai, Jianwei; Wang, Haibin; Lu, Zhongxian

    2015-01-01

    The male's ability to reproduce is completely dependent on Sertoli cells. However, the mechanisms governing the functional integrity of Sertoli cells have remained largely unexplored. Here, we demonstrate that deletion of Shp2 in Sertoli cells results in infertility in mice. In Shp2 knockout mice (SCSKO), a normal population of Sertoli cells was observed, but the blood-testis barrier (BTB) was not formed. Shp2 ablation initiated the untimely and excessive differentiation of spermatogonial stem cells (SSCs) by disturbing the expression of paracrine factors. As a consequence, the process of spermatogenesis was disrupted, and the germ cells were depleted. Furthermore, Shp2 deletion impaired the cell junctions of the primary Sertoli cells and failed to support the clonal formation of SSCs co-cultured with SCSKO Sertoli cells. As expected, Shp2 restoration largely restores the cell junctions of the primary Sertoli cells and the clonal formation of SSCs. To identify the underlying mechanism, we further demonstrated that the absence of Shp2 suppressed Erk phosphorylation, and thus, the expression of follicle-stimulating hormone (FSH)- and testosterone-induced target genes. These results collectively suggest that Shp2 is a critical signaling protein that is required to maintain Sertoli cell function and could serve as a novel target for male infertility therapies. PMID:26265072

  14. Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice

    PubMed Central

    Hu, Xiaopeng; Tang, Zhenzhou; Li, Yang; Liu, Wensheng; Zhang, Shuang; Wang, Bingyan; Tian, Yingpu; Zhao, Yinan; Ran, Hao; Liu, Wenjie; Feng, Gen-Sheng; Shuai, Jianwei; Wang, Haibin; Lu, Zhongxian

    2015-01-01

    The male’s ability to reproduce is completely dependent on Sertoli cells. However, the mechanisms governing the functional integrity of Sertoli cells have remained largely unexplored. Here, we demonstrate that deletion of Shp2 in Sertoli cells results in infertility in mice. In Shp2 knockout mice (SCSKO), a normal population of Sertoli cells was observed, but the blood-testis barrier (BTB) was not formed. Shp2 ablation initiated the untimely and excessive differentiation of spermatogonial stem cells (SSCs) by disturbing the expression of paracrine factors. As a consequence, the process of spermatogenesis was disrupted, and the germ cells were depleted. Furthermore, Shp2 deletion impaired the cell junctions of the primary Sertoli cells and failed to support the clonal formation of SSCs co-cultured with SCSKO Sertoli cells. As expected, Shp2 restoration largely restores the cell junctions of the primary Sertoli cells and the clonal formation of SSCs. To identify the underlying mechanism, we further demonstrated that the absence of Shp2 suppressed Erk phosphorylation, and thus, the expression of follicle-stimulating hormone (FSH)- and testosterone-induced target genes. These results collectively suggest that Shp2 is a critical signaling protein that is required to maintain Sertoli cell function and could serve as a novel target for male infertility therapies. PMID:26265072

  15. In vitro effects of simulated microgravity on Sertoli cell function

    NASA Astrophysics Data System (ADS)

    Masini, M. A.; Prato, P.; Scarabelli, L.; Lanza, C.; Palmero, S.; Pointis, G.; Ricci, F.; Strollo, F.

    2011-02-01

    With the advent of space flights questions concerning the effects of microgravity (0×G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0×G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1×G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0×G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1×G samples. Initially, 0×G seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0×G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.

  16. Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion

    PubMed Central

    Archambeault, Denise R.; Yao, Humphrey Hung-Chang

    2010-01-01

    Formation of tubular structures relies upon complex interactions between adjacent epithelium and mesenchyme. In the embryonic testes, dramatic compartmentalization leads to the formation of testis cords (epithelium) and the surrounding interstitium (mesenchyme). Sertoli cells, the epithelial cell type within testis cords, produce signaling molecules to orchestrate testis cord formation. The interstitial fetal Leydig cells, however, are thought only to masculinize the embryo and are not known to be involved in testis cord morphogenesis. Contrary to this notion, we have identified activin A, a member of the TGF-β protein superfamily, as a product of the murine fetal Leydig cells that acts directly upon Sertoli cells to promote their proliferation during late embryogenesis. Genetic disruption of activin βA, the gene encoding activin A, specifically in fetal Leydig cells resulted in a failure of fetal testis cord elongation and expansion due to decreased Sertoli cell proliferation. Conditional inactivation of Smad4, the central component of TGF-β signaling, in Sertoli cells led to testis cord dysgenesis and proliferative defects similar to those of Leydig cell-specific activin βA knockout testes. These results indicate that activin A is the major TGF-β protein that acts directly on Sertoli cells. Testicular dysgenesis in activin βA and Smad4 conditional knockout embryos persists into adulthood, leading to low sperm production and abnormal testicular histology. Our findings challenge the paradigm that fetal testis development is solely under the control of Sertoli cells, by uncovering an active and essential role of fetal Leydig cells during testis cord morphogenesis. PMID:20498064

  17. [Experimental Evidence of Proliferation and Reproduction of Highly Differentiated Sertoli Cells].

    PubMed

    Zakhidova, S T; Marshak, T L

    2015-01-01

    The main results of studies regarding the biology of Sertoli cells under various experimental conditions are considered. Possible potential mechanisms underlying the transition of highly differentiated Sertoli cells to dedifferentiation, limited by proliferation and reproduction and not accompanied by significant phenotypic changes, are discussed. PMID:26415275

  18. Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance.

    PubMed

    Eid, Nabil; Ito, Yuko; Otsuki, Yoshinori

    2012-02-01

    Although chronic ethanol consumption results in Sertoli cell vacuolization and augmented testicular germ cell apoptosis via death receptor and mitochondrial pathways, Sertoli cells are resistant to apoptosis. The aim of this study was to examine whether the activation of autophagy in the Sertoli cells of ethanol-treated rats (ETR) may have a role in their survival. Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. The TUNEL method demonstrated that Sertoli cells were always TUNEL-negative despite the presence of many apoptotic germ cells in ETR, supporting our previous studies. Electron microscopy revealed the presence of large numbers of autophagic vacuoles (AVs) in Sertoli cells of ETR compared to few AVs in control testes. Most of the AVs in Sertoli cells of ETR enveloped and sequestered damaged and abnormally shaped mitochondria, without cytoplasm, indicating mitochondrial autophagy (mitophagy). Immuno-electron microscopy showed the localization of LC3, a specific marker of early AVs (autophagosomes), around AVs sequestering mitochondria in Sertoli cells of ETR. Immunohistochemical staining of LC3 demonstrated a punctate pattern in Sertoli cells of ETR, confirming the formation of autophagosomes, while LC3 puncta were almost absent in control testes. Moreover, increased immunoreactivity of LAMP-2, a lysosomal membrane protein and marker of late AVs (autolysosomes), was mainly observed in Sertoli cells of ETR, with weaker expression in control testes. Via the deletion of pro-apoptotic damaged mitochondria, enhanced Sertoli cell mitophagy in ETR may be an anti-apoptotic mechanism that is essential for spermatogenesis. PMID:22076330

  19. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    SciTech Connect

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-{gamma}-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-{alpha} induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.

  20. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    SciTech Connect

    Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  1. Expression of Dominant-Negative Thyroid Hormone Receptor Alpha1 in Leydig and Sertoli Cells Demonstrates No Additional Defect Compared with Expression in Sertoli Cells Only

    PubMed Central

    Fumel, Betty; Froment, Pascal; Holzenberger, Martin; Livera, Gabriel; Monget, Philippe; Fouchcourt, Sophie

    2015-01-01

    Background In the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial. Methods The TR?AMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TR?1 (thyroid receptor ?1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter. Findings We showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TR?AMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TR?AMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TR?AMI-ARO males showed normal fertility. This phenotype is similar to TR?AMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TR?AMI-ARO. Conclusions/Significance We concluded that the presence of a mutant TR?AMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TR?1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis. PMID:25793522

  2. Interleukin 1alpha-induced disruption of the Sertoli cell cytoskeleton affects gap junctional communication.

    PubMed

    Chojnacka, Katarzyna; Bilinska, Barbara; Mruk, Dolores D

    2016-05-01

    Gap junctions (GJ) are transmembrane channels that connect the cytoplasms of adjacent cells, thereby facilitating the rapid exchange of ions, second messengers, and metabolites smaller than 1kDa. Connexin 43, the best-studied GJ protein, is a component of the Sertoli cell barrier/blood-testis barrier (BTB). To gain insight into the biology of the BTB, we investigated the effects of interleukin 1alpha (IL1A), a pro-inflammatory cytokine that disrupts BTB function, on gap junctional communication (GJC) in Sertoli cells. Compared with controls, the levels of connexin 43 and connexin 43 (Ser 368) increased ~30- and 20-fold, respectively, at 24h after IL1A treatment. To assess GJ function, we investigated fluorescence recovery in photobleached Sertoli cells after vehicle or IL1A treatment. Compared with the control, IL1A affected the ability of calcein to return to photobleached cells, indicating that GJC was compromised. To explain the effects of IL1A on GJ function, the involvement of the Sertoli cell cytoskeleton was investigated. Stress fibers aggregated at the periphery of Sertoli cells treated with IL1A. These results were substantiated by a biochemical assay that showed IL1A to disrupt the bundling of exogenous F-actin by Sertoli cells. In summary, IL1A regulates GJC in Sertoli cells, which is critical for BTB restructuring. PMID:26879129

  3. Expression of Genomic Functional Estrogen Receptor 1 in Mouse Sertoli Cells

    PubMed Central

    Lin, Jing; Zhu, Jia; Li, Xian; Li, Shengqiang; Lan, Zijian; Ko, Jay

    2014-01-01

    There is no consensus whether Sertoli cells express estrogen receptor 1 (Esr1). Reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence demonstrated that mouse Sertoli cell lines, TM4, MSC-1, and 15P-1, and purified primary mouse Sertoli cells (PSCs) contained Esr1 messenger RNA and proteins. Incubation of Sertoli cells with 17?-estradiol (E2) or ESR1 agonist stimulated the expression of an estrogen responsive gene Greb1, which was prevented by ESR inhibitor or ESR1 antagonist. Overexpression of Esr1 in MSC-1 enhanced E2-induced Greb1 expression, while knockdown of Esr1 by small interfering RNA in TM4 attenuated the response. Furthermore, E2-induced Greb1 expression was abolished in the PSCs isolated from Amh-Cre/Esr1-floxed mice in which Esr1 in Sertoli cells were selectively deleted. Chromatin immunoprecipitation assays indicated that E2-induced Greb1 expression in Sertoli cells was mediated by binding of ESR1 to estrogen responsive elements. In summary, ligand-dependent nuclear ESR1 was present in mouse Sertoli cells and mediates a classical genomic action of estrogens. PMID:24615934

  4. Male Differentiation of Germ Cells Induced by Embryonic Age-Specific Sertoli Cells in Mice1

    PubMed Central

    Ohta, Kohei; Yamamoto, Miyuki; Lin, Yanling; Hogg, Nathanael; Akiyama, Haruhiko; Behringer, Richard R.; Yamazaki, Yukiko

    2012-01-01

    ABSTRACT Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells. PMID:22262692

  5. Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice.

    PubMed

    Ohta, Kohei; Yamamoto, Miyuki; Lin, Yanling; Hogg, Nathanael; Akiyama, Haruhiko; Behringer, Richard R; Yamazaki, Yukiko

    2012-04-01

    Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells. PMID:22262692

  6. Cytokines produced by microwave-radiated Sertoli cells interfere with spermatogenesis in rat testis.

    PubMed

    Wu, H; Wang, D; Shu, Z; Zhou, H; Zuo, H; Wang, S; Li, Y; Xu, X; Li, N; Peng, R

    2012-05-01

    Microwave radiation resulted in degeneration, apoptosis or necrosis in germ cells at different stages. The molecular mechanisms by which microwaves induce spermatogenesis disorder have not been completely understood. Sertoli cells play crucial roles in mammalian spermatogenesis. Cytokines produced by Sertoli cells play pleiotropic roles in different conditions. At physiologically low concentration, TNFα, IL-1β and IL-6 behave as survival factors; while under pathological condition, these cytokines can induce apoptosis in testis. The effects of cytokines produced by microwave-radiated Sertoli cells on spermatogenesis are poorly understood. The purpose of this study was to determine the effect of cytokines produced by microwave-radiated Sertoli cells on the germ cells. We focused the effect of TNFα, IL-1β and IL-6 on the germ cells. The results showed that TNFα, IL-1β and IL-6 were increased in Sertoli cells after exposure to microwave radiation. These up-regulated cytokines can induce apoptosis and lipid peroxidation in the membrane of germ cells. In addition, germ cell apoptosis was associated with the up-regulation of Bax/Bcl-2 and caspase-3. These results suggest that cytokines produced by microwave-radiated Sertoli cells may disrupt spermatogenesis. Our data provided novel insight into the injury mechanism of germ cells induced by microwave radiation. PMID:22211786

  7. Disappearance of vimentin in Sertoli cells: a mono(2-ethylhexyl) phthalate effect.

    PubMed

    Tay, Tat Wei; Andriana, Bibin Bintang; Ishii, Maki; Tsunekawa, Naoki; Kanai, Yoshiakira; Kurohmaru, Masamichi

    2007-01-01

    The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V-FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V-FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells. PMID:17661219

  8. Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats

    PubMed Central

    2012-01-01

    Background Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. Methods Prepubertal rats received the dose of 5?mg/Kg of doxorubicin, which was fractioned in two doses: 3?mg/Kg at 15dpp and 2?mg/Kg at 22dpp. The testes were collected at 40, 64 and 127dpp, fixed in Bouins liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS?+?H-stained sections. Results The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats. Conclusions and discussion These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells. PMID:22967030

  9. The Sertoli cell: one hundred fifty years of beauty and plasticity.

    PubMed

    França, L R; Hess, R A; Dufour, J M; Hofmann, M C; Griswold, M D

    2016-03-01

    It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular 'nurse cell', not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cell's role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways. PMID:26846984

  10. Krppel-like factor 4 is involved in functional differentiation of testicular Sertoli cells

    PubMed Central

    Godmann, Maren; Katz, Jonathan P.; Guillou, Florian; Simoni, Manuela; Kaestner, Klaus H.; Behr, Rdiger

    2008-01-01

    Krppel-like factor 4 (KLF4) is a pleiotropic zinc finger transcription factor that regulates genes being involved in differentiation and cell-cycle control. Knock out studies revealed a critical function for KLF4 in the terminal differentiation of many epithelial cells. In testicular Sertoli cells, Klf4 is strongly inducible by the glycoprotein Follicle stimulating hormone (FSH). Since KLF4 is essential for postnatal survival in mice, we deleted Klf4 specifically in Sertoli cells using the Cre/loxP system. Importantly, around postnatal day 18, a critical period of terminal Sertoli cell differentiation, mutant seminiferous tubules exhibited a disorganized germinal epithelium and delayed lumen formation. The ultrastructural finding of highly vacuolized Sertoli cell cytoplasm and the identification of differentially expressed genes, which are known to play roles during vesicle transport and fusion or for maintenance of the differentiated cell state, suggest impaired apical secretion of the Sertoli cell. Interestingly, a high proportion of all identified genes was localized in a small subregion of chromosome 7 suggesting coordinated regulation. Intriguingly, adult mutant mice are fertile and show normal testicular morphology, although the testosterone levels are decreased. In summary, KLF4 plays a significant role for proper and timely Sertoli cell differentiation in pubertal mice. PMID:18243172

  11. Constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence.

    PubMed

    Garcia, Thomas Xavier; DeFalco, Tony; Capel, Blanche; Hofmann, Marie-Claude

    2013-05-01

    Notch signaling components have long been detected in Sertoli and germ cells in the developing and mature testis. However, the role of this pathway in testis development and spermatogenesis remains unknown. Using reporter mice expressing green fluorescent protein following Notch receptor activation, we found that Notch signaling was active in Sertoli cells at various fetal, neonatal, and adult stages. Since Notch signaling specifies stem cell fate in many developing and mature organ systems, we hypothesized that maintenance and differentiation of gonocytes and/or spermatogonial stem cells would be modulated through this pathway in Sertoli cells. To this end, we generated mutant mice constitutively expressing the active, intracellular domain of NOTCH1 (NICD1) in Sertoli cells. We found that mutant Sertoli cells were morphologically normal before and after birth, but presented a number of functional changes that drastically affected gonocyte numbers and physiology. We observed aberrant exit of gonocytes from mitotic arrest, migration toward cord periphery, and premature differentiation before birth. These events, presumably unsupported by the cellular microenvironment, were followed by gonocyte apoptosis and near complete disappearance of the gonocytes by day 2 after birth. Molecular analysis demonstrated that these effects are correlated with a dysregulation of Sertoli-expressed genes that are required for germ cell maintenance, such as Cyp26b1 and Gdnf. Taken together, our results demonstrate that Notch signaling is active in Sertoli cells throughout development and that proper regulation of Notch signaling in Sertoli cells is required for the maintenance of gonocytes in an undifferentiated state during fetal development. PMID:23391689

  12. Implications of Sertoli cell induced germ cell apoptosis to testicular pathology

    PubMed Central

    Murphy, Caitlin J; Richburg, John H

    2014-01-01

    After exposure to toxicants, degenerating germ cells represents the most common testicular histopathological alteration, regardless of the mechanism of toxicity. Therefore, deciphering the primary toxicant cellular target and mechanism of action can be extremely difficult. However, most testicular toxicants display a cell-specific and a stage-specific pattern of damage, which is the best evidence for identifying the primary cellular target (i.e. germ cell, Sertoli cell, peritubular myoid cell, or Leydig cell). Some toxicant-induced Sertoli cell injury presents with germ cell apoptosis occurring primarily in spermatocytes in rats in stages XI-XIV, I and II. Although some toxicants result in spermatid degeneration and apoptosis, it is still unclear if spermatid apoptosis is a result of Sertoli cell-selective apoptosis or a direct effect of toxicants on spermatids, therefore if this is seen as the earliest change, one cannot infer the mechanism of apoptosis. This review summarizes some of the distinguishing features of Sertoli cell-induced germ cell apoptosis and the associated mechanisms of cell death to provide the toxicologist observing similar cell death, with evidence about a potential mode of action. PMID:26413394

  13. Sertoli cells in the testis of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): light and electron microscopic perspective.

    PubMed

    Smita, Mathew; Oommen, Oommen V; George, Jancy M; Akbarsha, M A

    2003-12-01

    The caecilians have evolved a unique pattern of cystic spermatogenesis in which cysts representing different stages in spermatogenesis coexist in a testis lobule. We examined unsettled issues relating to the organization of the caecilian testis lobules, including the occurrence of a fatty matrix, the possibility of both peripheral and central Sertoli cells, the origin of Sertoli cells from follicular cells, and the disengagement of older Sertoli cells to become loose central Sertoli cells. We subjected the testis of Ichthyophis tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae) from the Western Ghats of Kerala, India, to light and transmission electron microscopic studies. Irrespective of the functional state of the testis, whether active or regressed, Sertoli cells constitute a permanent feature of the lobules. The tall Sertoli cells adherent to the basal lamina with basally located pleomorphic nuclei extend deeper into the lobule to meet at the core. There they provide for association of germ cells at different stages of differentiation, an aspect that has earlier been misconceived as the fatty matrix. Germ cells up to the 4-cell stage remain in the intercalating region of the Sertoli cells and they are located at the apices of the Sertoli cells from the 8-cell stage onwards. The developing germ cells are intimately associated with the Sertoli cell adherent to the basal lamina until spermiation. There are ameboid cells in the core of the lobules that appear to interact with the germ cells at the face opposite to their attachment with the Sertoli cells. Adherence of the Sertoli cells to the basal lamina is a permanent feature of the caecilian testicular lobules. The ameboid cells in the core are neither Sertoli cells nor their degeneration products. PMID:14584033

  14. Testicular cytology indicates differences in Sertoli cell counts between "good freezer" and "poor freezer" bulls.

    PubMed

    Rajak, Shailendra Kumar; Thippeswamy, Vijetha Bajjalli; Kumaresan, Arumugam; Layek, Siddhartha Shankar; Mohanty, Tushar Kumar; Gaurav, Mukesh Kumar; Chakravarty, Atish Kumar; Datta, Tirtha Kumar; Manimaran, Ayyasamy; Prasad, Shiv

    2016-01-01

    In artificial insemination, poor quality of semen unsuitable for cryopreservation and susceptibility of spermatozoa to cryodamage in crossbred bulls have been a matter of concern. Present study was designed to identify the testicular cytology indices that might be used to predict the semen quality and cryotolerance of spermatozoa in bulls. Based on the ejaculate rejection rate and sperm cryotolerance, bulls (Holstein Friesian X Tharparkar crossbred) were classified into either good (producing good quality semen with spermatozoa having good cryotolerance; n = 4) or poor (producing poor quality semen with spermatozoa having poor cryotolerance; n = 4). Testicular cytology was studied in all the 8 bulls using fine needle aspiration technique. Testicular cytology of good bulls and poor bulls differed significantly. The proportion of Sertoli cells was significantly higher in good bulls (25.3 1.6) compared to poor bulls (11.0 0.8). The Sertoli cell index was 46.1 5.0 in good bulls while it was only 13.8 1.3 in poor bulls. The cut off values, as determined using Receiver Operating Characteristics analysis, indicate that the bulls having testicular cytogram comprising of < 15.5% Sertoli cells, < 24.3 Sertoli cell index and > 4.0 spermatogenic cells to Sertoli cell ratio might be a poor bull in terms of semen quality and cryotolerance of spermatozoa. The proportion of Sertoli cells in the testicular cytology had positive (P < 0.05) relationship with semen quality and cryotolerance of spermatozoa. PMID:26891549

  15. Depletion of the p43 Mitochondrial T3 Receptor Increases Sertoli Cell Proliferation in Mice

    PubMed Central

    Fumel, Betty; Roy, Stéphanie; Fouchécourt, Sophie; Livera, Gabriel; Parent, Anne-Simone; Casas, François; Guillou, Florian

    2013-01-01

    Among T3 receptors, TRα1 is ubiquitous and its deletion or a specific expression of a dominant-negative TRα1 isoform in Sertoli cell leads to an increase in testis weight and sperm production. The identification of a 43-kDa truncated form of the nuclear receptor TRα1 (p43) in the mitochondrial matrix led us to test the hypothesis that this mitochondrial transcription factor could regulate Sertoli cell proliferation. Here we report that p43 depletion in mice increases testis weight and sperm reserve. In addition, we found that p43 deletion increases Sertoli cell proliferation in postnatal testis at 3 days of development. Electron microscopy studies evidence an alteration of mitochondrial morphology observed specifically in Sertoli cells of p43−/− mice. Moreover, gene expression studies indicate that the lack of p43 in testis induced an alteration of the mitochondrial-nuclear cross-talk. In particular, the up-regulation of Cdk4 and c-myc pathway in p43−/− probably explain the extended proliferation recorded in Sertoli cells of these mice. Our finding suggests that T3 limits post-natal Sertoli cell proliferation mainly through its mitochondrial T3 receptor p43. PMID:24040148

  16. Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production

    PubMed Central

    Hayrabedyan, Soren; Todorova, Krassimira; Jabeen, Asma; Metodieva, Gergana; Toshkov, Stavri; Metodiev, Metodi V.; Mincheff, Milcho; Fernández, Nelson

    2016-01-01

    Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\\NOD1 and NOD2 crosstalk converged in NFκB activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1β secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1β restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1β secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1β expression, while NOD2 inversely promoted IL-1β. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction. PMID:26744177

  17. Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production.

    PubMed

    Hayrabedyan, Soren; Todorova, Krassimira; Jabeen, Asma; Metodieva, Gergana; Toshkov, Stavri; Metodiev, Metodi V; Mincheff, Milcho; Fernndez, Nelson

    2016-01-01

    Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\\NOD1 and NOD2 crosstalk converged in NF?B activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1? secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1? restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1? secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1? expression, while NOD2 inversely promoted IL-1?. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction. PMID:26744177

  18. Co-culture of Mouse Embryonic Stem Cells with Sertoli Cells Promote in vitro Generation of Germ Cells

    PubMed Central

    Miryounesi, Mohammad; Nayernia, Karim; Dianatpour, Mahdi; Mansouri, Fatemeh; Modarressi, Mohammad Hossein

    2013-01-01

    Objective(s): Sertoli cells support in vivo germ cell production; but, its exact mechanism has not been well understood. The present study was designed to analyze the effect of Sertoli cells in differentiation of mouse embryonic stem cells (mESCs) to germ cells. Materials and Methods: A fusion construct composed of a Stra8 gene promoter and the coding region of enhanced green fluorescence protein was produced to select differentiated mESCs. To analyze sertoli cells effect in differentiation process, mESCs were separated into two groups: the first group was cultured on gelatin with retinoic acid treatment and the second group was co-cultured with sertoli cell feeder without retinoic acid induction. Expressions of pre-meiotic (Stra8), meiotic (Dazl and Sycp3) and post-meiotic (Prm1) genes were evaluated at different differentiation stages (+7, +12 and +18 days of culture). Results: In the first group, expressions of meiotic and post-meiotic genes started 12 and 18 days after induction with retinoic acid, respectively. In the second group, 7 days after co-culturing with Sertoli cells, expression of meiotic and post-meiotic genes was observed. Conclusion: These results show that differentiation process to germ cells is supported by Sertoli cells. Our findings provide a novel effective approach for generation of germ cell in vitro and studying the interaction of germ cells with their niche. PMID:23997904

  19. Primary rat Sertoli and interstitial cells exhibit a differential response to cadmium

    SciTech Connect

    Clough, S.R.; Welsh, M.J.; Payne, A.H.; Brown, C.D.; Brabec, M.J. )

    1990-01-01

    Two cell types central to the support of spermatogenesis, the Sertoli cell and the interstitial (Leydig) cell, were isolated from the same cohort of young male rats and challenged with cadmium chloride to compare their susceptibility to the metal. Both cell types were cultured under similar conditions, and similar biochemical endpoints were chosen to minimize experimental variability. These endpoints include the uptake of 109Cd, reduction of the vital tetrazolium dye MTT, incorporation of 3H-leucine, change in heat-stable cadmium binding capacity, and production of lactate. Using these parameters, it was observed that the Sertoli cell cultures were adversely affected in a dose-and time-dependent manner, while the interstitial cell cultures, treated with identical concentrations of CdCl2, were less affected. The 72-hr LC50's for Sertoli cells and interstitial cells were 4.1 and 19.6 microM CdCl2, respectively. Thus, different cell populations within the same tissue may differ markedly in susceptibility to a toxicant. These in vitro data suggest that the Sertoli cell, in relation to the interstitium, is particularly sensitive to cadmium. Because the Sertoli cell provides functional support for the seminiferous epithelium, the differential sensitivity of this cell type may, in part, explain cadmium-induced testicular dysfunction, particularly at doses that leave the vascular epithelium intact.

  20. Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

    2015-11-01

    The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

  1. The Sertoli cell of the water buffalo--an electron microscopic study.

    PubMed Central

    Azmi, T I; Bongso, T A; Harisah, M; Basrur, P K

    1990-01-01

    The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9a. Fig. 9b. Fig. 10. Fig. 11. PMID:2306676

  2. Connexin-43: A possible mediator of heat stress effects on ram Sertoli cells

    PubMed Central

    Hassanpour, Hossain; Kadivar, Ali; Mirshokraei, Pejman; Nazari, Hassan; Afzali, Azita; Badisanaye, Maryam

    2015-01-01

    Sertoli cells are an essential group of cells in seminiferous epithelium which provide nutritional and structural supports for spermatogenic cells via cell junctions. In this study, the gene expression of connexin-43, the most abundantly distributed gap junction protein of cells, was investigated in ram Sertoli cells under mild and severe heat stresses with real-time quantitative PCR. Sertoli cells were isolated from testes of 10 lambs. After culture and 3 passages, they were treated with mild (39 ?C) and severe (42 ?C) heat stress for 6 hr. The results showed a significant reduction in the percentage of live cells under severe heat stress compared to the control group (32 ?C), (p <0.05). Relative quantification analysis revealed significantly higher (3.80 fold increase) values of connexin-43 transcripts in severely heat stressed group than control group (p <0.05). It is concluded that challenging Sertoli cells with 42 ?C heat could threaten their survival, and overexpression of connexin-43 may cause dysfunction of Sertoli cells due to heat stress. These findings can be useful to identify the molecular mechanisms involved in adverse effects of heat stress on male reproduction and enhance our understanding of its pathogenesis. PMID:26261707

  3. The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro

    PubMed Central

    Rastegar, Tayebeh; Habibi Roudkenar, Mehryar; Parvari, Soraya; Baazm, Maryam

    2015-01-01

    Background: Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs) self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. Objective: This study investigated the role of luekemia inhibitory factor (LIF) on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. Materials and Methods: SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Results: Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05). Conclusion: Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment. PMID:26730242

  4. Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia

    PubMed Central

    Vallés, Ana S.; Aveldaño, Marta I.; Furland, Natalia E.

    2014-01-01

    Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC). Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days) of rat testes to 43°C led to accumulation of neutral lipids. This SC-specific lipid function took 1–2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43°C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (α-tubulin) and microfilament (f-actin) organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster. PMID:24690895

  5. Uptake and metabolism of retinol in cultured sertoli cells: evidence for a kinetic model

    SciTech Connect

    Bishop, P.D.; Griswold, M.D.

    1987-11-17

    When cultured Sertoli cells derived from 20-day-old weanling rats were supplied (/sup 3/H) retinol bound to serum retinol binding protein-transthyretin complex, (/sup 3/H) retinol was rapidly incorporated and (/sup 3/H) retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied (/sup 3/H) retinol in culture under identical conditions likewise incorporated (/sup 3/H) retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular (/sup 3/H) retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of (/sup 3/H) retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of (/sup 3/H) retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of (/sup 3/H) retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of (/sup 3/H) retinol. During the time course the specific activity of (/sup 3/H) retinyl palmitate eventually exceeded that of intracellular (/sup 3/H) retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.

  6. Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells.

    PubMed

    Sipione, Simonetta; Simmen, Katia C; Lord, Sarah J; Motyka, Bruce; Ewen, Catherine; Shostak, Irene; Rayat, Gina R; Dufour, Jannette M; Korbutt, Greg S; Rajotte, Ray V; Bleackley, R Chris

    2006-10-15

    Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection. PMID:17015688

  7. Receptors and signaling pathways involved in proliferation and differentiation of Sertoli cells

    PubMed Central

    Lucas, Thas FG; Nascimento, Aline R; Pisolato, Raisa; Pimenta, Maristela T; Lazari, Maria Fatima M; Porto, Catarina S

    2014-01-01

    The identification of the hormones and other factors regulating Sertoli cell survival, proliferation, and maturation in neonatal, peripubertal, and pubertal life remains one of the most critical questions in testicular biology. The regulation of Sertoli cell proliferation and differentiation is thought to be controlled by cellcell junctions and a set of circulating and local hormones and growth factors. In this review, we will focus on receptors and intracellular signaling pathways activated by androgen, follicle-stimulating hormone, thyroid hormone, activin, retinoids, insulin, insulin-like growth factor, relaxin, and estrogen, with special emphasis on estrogen receptors. Estrogen receptors activate intracellular signaling pathways that converge on cell cycle and transcription factors and play a role in the regulation of Sertoli cell proliferation and differentiation. PMID:25225624

  8. Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection

    PubMed Central

    Wang, Xiu-Xing; Ying, Pu; Diao, Fan; Wang, Qiang; Ye, Dan; Jiang, Chen; Shen, Ning; Xu, Na; Chen, Wei-Bo; Lai, Shan-Shan; Jiang, Shan; Miao, Xiao-Li; Feng, Jin; Tao, Wei-Wei; Zhao, Ning-Wei; Yao, Bing; Xu, Zhi-Peng; Sun, Hai-Xiang; Sha, Jia-Hao; Huang, Xing-Xu; Shi, Qing-Hua; Tang, Hong

    2013-01-01

    Mumps commonly affects children 59 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cellonly syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-?B signaling pathways were constitutively activated in GGPPS?/? Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS?/? Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility. PMID:23825187

  9. Clusterin produced by Sertoli cells inhibits heat stress-induced apoptosis in the rat testis.

    PubMed

    Matsushita, K; Miyake, H; Chiba, K; Fujisawa, M

    2016-02-01

    The objectives of this study were to determine whether the inhibition of clusterin expression in rat Sertoli cells enhances heat stress-induced apoptosis. The scrotums of rats were immersed in a water bath of 43C for 15min. Testicular weight and germ cell number markedly decreased after the heat treatment in a time-dependent manner. In contrast, clusterin mRNA and protein expression levels were significantly up-regulated and peaked on day 21. The apoptotic index was markedly increased 1day after the heat treatment. We then purified Sertoli cells from the rat testes, and an expression vector containing siRNA targeting the clusterin gene was transiently transfected into Sertoli cells. Following exposure to heat stress at 41C for 12h, clusterin mRNA was markedly up-regulated after transfection with the control vector; however, the transfection of siRNA targeting the clusterin resulted in >70% reduction in the expression of clusterin mRNA. Furthermore, the apoptotic index in these Sertoli cells was significantly higher after the treatment with siRNA targeting the clusterin than control, and the most prominent difference was observed within 24h after the heat treatment. These results suggest that an increase in the secretion of clusterin by Sertoli cells protects the testes from heat stress-induced injury. PMID:25661013

  10. 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. PMID:22200512

  11. Experimental Sertoli cell tumors in the mouse and their progression into a mixed germ cell-sex cord proliferation.

    PubMed Central

    Paquis-Flucklinger, V.; Rassoulzadegan, M.; Michiels, J. F.

    1994-01-01

    Males of transgenic families where the large T protein of polyoma virus is expressed in the seminiferous epithelium of the testis (Sertoli and germ cells) develop bilateral testicular tumors when they become old (15 to 18 months). The histological features of these tumors revealed a neoplastic proliferation of Sertoli cell origin. Occasional isolated germ cells arrested at premeiotic stages were seen in the tumor. They did not participate in tumoral proliferation and their malignant character could not at first be established. Tumor cells injected in athymic (nu/nu) mice generated secondary tumors. In this case, a proliferative component of non-Sertoli origin was clearly evident. Its ultrastructural characteristics and the expression of genes that are transcribed in vivo in male germ cells (c-kit, LDH-X, and Hox a-4) suggest the progression of an initial, apparently pure Sertoli cell tumor into a mixed proliferation. Images Figure 1 Figure 2 Figure 3 PMID:7510454

  12. SertoliLeydig cell tumor of the ovary: A diagnostic dilemma

    PubMed Central

    Liggins, Casandra A.; Ma, Ly T.; Schlumbrecht, Matthew P.

    2015-01-01

    Background SertoliLeydig cell tumors are rare sex-cord stromal tumors of the ovary that can present with a variety of histological elements, which may complicate diagnosis and treatment. Case A 40-year-old female presenting with pelvic pain is found to have a large complex right adnexal mass and elevated alpha-fetoprotein. The mass was diagnosed as a SertoliLeydig cell tumor with heterologous elements including carcinoid and hepatoid components. She was treated with surgical resection followed by adjuvant chemotherapy and remains clear of disease. Conclusion Prognostic indicators for SertoliLeydig cell tumors include degree and type of heterologous element differentiation. Thorough characterization of such elements is crucial for adequate diagnosis and treatment.

  13. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells

    PubMed Central

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  14. Specific deletion of AMP-activated protein kinase (?1AMPK) in mouse Sertoli cells modifies germ cell quality.

    PubMed

    Bertoldo, Michael J; Guibert, Edith; Faure, Melanie; Guillou, Florian; Ram, Christelle; Nadal-Desbarats, Lydie; Foretz, Marc; Viollet, Benoit; Dupont, Jolle; Froment, Pascal

    2016-03-01

    The AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis which plays a role in fertility. Complete disruption of the AMPK catalytic subunit ?1 gene (?1AMPK KO) in male mice results in a decrease in litter size which is associated with the production of altered sperm morphology and motility. Because of the importance of Sertoli cells in the formation of germ cells, we have chosen to selectively disrupt ?1AMPK only in the Sertoli cells in mice (Sc-?1AMPK-KO mice). Specific deletion of the ?1AMPK gene in Sertoli cells resulted in a 25% reduction in male fertility associated with abnormal spermatozoa with a thin head. No clear alterations in testis morphology or modification in the number of Sertoli cells invivo were observed, but a dysregulation in energy metabolism in Sertoli cells occurred. We have reported an increase in lactate production, in lipid droplets, and a reduction in ATP production in Sc-?1AMPK-KO Sertoli cells. These perturbations were associated with lower expression of mitochondrial markers (cytochrome c and PGC1-?). In addition another metabolic sensor, the deacetylase SIRT1, had a reduction in expression which is correlated with a decline in deacetylase activity. Finally, expression and localization of junctions forming the blood-testis barrier between Sertoli cells themselves and with germ cells were deregulated in Sc-?1AMPK-KO. In conclusion, these results suggest that dysregulation of the energy sensing machinery exclusively through disruption of ?1AMPK in Sertoli cells translates to a reduction in the quality of germ cells and fertility. PMID:26772142

  15. Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice

    PubMed Central

    Jiang, Xiaohua; Zhang, Huan; Yin, Shi; Zhang, Yuanwei; Yang, Weimei; Zheng, Wei; Wang, Liu; Wang, Zheng; Bukhari, Ihtisham; Cooke, Howard J.; Iqbal, Furhan; Shi, Qinghua

    2014-01-01

    Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules. PMID:25395169

  16. Characteristics of retinol accumulation from serum retinol-binding protein by cultured sertoli cells

    SciTech Connect

    Shingleton, J.L.; Skinner, M.K.; Ong, D.E. )

    1989-12-12

    The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoil cells accumulated ({sup 3}H)retinol in a time- and temperature-dependent manner. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/{mu}g of cellular DNA. Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with ({sup 3}H)retinol-RBP for ({sup 3}H)retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free ({sup 3}H)retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 {mu}M. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. Competition studies indicated, however, that protein recognition is important in the retinol uptake process. RBP, CRBP, and CRBP(II) competed with ({sup 3}H)retinol-RBP for ({sup 3}H)retinol accumulation, but free retinol, retinol-bovine serum albumin, and retinol-{beta}-lactoglobulin did not. These studies indicated that Sertoli cell uptake of retinol involved recognition of the retinol-RBP complex at the cell surface with subsequent internalization of retinol, but not RBP.

  17. Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

    PubMed

    Wang, Lingzhi; Peng, Jianxin; Huang, Huansen; Wang, Qin; Yu, Meiling; Tao, Liang

    2015-10-01

    Cisplatin, an important chemotherapeutic agent against testicular germ cell cancer, induces testicular toxicity on Leydig and Sertoli cells, leading to serious side-effects such as azoospermia and infertility. In a previous study, it was found that simvastatin enhanced the sensitivity of Leydig tumor cells to chemotherapeutic toxicity through the enhancement of gap junction functions. In the present study, the effect of simvastatin on the sensitivity of normal Sertoli cells to cisplatin and the role of gap junctions in such effects was investigated. The results showed that, simvastatin attenuated cisplatin toxicity only when cells exhibited high-density culture where gap junctional formation was possible. When gap junction function was decreased by the gap junction inhibitor or by siRNA targeting connexin 43, the protective effect of simvastatin to cisplatin toxicity was substantially attenuated. Simvastatin also enhanced gap junction functions between Sertoli cells. This effect was mediated by the reduction of PKC-mediated connexin phosphorylation, thereby increasing connexin 43 membrane localization. Thus, simvastatin-induced enhancement of gap junction?mediated intercellular communication attenuated cisplatin toxicity on Sertoli cells. This result indicated that enhancement of gap junction function by simvastatin may have bilateral beneficial effects on cisplatin?based chemotherapy, enhancing cisplatin killing on cancer while ameliorating the reproduction toxicity. PMID:26260290

  18. Sertoli cell proliferation in the adult testis is induced by unilateral gonadectomy in African catfish.

    PubMed

    Schulz, Rdiger W; van Dijk, Wytske; Chaves-Pozo, Elena; Garca-Lpez, Angel; de Frana, Luiz R; Bogerd, Jan

    2012-05-15

    Survival and development of male germ cells depends on their close contact with Sertoli cells. In the cystic spermatogenesis found in fish, one germ cell clone, initially a single undifferentiated spermatogonium type A, is enclosed by and accompanied through spermatogenesis by a group of Sertoli cells. Previous work showed that after forming such spermatogenic cysts, Sertoli cells proliferated mainly during the mitotic expansion of the spermatogonial clone in the cyst. Here, we used unilateral gonadectomy (ULG) as experimental model to study Sertoli cell proliferation at the start of cyst development in adult African catfish testis. Four days after surgery, we observed a particularly strong increase in the number of mitotic Sertoli cells along with a significant increase in the number of mitotic single type A spermatogonia. Proliferation of pairs of spermatogonia or of larger germ cell clones, however, did not change. At the same time, pituitary transcript levels of the three gonadotropin-subunits (cga, glycoprotein hormones, alpha polypeptide; fshb, follicle stimulating hormone, beta polypeptide; lhb, luteinizing hormone, beta polypeptide) were not different between sham-operated and ULG males. However, expression of the gonadotropin-releasing hormone receptor gene gnrhr1 was significantly reduced after ULG, and Lh plasma levels were slightly elevated. In the testis remaining after ULG, Fsh receptor (fshr) mRNA levels increased significantly but luteinizing hormone/choriogonadotropin receptor (lhcgr) mRNA levels did not change. Circulating androgen levels did not differ between groups, but testicular androgen release increased significantly 2- to 3-fold after ULG. Considering the strong steroidogenic potency of Fsh and the expression of the fshr gene by Leydig cells in catfish, we explain the absence of an effect of ULG on circulating androgen levels by an Fshr-mediated, compensatory increase in the steroid production of the remaining testis, perhaps supported in addition by the increased Lh plasma levels. Since Fsh is a major stimulator of mammalian Sertoli cell proliferation, we propose that ULG-induced activation of the Fsh signalling system also promoted Sertoli cell proliferation and - possibly as a consequence of that - proliferation of single type A spermatogonia, providing the basis for an increased spermatogenic capacity. PMID:22465554

  19. Androgen-dependent sertoli cell tight junction remodeling is mediated by multiple tight junction components.

    PubMed

    Chakraborty, Papia; William Buaas, F; Sharma, Manju; Smith, Benjamin E; Greenlee, Anne R; Eacker, Stephen M; Braun, Robert E

    2014-07-01

    Sertoli cell tight junctions (SCTJs) of the seminiferous epithelium create a specialized microenvironment in the testis to aid differentiation of spermatocytes and spermatids from spermatogonial stem cells. SCTJs must be chronically broken and rebuilt with high fidelity to allow the transmigration of preleptotene spermatocytes from the basal to adluminal epithelial compartment. Impairment of androgen signaling in Sertoli cells perturbs SCTJ remodeling. Claudin (CLDN) 3, a tight junction component under androgen regulation, localizes to newly forming SCTJs and is absent in Sertoli cell androgen receptor knockout (SCARKO) mice. We show here that Cldn3-null mice do not phenocopy SCARKO mice: Cldn3(-/-) mice are fertile, show uninterrupted spermatogenesis, and exhibit fully functional SCTJs based on imaging and small molecule tracer analyses, suggesting that other androgen-regulated genes must contribute to the SCARKO phenotype. To further investigate the SCTJ phenotype observed in SCARKO mutants, we generated a new SCARKO model and extensively analyzed the expression of other tight junction components. In addition to Cldn3, we identified altered expression of several other SCTJ molecules, including down-regulation of Cldn13 and a noncanonical tight junction protein 2 isoform (Tjp2iso3). Chromatin immunoprecipitation was used to demonstrate direct androgen receptor binding to regions of these target genes. Furthermore, we demonstrated that CLDN13 is a constituent of SCTJs and that TJP2iso3 colocalizes with tricellulin, a constituent of tricellular junctions, underscoring the importance of androgen signaling in the regulation of both bicellular and tricellular Sertoli cell tight junctions. PMID:24825397

  20. Loss of Dicer in Sertoli Cells Has a Major Impact on the Testicular Proteome of Mice

    PubMed Central

    Papaioannou, Marilena D.; Lagarrigue, Mlanie; Vejnar, Charles E.; Rolland, Antoine D.; Khne, Franoise; Aubry, Florence; Schaad, Olivier; Fort, Alexandre; Descombes, Patrick; Neerman-Arbez, Marguerite; Guillou, Florian; Zdobnov, Evgeny M.; Pineau, Charles; Nef, Serge

    2011-01-01

    Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3?UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control. PMID:20467044

  1. Basement membrane increases G-protein levels and follicle-stimulating hormone responsiveness of Sertoli cell adenylyl cyclase activity.

    PubMed

    Dym, M; Lamsam-Casalotti, S; Jia, M C; Kleinman, H K; Papadopoulos, V

    1991-02-01

    On a basement membrane substrate, Sertoli cells in culture have been shown to assume a phenotype similar to that of the in vivo differentiated cells. Sertoli cells from 10-day-old rats were cultured on plastic and on different extracellular matrix substrates [laminin, a reconstituted basement membrane (Matrigel), and a synthetic laminin peptide containing the arginine-glycine-aspartic acid (RGD) tripeptide sequence] to investigate the effects of the extracellular matrix on FSH responsiveness. Both laminin and Matrigel markedly enhanced the cAMP response to FSH and cholera toxin, indicating modifications at the level of guanine nucleotide-binding regulatory (G) proteins. Furthermore, Sertoli cell grown on either of these two substrates responded to physiological levels of FSH (25-50 ng/ml), whereas pharmacological levels of FSH (500 ng/ml) were required for cells grown on either plastic or on the RGD-containing laminin peptide. Immunoblotting of Sertoli cell plasma membranes with antibodies directed against the alpha-subunit of the stimulatory G-protein (Gs alpha) of adenylyl cyclase indicated that Sertoli cell culture on either laminin or Matrigel increased the amounts of Gs alpha. These results were further confirmed by immunoprecipitating the Gs alpha protein from the particulate fraction of [35S]methionine metabolically labeled Sertoli cells. However, Northern blot analysis using a cDNA probe for Gs alpha did not demonstrate changes in gene expression when Sertoli cells were grown on the various substrates. Immunofluorescent studies revealed that the Gs complex of adenylyl cyclase was preferentially located at the base of the Sertoli cells at the site of contact with the extracellular matrix. These data suggest that culture of epithelial Sertoli cells on basement membrane substrates enhances the Gs complex of adenylyl cyclase and the cAMP response to FSH, consistent with the more differentiated morphology and function of the cells. PMID:1846579

  2. Management of an invasive and metastatic Sertoli cell tumor with associated myelotoxicosis in a dog.

    PubMed

    Withers, Sita S; Lawson, Corinne M; Burton, Andrew G; Rebhun, Robert B; Steffey, Michele A

    2016-03-01

    We describe the surgical and post-operative management of a large, invasive, and metastatic functional Sertoli cell tumor in a 9-year-old cryptorchid male Labrador retriever dog. Despite residual disease after surgery, bone marrow recovery occurred without administration of bone marrow stimulants and serum estradiol accurately predicted tumor recurrence. PMID:26933269

  3. MICRODISSECTION TESTICULAR SPERM EXTRACTION IN MEN WITH SERTOLI CELL ONLY TESTICULAR HISTOLOGY

    PubMed Central

    Berookhim, Boback M.; Palermo, Gianpiero D.; Zaninovic, Nikica; Rosenwaks, Zev; Schlegel, Peter N.

    2015-01-01

    Objective To study the outcomes of microdissection testicular sperm extraction (microTESE) among men with pure Sertoli cell only histology on diagnostic testicular biopsy. Design Retrospective cohort study. Setting Tertiary referral center. Patients 640 patients with pure Sertoli cell only histology on testicular biopsy who underwent microTESE by a single surgeon. Intervention MicroTESE. Main Outcome Measure Sperm retrieval rates. Results Overall, 44.5% of patients with Sertoli cell-only had sperm retrieved with microTESE. No difference was noted in sperm retrieval rates based on testis volume (? 15cc versus <15cc, 35.3% versus 46.1%, respectively). Patients with ? 15cc testicular volume and FSH 10-15 mU/mL had the worst prognosis, with a sperm retrieval rate of 6.7%. Conclusions Patients with previous testicular biopsy demonstrating Sertoli cell only histology can be counseled that they have a reasonable likelihood of sperm retrieval with the contemporary delivery of microTESE. Given this finding, the utility of testicular biopsy prior to microTESE is further questioned. PMID:25441063

  4. Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells

    SciTech Connect

    Otsuka, Atsushi; Abe, Tadashi; Watanabe, Masami; Yagisawa, Hitoshi; Takei, Kohji; Yamada, Hiroshi

    2009-01-16

    Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

  5. Zearalenone impairs the male reproductive system functions via inducing structural and functional alterations of sertoli cells.

    PubMed

    Zheng, WangLong; Pan, ShunYe; Wang, Guangguang; Wang, Ya Jun; Liu, Qing; Gu, JianHong; Yuan, Yan; Liu, Xue Zhong; Liu, Zong Ping; Bian, Jian Chun

    2016-03-01

    The aim of this study was to investigate the effects of ZEA on the cytoskeletal structure, and factors specifically expressed by Sertoli cells. Primary Sertoli cells from rats aged 18-21 days were exposed to increasing ZEA concentrations (0, 5, 10, 20μgmL(-1)) for 24h. The results of immunofluorescence showed disruption of α-tubulin filaments and F-actin bundles, and damage to the nucleus of Sertoli cells on exposure to ZEA. In the control group, the protein level expression of androgen-binding protein (ABP), transferrin, vimentin, N-cadherin, and follicle-stimulating hormone receptor (FSHR) were decreased significantly (p<0.05, p<0.01). The mRNA levels of ABP, transferrin, vimentin, N-cadherin, and FSHR varied significantly in the experimental group (p<0.05). The results of enzyme-linked immunosorbent assay indicated a significant decrease in the levels of inhibin-β and transferrin in the cultural supernatants (p<0.05). Additionally, the ultrastructural analysis indicated the absence of mitochondria and Golgi apparatus, and presence of vacuoles in the cytoplasm. These findings showed that ZEA treatment can damage the cytoskeletal structure and affect specific secretory functions of Sertoli cells, which may be an underlying cause of ZEA-induced reproductive toxicity. PMID:26851377

  6. Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response.

    PubMed Central

    Raychoudhury, Samir S; Kubinski, Dana

    2003-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contaminants. Some PAHs are carcinogens and may affect the male reproductive system. Therefore, we exposed cultured rat Sertoli cells to a variety of PAHs to determine possible direct toxic effects on the cells of the seminiferous epithelium. Sertoli cells were chosen because they support germ cell development and maintain spermatogenesis. Sertoli cells were isolated from 19-21-day-old male rats and cultured in medium containing 0.08% dimethylsulfoxide as vehicle or in the presence of a variety of PAHs. In the first set of experiments, cultured Sertoli cells were incubated in the presence of 10(-4) M, 10(-6 )M, 10(-8) M, 10(-12) M, and 10(-16) M fluoranthene (FL) for 24 hr. After 24 hr, FL at 10(4), 10(-6), and 10(-8) M killed significant numbers of Sertoli cells as revealed by cell viability determinations. Sertoli cells cultured in the presence of 10(-6) M and 10(-8) M FL showed morphologic changes. Cell protein levels were decreased and lactate production in the medium increased in a concentration-dependent manner. In addition, Sertoli cells exposed to 10(6) M and 10(-8) M FL exhibited altered F-actin and alpha-tubulin distributions compared with untreated controls. Because FL killed about 62% of cells at 10(-4) M (100 micro g/mL) and 48% of cells at 10(-6) M (1 micro g/mL), increased lactate production about 3-fold at both concentrations, and decreased cell protein by half at 10(-4) M (100 micro g/mL), we decided to use a range of concentrations between 10 and 100 micro g/mL for the second set of experiments using benz[a]anthracene (BaA), benzo[a]pyrene (BaP), or benzo[b]fluoranthene (BbF). After 24 hr, BaA (100 micro g/mL), BaP (50 and 100 micro g/mL), and BbF (100 micro g/mL) significantly increased lactate level in the medium in a concentration-dependent manner. In a third set of experiments, cells were treated in culture uniformly with only 10 micro g/mL FL, BaA, BaP, or BbF for 24 hr. The cytotoxic effects exerted by these PAHs tested resulted in different apoptotic responses as characterized by in situ fluorescence staining. Microscopic analysis of apoptotic cells demonstrated nuclei of reduced size and labeled 3 -OH DNA ends when Sertoli cells had been incubated for 24 hr with 10 micro g BaP or BbF, but not with vehicle, media, FL, or BaA. Thus, our results demonstrate that the toxic effects of BaA and BbF on Sertoli cells are exerted through apoptosis, whereas FL and BaA do not elicit the apoptotic response. PMID:12515676

  7. AB46. Screening and identification for the target genes of androgen receptor in mouse Sertoli cells

    PubMed Central

    Gui, Yaoting; Mou, Lisha; Zhang, Qiaoxia; Yang, Lihua; Wang, Yadong; Cai, Zhiming

    2014-01-01

    Androgen and androgen receptor (AR) play important roles in spermatogenesis, yet detailed androgen/AR signals in Sertoli cells remain unclear. To identify AR target genes in Sertoli cells, we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR) and their littermate wild-type (WT) mice. Digital gene expression analysis identified 2,276 genes downregulated and 2,865 genes upregulated in the S-AR mice testis compared to WT ones. To further nail down the difference within Sertoli cells, we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells. Interestingly, additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing ten times more androgen sensitivity than TM4 cells. In the condition where maximal androgen response was demonstrated, we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone. Among these genes, 603 androgen-/AR-regulated genes, including 164 up-regulated and 439 down-regulated, were found in both S-AR mice testis and TM4/AR cells. Ubiquitin-conjugating enzyme E2B (Ube2b) is one of the regulated genes from the digital gene expression analysis. The expression of UBE2B was decreased in the testes of the S-AR mice analyzed by quantitative RT-PCR (qRT-PCR) and immunofluorescence. The up-regulation of Ube2b gene by testosterone was further demonstrated by Western blot and qRT-PCR in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay validated that the ligand-bound AR activated Ube2b transcription via directly binding to the androgen-responsive element of the Ube2b promoter. In vitro analyses showed that testosterone increased UBE2B expression and activated H2A ubiquitylation, while downregulation of UBE2B blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly down-regulated and 71 were up-regulated by UBE2B in TM4 cells. These results suggest that Ube2b, as a direct AR transcriptional target in Sertoli cells, mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation. Our previous digital gene expression analysis data also showed that heat shock transcription factors 1 (HSF1) was regulated by androgen in mouse Sertoli cells. We found that the expression of Hsf1 was increased in the testes of S-AR mice compared with wild-type mice by quantitative real-time PCR and the expression of HSF1 in the S-ARSertoli cells was significantly increased as examined by immunofluorescence analysis. Besides, in vitro study showed that testosterone repressed the expression of Hsf1 in TM4 cells. Moreover, luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to Hsf1 promoter. Our experiment also demonstrated that testosterone downregulated the expression of heat shock protein HSP105 and HSP60 by inhibiting the transcription of Hsf1. Taken together, these results reveal that HSF1 is a novel target of androgen receptor in mouse Sertoli Cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting HSF1 expression.

  8. Bilateral retiform variant of sertoli leydig cell tumour of ovary: An uncommon tumor with review of literature

    PubMed Central

    Rathi, Monika; Budania, Satish Kumar; Khalid, Mohammad; Mittal, Ankur

    2015-01-01

    Sertoli-leydig cell tumors are the uncommon sex-cord stromal tumors of the ovary. We report a case of 42-year-old female with retiform variant of sertoli-leydig cell tumour. She presented with the complaint of mass in abdomen for 7 years. Ultrasound revealed bilateral ovarian mass suggestive of malignancy. Bilateral salpingo-oopherectomy with surgical staging was done. The tumor was diagnosed as stage I retiform variant of sertoli-leydig cell tumor on histopathology and immunohistochemistry. PMID:25861207

  9. Condensation behavior of the human x chromosome in male germ cells and Sertoli cells examined by flourescence in situ hybridisation

    SciTech Connect

    Kofman-Alfaro, S.; Cervantes, A.; Speed, R.M.

    1994-09-01

    The chromatin condensation behavior of the human x chromosome has been studied by FISH analysis in germ cells and Sertoli cells of the adult testes. Comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this state of meiosis, could be a prerequisite for XY pairing crossing-over. In patients with {open_quotes}Sertoli-cell-only{close_quotes} syndrome, the sex chromosomes, by in situ hybridization analysis, appear extremely contracted compared with their normal extended state seen in adult Sertoli cells of fertile men. By contrast, the state of expansion of chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to pattern of expression noted for sex-linked genes in the human testis.

  10. Age-dependent changes in the in-vitro response of a pig Sertoli cell-enriched population to FSH.

    PubMed

    Monet-Kuntz, C; Fontaine, I

    1989-07-01

    The response of pig Sertoli cell-enriched cultures to FSH was investigated by measuring plasminogen activator (PA) secretion in culture, throughout the nonpubertal and prepubertal periods. Sertoli cell-enriched populations could be isolated from birth until a testicular weight of 56 g. FSH elicited a dose-dependent increase in PA secretion by pig Sertoli cell-enriched cultures. The ED50 was minimal for cells coming from testes weighing 10-22 g, and increased more than 2-fold for cells from heavier testes. This suggests that, at the end of the non-pubertal period, an increased FSH sensitivity is important for initiation of spermatogenesis in this species, and that during the prepubertal period Sertoli cells become less sensitive to FSH. The FSH-stimulated PA secretion increased about 10-fold from a testicular weight of 25 g onwards, i.e. when primary spermatocytes appear in seminiferous tubules. PMID:2503609

  11. Environmentally induced epigenetic transgenerational inheritance of altered Sertoli cell transcriptome and epigenome: molecular etiology of male infertility.

    PubMed

    Guerrero-Bosagna, Carlos; Savenkova, Marina; Haque, Md Muksitul; Nilsson, Eric; Skinner, Michael K

    2013-01-01

    Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. PMID:23555832

  12. Sertoli-germ cell junctions in the testis: a review of recent data.

    PubMed

    Kopera, Ilona A; Bilinska, Barbara; Cheng, C Yan; Mruk, Dolores D

    2010-05-27

    Spermatogenesis is a process that involves an array of cellular and biochemical events, collectively culminating in the formation of haploid spermatids from diploid precursor cells known as spermatogonia. As germ cells differentiate from spermatogonia into elongated spermatids, they also progressively migrate across the entire length of the seminiferous epithelium until they reach the luminal edge in anticipation of spermiation at late stage VIII of spermatogenesis. At the same time, these germ cells must maintain stable attachment with Sertoli cells via testis-unique intermediate filament- (i.e. desmosome-like junctions) and actin- (i.e. ectoplasmic specializations, ESs) based cell junctions to prevent sloughing of immature germ cells from the seminiferous epithelium, which may result in infertility. In essence, both desmosome-like junctions and basal ESs are known to coexist between Sertoli cells at the level of the blood-testis barrier where they cofunction with the well-studied tight junction in maintaining the immunological barrier. However, the type of anchoring device that is present between Sertoli and germ cells depends on the developmental stage of the germ cell, i.e. desmosome-like junctions are present between Sertoli and germ cells up to, but not including, step 8 spermatids after which this junction type is replaced by the apical ES. While little is known about the biology of the desmosome-like junction in the testis, we have a relatively good understanding of the molecular architecture and the regulation of the ES. Here, we discuss recent findings relating to these two junction types in the testis, highlighting prospective areas that should be investigated in future studies. PMID:20403872

  13. A spontaneously occurring malignant ovarian Sertoli cell tumor in a young Sprague Dawley rat

    PubMed Central

    Kinoshita, Yuichi; Yoshizawa, Katsuhiko; Emoto, Yuko; Yuki, Michiko; Yuri, Takashi; Shikata, Nobuaki; Elmore, Susan A.; Tsubura, Airo

    2015-01-01

    Primary ovarian tumors are generally uncommon in rats used in toxicologic studies. A malignant Sertoli cell tumor was present in the ovary of a 19-week-old female Sprague Dawley rat. Macroscopically, the mass was white and firm, 10 × 13 × 17 mm in size, and located in the right ovary. Histopathologically, the mass was composed of nests of pleomorphic cells, which formed seminiferous-like tubules separated by a thin fibrovascular stroma. The tubules were lined by tumor cells, which had basally located nuclei and abundant eosinophilic and vacuolated cytoplasm. In some areas, the tumor cells were arranged in a retiform growth pattern, mimicking a rete testis/ovarii. Disseminated metastases to the surfaces of the mesentery, spleen and liver were also present. Immunohistochemically, many tumor cells were strongly positive for vimentin, estrogen receptor α and Ki 67. Some tumor cells were positive for pancytokeratin and inhibin α. These findings closely resemble those of an ovarian-derived human malignant Sertoli cell tumor. From our review of the literature, we believe this is the first report of a spontaneous malignant Sertoli cell tumor in the ovary of a young laboratory rat. This case might provide useful historical control information for rat toxicity studies. PMID:26989303

  14. Aromatase gene expression in immature rat Sertoli cells: age-related changes in the FSH signalling pathway.

    PubMed

    Bourama-Lelong, Hlne; Vanneste, Marion; Delalande, Christelle; Zanatta, Lela; Wolczynski, Slawomir; Carreau, Serge

    2010-01-01

    Aromatase, the enzyme responsible for the transformation of androgens into oestrogens, is encoded by the cyp19 gene expressed in the testis. The aim of the present study was to analyse the evolution of aromatase gene expression under FSH control in rat Sertoli cells between 10 and 30 days post partum, corresponding to the end of the proliferative period of Sertoli cells, establishment of the blood-testis barrier and acquisition of the mature phenotype. The maximum stimulatory effect of FSH on aromatase gene expression was obtained in 20-day-old rat Sertoli cells, compared with cells from 10- and 30-day-old rats, in parallel with the differentiation of Sertoli cells. Using two effectors of the protein kinase A pathway (i.e. forskolin and dibutyryl-cAMP) revealed differential effects between cells from rats aged 20 and 30 days, implying the involvement of another signalling pathway. Experiments using the specific phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 revealed that PI3-K is strongly involved in FSH-induced aromatase expression in Sertoli cells from both 20- and 30-day-old rats. In vivo, this decrease could be explained by a negative effect exerted by germ cells because, in coculture, aromatase gene expression in 20-day-old Sertoli cells is greatly diminished. PMID:20188023

  15. Large moderately-differentiated ovarian Sertoli-Leydig cell tumor in a 13-year-old female: A case report

    PubMed Central

    ZHANG, HUI; HAO, JING; LI, CHUN-YAN; LI, TAO; MU, YU-LAN

    2016-01-01

    Sertoli-Leydig cell tumor of the ovary, also known as androblastoma, is a rare neoplasm from the group of sex cord-stromal tumors of the ovary. The tumor accounts for <0.5% of all primary ovarian neoplasms. The clinical signs and symptoms of Sertoli-Leydig cell tumors can be associated with either hormonal production or the presence of a mass-occupying lesion. In the current study, a 13-year-old female was diagnosed with a stage Ic ovarian Sertoli-Leydig cell tumor following abdominal pain and distension. One month after a right oophorectomy, the follow-up magnetic resonance imaging scan was negative for residual or recurrent tumor. The overall 5-year survival rate for moderately-differentiated (grade 2) and poorly-differentiated (grade 3) Sertoli-Leydig cell tumors is 80%, and long-term follow-up is therefore highly advised in this patient.

  16. Case Report of a Man With Sertoli Cell Only, Transverse Testicular Ectopia, and External Auditory Canal Atresia

    PubMed Central

    zdemir, Enver; Koan, Hseyin; Yaz?c?, Mehmet; Alt?ok, Erin; Su, Fuat E.; Gne?, Mehmet N.

    2014-01-01

    Transverse testicular ectopia (TTE) is an uncommon congenital anomaly, reported mostly as pediatric case reports. Herewith we report a 43-year old man presenting with sertoli cell only, Transverse testicular ectopia and external auditory canal atresia.

  17. Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane.

    PubMed

    Guillou, F; Monet-Kuntz, C; Fontaine, I; Flechon, J E

    1990-05-01

    The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system, qualitative changes in the expression of intermediate filament proteins. PMID:1694107

  18. Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells.

    PubMed

    Wu, Han; Shi, Lili; Wang, Qing; Cheng, Lijing; Zhao, Xiang; Chen, Qiaoyuan; Jiang, Qian; Feng, Min; Li, Qihan; Han, Daishu

    2016-01-01

    Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-?, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-? and IFN-?). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis. PMID:26776505

  19. Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells

    PubMed Central

    Wu, Han; Shi, Lili; Wang, Qing; Cheng, Lijing; Zhao, Xiang; Chen, Qiaoyuan; Jiang, Qian; Feng, Min; Li, Qihan; Han, Daishu

    2016-01-01

    Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis. PMID:26776505

  20. Prenatal and lactation nicotine exposure affects Sertoli cell and gonadotropin levels in rats.

    PubMed

    Paccola, C C; Miraglia, S M

    2016-02-01

    Nicotine is largely consumed in the world as a component of cigarettes. It can cross the placenta and reach the milk of smoking mothers. This drug induces apoptosis, affects sex hormone secretion, and leads to male infertility. To investigate the exposure to nicotine during the whole intrauterine and lactation phases in Sertoli cells, pregnant rats received nicotine (2 mg/kg per day) through osmotic minipumps. Male offsprings (30, 60, and 90 days old) had blood collected for hormonal analysis (FSH and LH) and their testes submitted for histophatological study, analysis of the frequency of the stages of seminiferous epithelium cycle, immunolabeling of apoptotic epithelial cells (TUNEL and Fas/FasL), analysis of the function and structure of Sertoli cells (respectively using transferrin and vimentin immunolabeling), and analysis of Sertoli-germ cell junctional molecule (β-catenin immunolabeling). The exposure to nicotine increased the FSH and LH plasmatic levels in adult rats. Although nicotine had not changed the number of apoptotic cells, neither in Fas nor FasL expression, it provoked an intense sloughing of epithelial cells and also altered the frequency of some stages of the seminiferous epithelium cycle. Transferrin and β-catenin expressions were not changed, but vimentin was significantly reduced in the early stages of the seminiferous cycle of the nicotine-exposed adult rats. Thus, we concluded that nicotine exposure during all gestational and lactation periods affects the structure of Sertoli cells by events causing intense germ cell sloughing observed in the tubular lumen and can compromise the fertility of the offspring. PMID:26556892

  1. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors

    PubMed Central

    Sheng, Chao; Zheng, Qinyuan; Wu, Jianyu; Xu, Zhen; Wang, Libin; Li, Wei; Zhang, Haijiang; Zhao, Xiao-Yang; Liu, Lei; Wang, Ziwei; Guo, Changlong; Wu, Hua-Jun; Liu, Zhonghua; Wang, Liu; He, Shigang; Wang, Xiu-Jie; Chen, Zhiguo; Zhou, Qi

    2012-01-01

    Multipotent neural stem/progenitor cells hold great promise for cell therapy. The reprogramming of fibroblasts to induced pluripotent stem cells as well as mature neurons suggests a possibility to convert a terminally differentiated somatic cell into a multipotent state without first establishing pluripotency. Here, we demonstrate that Sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties. The induced neural stem/progenitor cells (iNSCs) express multiple NSC-specific markers, exhibit a global gene-expression profile similar to normal NSCs, and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons. iNSC-derived neurons stain positive for tyrosine hydroxylase (TH), ?-aminobutyric acid, and choline acetyltransferase. In addition, iNSCs can survive and generate synapses following transplantation into the dentate gyrus. Generation of iNSCs may have important implications for disease modeling and regenerative medicine. PMID:22064700

  2. Hormonal regulation of cytochrome oxidase subunit messenger RNAs in rat Sertoli cells.

    PubMed

    Ku, C Y; Lu, Q; Ussuf, K K; Weinstock, G M; Sanborn, B M

    1991-11-01

    Using differential hybridization to screen a rat Sertoli cell cDNA library for hormonally regulated gene products, we isolated a clone, designated 13-10, which contained a 1.0-kilobase insert and hybridized to a 1.7-kilobase message in total testis, Sertoli cells, and peritubular cells. This mRNA was decreased relative to untreated control levels in total testicular RNA from hypophysectomized rats, but was increased by FSH treatment begun on the day of hypophysectomy. FSH caused a transient rise in 13-10 mRNA at 24 h in cultured Sertoli cells. There was no comparable rise in beta-actin RNA or the RNA/DNA ratio at this time, suggesting that the effect on 13-10 was specific. Testosterone had no effect at any time interval studied. The 13-10 mRNA was not increased in peritubular cells treated in vitro with FSH or testosterone. Sequence analysis of 13-10 revealed more than 99% homology with a portion of the sequence of rat liver cytochrome oxidase subunit I (COX I). The clone included 58% of the open reading frame of COX I as well as that for the adjacent Ser-tRNA. COX I is a mitochondrial gene, and Southern analysis confirmed 13-10 sequence in testicular mitochondrial DNA. In addition to FSH, forskolin and (Bu)2cAMP also increased COX I steady state mRNA in Sertoli cells (3.8-, 4.1-, and 9.2-fold, respectively). (Bu)2cAMP increased mRNA for other mitochondrial gene products, COX subunit II and 16S rRNA (6.9- and 5.4-fold, respectively), whereas the smaller effects elicited by forskolin and FSH were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1664046

  3. Role of Endoplasmic reticulum apoptotic pathway in testicular Sertoli cells injury induced by Carbon disulfide.

    PubMed

    Guo, Yinsheng; Ji, Jiajia; Wang, Wei; Dong, Yu; Zhang, Zhen; Zhou, Yijun; Chen, Guoyuan; Cheng, Jinquan

    2015-08-01

    The exposure of Carbon disulfide (CS2) is associated with germ cell injury and male infertility in animals and humans. However, the molecular mechanism is currently unknown. This study show here that CS2-induced Sertoli cells injury via Endoplasmic reticulum (ER) apoptotic pathway. SD male rats were exposed to doses of CS2 (0, 50, 250, 1250mgm(-3)) for 4weeks. After treatment, loose structures of seminiferous tubules and disordered cell arrangements were observed by light microscopy. Ultrastructural lesions, deformed chromatins and vacuoles formed from swollen ER were observed by electron microscopy. After primary culture of Sertoli cells, a dose-dependent increased apoptosis were found. The increased activity of Caspase 3, accumulation of intracellular Ca(2+), up-regulation of mRNA and protein expressions of ER apoptotic relative molecules (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) were also found in this study. Altogether, our findings indicated that ER apoptotic pathway played an important role in CS2-induced Sertoli cell impairment. PMID:25816788

  4. TiO2 nanoparticles-induced apoptosis of primary cultured Sertoli cells of mice.

    PubMed

    Hong, Fashui; Zhao, Xiaoyang; Chen, Ming; Zhou, Yingjun; Ze, Yuguan; Wang, Ling; Wang, Yajing; Ge, Yushuang; Zhang, Qi; Ye, Lingqun

    2016-01-01

    Titanium dioxide nanoparticles (TiO2 NPs), as largest production and use of nanomaterials, have been demonstrated to have a potential toxicity on reproductive system. However, the mechanism underlying male reproductive toxicity of TiO2 NPs remains limited. Thus, our study was designed to examine the cellular viability, apoptosis, oxidative stress, antioxidant capacity, and expression of apoptotic cytokines in primary cultured Sertoli cells isolated from mice under TiO2 NPs exposure. Results showed that TiO2 NPs exposure from 5 to 30 ?g/mL resulted in reduction of cell viability, lactate dehydrogenase release, and induction of apoptosis or death on Sertoli cells. TiO2 NPs could migrate to Sertoli cells, which induced mitochondria-mediated or endoplasmic-reticulum-mediated apoptotic changes including elevation in reactive oxygen species (ROS) generation and reductions in superoxide dismutase, catalase, and glutathione peroxidase activities, decreases in mitochondrial membrane potential (??m), and releases of cytochrome c into the cytosol. In addition, upregulation of cytochrome c, Bax, caspase-3, glucose-regulated protein 78, and C/EBP homologous protein and caspase-12 protein expression, and downregulation of bcl-2 protein expression in primary cultured Sertoli cells induced by TiO2 NPs treatment. All of the results suggested that ROS generation may play a critical role in the initiation of TiO2 NPs-induced apoptosis by mediation of the disruption of ??m, the cytochrome c release, and further the activation of caspase cascade and unfolded protein response signaling pathway. 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 124-135, 2016. PMID:26238530

  5. Androgen-Dependent Sertoli Cell Tight Junction Remodeling Is Mediated by Multiple Tight Junction Components

    PubMed Central

    Chakraborty, Papia; William Buaas, F.; Sharma, Manju; Smith, Benjamin E.; Greenlee, Anne R.; Eacker, Stephen M.

    2014-01-01

    Sertoli cell tight junctions (SCTJs) of the seminiferous epithelium create a specialized microenvironment in the testis to aid differentiation of spermatocytes and spermatids from spermatogonial stem cells. SCTJs must be chronically broken and rebuilt with high fidelity to allow the transmigration of preleptotene spermatocytes from the basal to adluminal epithelial compartment. Impairment of androgen signaling in Sertoli cells perturbs SCTJ remodeling. Claudin (CLDN) 3, a tight junction component under androgen regulation, localizes to newly forming SCTJs and is absent in Sertoli cell androgen receptor knockout (SCARKO) mice. We show here that Cldn3-null mice do not phenocopy SCARKO mice: Cldn3−/− mice are fertile, show uninterrupted spermatogenesis, and exhibit fully functional SCTJs based on imaging and small molecule tracer analyses, suggesting that other androgen-regulated genes must contribute to the SCARKO phenotype. To further investigate the SCTJ phenotype observed in SCARKO mutants, we generated a new SCARKO model and extensively analyzed the expression of other tight junction components. In addition to Cldn3, we identified altered expression of several other SCTJ molecules, including down-regulation of Cldn13 and a noncanonical tight junction protein 2 isoform (Tjp2iso3). Chromatin immunoprecipitation was used to demonstrate direct androgen receptor binding to regions of these target genes. Furthermore, we demonstrated that CLDN13 is a constituent of SCTJs and that TJP2iso3 colocalizes with tricellulin, a constituent of tricellular junctions, underscoring the importance of androgen signaling in the regulation of both bicellular and tricellular Sertoli cell tight junctions. PMID:24825397

  6. AB081. Study on the secretory function of Sertoli cells and testicular damage in diabetic rats

    PubMed Central

    Liu, Baoxing; Zhang, Xiuping; Qin, Mao; Song, Aiping; Cui, Tianwei

    2015-01-01

    Objective To investigate the changes of secretory function of Sertoli cells and testicular damage in diabetic rats. Methods Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 55 mg/kg/i.p.) in adult rats. Eight weeks later testicular tissue was harvested; serum INHB and testosterone (T) levels were measured by ELISA. The secretory proteins such as ABP, TF were measured by western blot. Testicular damage was detected by using hematoxylin and eosin (HE) staining and apoptosis was identified by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL). Seminiferous tubular sperm formation was evaluated using Johnsens score. Results In diabetic rats, ABP protein levels, serum INHB and T levels were found to be decreased greatly. Meanwhile, TF protein levels decreased without significantly. Additionally, TUNEL-positive cells were increased and Johnsens score was decreased in diabetic testes. Conclusions These results suggest that the secretory function of Sertoli cell in diabetic rats was damaged. The altered secretory function on Sertoli cell may be responsible for the disturbed spermatogenesis.

  7. Identification of the Functions of Liver X Receptor-? in Sertoli Cells Using a Targeted Expression-Rescue Model.

    PubMed

    Maqdasy, Salwan; El Hajjaji, Fatim-Zohra; Baptissart, Marine; Viennois, Emilie; Oumeddour, Abdelkader; Brugnon, Florence; Trousson, Amalia; Tauveron, Igor; Volle, David; Lobaccaro, Jean-Marc A; Baron, Silvre

    2015-12-01

    Liver X receptors (LXRs) are key regulators of lipid homeostasis and are involved in multiple testicular functions. The Lxr?(-/-);Lxr?(-/-) mice have illuminated the roles of both isoforms in maintenance of the epithelium in the seminiferous tubules, spermatogenesis, and T production. The requirement for LXR? in Sertoli cells have been emphasized by early abnormal cholesteryl ester accumulation in the Lxr?(-/-) and Lxr?(-/-);Lxr?(-/-) mice. Other phenotypes, such as germ cell loss and hypogonadism, occur later in life in the Lxr?(-/-);Lxr?(-/-) mice. Thus, LXR? expression in Sertoli cells seems to be essential for normal testicular physiology. To decipher the roles of LXR? within the Sertoli cells, we generated Lxr?(-/-);Lxr?(-/-):AMH-Lxr? transgenic mice, which reexpress Lxr? in Sertoli cells in the context of Lxr?(-/-);Lxr?(-/-) mice. In addition to lipid homeostasis, LXR? is necessary for maintaining the blood-testis barrier and the integrity of the germ cell epithelium. LXR? is also implicated in the paracrine action of Sertoli cells on Leydig cells to modulate T synthesis. The Lxr?(-/-);Lxr?(-/-) and Lxr?(-/-);Lxr?(-/-):AMH-Lxr? mice exhibit lipid accumulation in germ cells after the Abcg8 down-regulation, suggesting an intricate LXR?-dependent cooperation between the Sertoli cells and germ cells to ensure spermiogenesis. Further analysis revealed also peritubular smooth muscle defects (abnormal lipid accumulation and disorganized smooth muscle actin) and spermatozoa stagnation in the seminiferous tubules. Together the present work elucidates specific roles of LXR? in Sertoli cell physiology in vivo beyond lipid homeostasis. PMID:26402841

  8. miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol

    PubMed Central

    2011-01-01

    Background It is known that some environmental chemicals affect the human endocrine system. The harmful effects of endocrine disrupting chemical (EDC) nonylphenol (NP) have been studied since the 1980s. It is known that NP adversely affects physiological functions by mimicking the natural hormone 17 beta-estradiol. In the present study, we analyzed the expression of miRNAs and their target genes in mouse Sertoli TM4 cells to better understand the regulatory roles of miRNAs on Sertoli cells after NP exposure. Methods Mouse TM4 Sertoli cells were treated with NP for 3 or 24 h, and global gene and miRNA expression were analyzed using Agilent mouse whole genome and mouse miRNA v13 arrays. Results We identified genes that were > 2-fold differentially expressed in NP-treated cells and control cells (P < 0.05) and analyzed their functions through Gene Ontology analysis. We also identified miRNAs that were differentially expressed in NP-treated and control cells. Of the 186 miRNAs the expression of which differed between NP-treated and control cells, 59 and 147 miRNAs exhibited 1.3-fold increased or decreased expression at 3 and 24 h, respectively. Network analysis of deregulated miRNAs suggested that Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNA in NP-treated TM4 cells. Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. Finally, miR-135a* target gene analysis suggests that the generation of reactive oxygen species (ROS) following exposure to NP exposure may be mediated by miR-135a* through regulation of the Wnt/beta-catenin signaling pathway. Conclusions Collectively, these data help to determine NP's actions on mouse TM4 Sertoli cells and increase our understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system. PMID:21914226

  9. The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus).

    PubMed

    Seco-Rovira, Vicente; Beltrn-Frutos, Esther; Ferrer, Concepcin; Sez, Francisco Jos; Madrid, Juan Francisco; Pastor, Luis Miguel

    2014-05-01

    In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells. PMID:24719257

  10. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytotoxicity accompanied by oxidative stress in rat Sertoli cells: Possible role of mitochondrial fractions of Sertoli cells

    SciTech Connect

    Aly, Hamdy A.A.; Khafagy, Rasha M.

    2011-05-01

    TCDD, as an endocrine disruptor, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underlying the testicular effects of TCDD, the potential toxicity of TCDD on Sertoli cells was investigated. Furthermore, the study aims to delineate whether mitochondrial fractions of Sertoli cells are involved in mediating the testicular effects of TCDD. Adult rat Sertoli cells were incubated with (5, 10 or 15 nM) of TCDD for 6, 12 or 24 h. Cell viability, lactate and LDH leakage into media along with lipid peroxidation, ROS generation, SOD, CAT, GPx, GR, {gamma}-GT and {beta}-glucuronidase activities, GSH content and {Delta}{psi}{sub m} were measured. Superoxide anion production, COX and cardiolipin content were measured in mitochondrial fractions. Cell viability was significantly decreased while lactate and LDH leakage into media were increased. ROS generation along with lipid peroxidation was also increased. SOD, CAT, GPx, GR activities and GSH content were significantly decreased. {gamma}-GT and {beta}-glucuronidase activities were also decreased. Superoxide anion production was increased while COX activity and cardiolipin content were decreased in mitochondrial fractions. Moreover, the {Delta}{psi}{sub m} was significantly decreased as measured in Sertoli cells. In conclusion, TCDD impairs Sertoli cell functions and this effect is, at least in part, attributed to oxidative stress. We have also found that TCDD increases mitochondrial superoxide anion production and decreases {Delta}{psi}{sub m}, COX activity and mitochondrial cardiolipin content. Our findings suggest that mitochondria may play an important role in ROS production, leading to the TCDD-induced oxidative stress response and resulting toxicological consequences in rat Sertoli cells.

  11. Sertoli cell-secreted protein(s) stimulates DNA synthesis in purified rat Leydig cells in vitro.

    PubMed

    Ojeifo, J O; Byers, S W; Papadopoulos, V; Dym, M

    1990-09-01

    We have examined the effects of Sertoli cell-secreted proteins (SCSP) on [3H]thymidine incorporation by purified preparations (greater than 96%) of rat Leydig cells to determine whether Sertoli cells influence DNA synthesis in these cells in vitro. Incubation of Leydig cells isolated from testes of rats of ages 16 to 90 days with SCSP (Mr greater than 10,000) induced significant dose-, time- and age-related increases in [3H]thymidine incorporation by the cells. A dose-response curve to SCSP showed that as little as 0.2 micrograms SCSP/ml consistently induced a small but significant increase (31% and 10% above control; P less than 0.001) in [3H]thymidine incorporation by Leydig cells isolated from immature (26 days) and mature (70 days) rats, respectively. The maximum response (230% and 48% above control) was obtained with a concentration of 18 micrograms SCSP/ml in cells isolated from immature and mature rats, respectively. Hydroxyurea, a specific inhibitor of replicative DNA synthesis, significantly (P less than 0.001) inhibited both basal and SCSP-induced [3H]thymidine incorporation in Leydig cells from immature and adult rats without affecting the viability of the cells. Incubation of immature rat Leydig cells in SCSP for 48 h also stimulated a 3-fold increase in cell number. The component of the crude SCSP which stimulated Leydig cell [3H]thymidine incorporation is trypsin-sensitive, heat-stable, and adsorbs to a heparin-agarose affinity column but not to concanavalin A-Sepharose. The secretion of this factor(s) by Sertoli cells is stimulated independently by FSH and testosterone. These results demonstrate for the first time that cultured Sertoli cells secrete a protein(s) which, in vitro, stimulates rat Leydig cell replicative DNA synthesis. PMID:2231558

  12. Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression.

    PubMed

    Willems, A; De Gendt, K; Allemeersch, J; Smith, L B; Welsh, M; Swinnen, J V; Verhoeven, G

    2010-06-01

    Evidence from several models of hormone depletion and/or replacement and from knockout animals points to a key role of androgens in the control of spermatogenesis. In testes of mice with a Sertoli cell-selective ablation of the androgen receptor (SCARKO), transcriptional profiling, using microarray technology, revealed that, already on postnatal day 10,692 genes are differentially expressed compared with testes of control mice. Further evaluation of a subset of these genes by quantitative RT-PCR suggested that differences in expression may already be evident on day 8 or earlier. As the androgen receptor in mouse Sertoli cells becomes immunologically detectable around day 5, we tried to identify the earliest responses to androgens by a new transcriptional profiling study on testes from 6-day-old SCARKO and control mice. No obvious and novel early androgen response genes, potentially acting as mediators of subsequent indirect androgen actions, could be identified. However, several genes differentially expressed on day 10 already displayed a response to androgen receptor ablation on day 6. Quantitative RT-PCR studies for 12 of these genes on 10 paired SCARKO and control testes from 4-, 6-, 8-, 10-, 20- and 50-day-old mice revealed significant differences in expression level from day 4 onwards for three genes (Eppin, PCI, Cldn11) and from day 6 onwards for one more gene (Rhox5). For at least two of these genes (Rhox5 and Eppin), there is evidence for direct regulation via the androgen receptor. For three additional genes (Gpd1, Tubb3 and Tpd52l1) significantly lower expression in the SCARKO was noted from day 8 onwards. For all the studied genes, an impressive increase in transcript levels was observed between day 4-50 and differential expression was maintained in adulthood. It is concluded that the SCARKO model indicates incipient androgen action in mouse Sertoli cells from day 4 onwards. PMID:19392831

  13. Ovarian Sertoli-Leydig Cell Tumour with Heterologus Elements Masquerading as Mucinous Tumour on Frozen Section: A Case Report

    PubMed Central

    Valiathan, Manna; Kumar, Sandeep; Kapoor, Sukriti

    2015-01-01

    Sertoli-Leydig cell tumour (SLCT) is an extremely rare ovarian neoplasm. This tumour is characterized by excessive proliferation of normal testicular structures sertoli and leydig cells. These cells are seen in varying proportions and exhibit varying degrees of differentiation. We report a case of primary ovarian SLCT with heterologus elements in a 17-year-old girl which was misdiagnosed on frozen section as mucinous cystic neoplasm. We discuss the clinicopathologic features of SLCT along with the unusual features seen in this case. PMID:26393134

  14. Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis.

    PubMed

    Gelly, J L; Richoux, J P; Grignon, G

    1994-05-01

    The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV-VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis. PMID:8020066

  15. Expression of P450 Aromatase in Granulosa Cell Tumors and Sertoli-Stromal Cell Tumors of the Ovary: Which Cells Are Responsible for Estrogenesis?

    PubMed Central

    Uchigasaki, Shinya; Fukase, Masayuki; Kurose, Akira

    2016-01-01

    Granulosa cell tumors are representative of estrogenic ovarian tumors, and some Sertoli-stromal cell tumors are also estrogenic. The exact cells that are responsible for estrogenesis, however, have yet to be identified. In the present study, 25 sex cord-stromal tumors (20 granulosa cell tumors, 4 Sertoli-Leydig cell tumors, and a Sertoli cell tumor) were immunohistochemically examined for expression of P450 aromatase, which is critical for estrogenesis. All of the tumors had been evaluated for estrogenic function, including contemporaneous endometrial hyperplasia and/or elevation of serum estradiol. Eleven of 14 estrogenic granulosa cell tumors showed sparse or aggregated immunoreactivity for aromatase, whereas 5 of 6 nonestrogenic tumors did not. Aromatase was selectively expressed by plump granulosa cells with eosinophilic or vacuolated cytoplasm, resembling luteinized granulosa cells. Such a localization of aromatase is analogous to that in normal ovaries. Aromatase expression in primary tumors was recapitulated by recurrent tumors. In Sertoli-stromal cell tumors, either undifferentiated plump cells or well-differentiated Sertoli cells expressed aromatase. In conclusion, the expression of P450 aromatase corresponds to specific cell morphology in sex cord-stromal tumors, including recurrent tumors. Aromatase status in granulosa cell tumors provides helpful information on whether serum estradiol could be a marker for recurrence. PMID:26166720

  16. Follicle-stimulating hormone stimulates estradiol-17beta synthesis in cultured Sertoli cells.

    PubMed Central

    Dorrington, J H; Armstrong, D T

    1975-01-01

    Sertoli cells isolated from testes of 20-day-old rats and maintained in primary culture synthesized estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol] (measured by specific radioimmunoassay) when testosterone (17beta-hydroxy-4-androsten-3-one) 0.5 muM, was added to the culture medium. No detectable estradiol synthesis occurred when cells were incubated in medium containing pregnenolone (3beta-hydroxypregn-5-en-20-one), 0.5 muM, or containing no added steroid substrate. Follicle-stimulating hormone (FSH) (NIH-FSH-S10, 5 mug/ml) stimulated estradiol synthesis 12- to 80-fold when added to medium containing testosterone, but not when added to medium containing pregnenolone or no exogenous steroid substrate. A highly purified FSH preparation, with FSH potency 50 times that of the NIH-FSH, caused a similar stimulation at a concentration of 0.25 mug/ml of medium, whereas luteinizing hormone (NIH-LH-S18, 5 MUG/ML) Caused only marginal stimulation. Dibutyryl-adenosine 3':5' cyclic monophosphate, 0.1 mM, caused a 30-fold increase in estradiol synthesis by Sertoli cells cultured in medium containing testosterone. These studies provide direct demonstration of estradiol-17beta production by Seroli cells from normal animals, and offer evidence that the synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and adenosine 3':5' cyclic monophosphate. PMID:170613

  17. Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells.

    PubMed

    Xu, Bo; Chen, Minjian; Ji, Xiaoli; Yao, Mengmeng; Mao, Zhilei; Zhou, Kun; Xia, Yankai; Han, Xiao; Tang, Wei

    2015-10-01

    Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment. PMID:26165742

  18. Equine follicle-stimulating hormone action in cultured Sertoli cells from rat, sheep and pig.

    PubMed

    Monet-Kuntz, C; Guillou, F; Fontaine, I; Combarnous, Y

    1991-07-01

    Using a suspension of seminiferous tubule cells, we had previously shown that equine FSH is superactive in the male rat, i.e. that it exhibits a higher biological potency than expected from its binding activity. In this work we investigated equine FSH superactivity in rat, pig and sheep, by comparing in each species the equine FSH with the homologous FSH, both for their binding activities (in a radioreceptor assay using a testicular membrane fraction) and for their in vitro biological potencies (in a plasminogen activator assay using a Sertoli cell-enriched population cultured on plastic). In the rat, the binding activity of equine FSH was identical to that of rat FSH, and the biological potency of equine FSH was 47 times higher than that of rat FSH. Hence, superactivity of equine FSH was confirmed in the rat. In the pig, equine FSH was not superactive, since it exhibited binding activity and biological potency identical to those of porcine FSH. In the sheep, the binding activity of equine FSH was 6 times higher than that of ovine FSH, and its biological potency was also higher (14 times). Therefore, equine FSH cannot be considered superactive in this species. In conclusion, equine FSH superactivity is closely related to the species from which Sertoli cells are isolated. PMID:1908169

  19. Sertoli-Leydig cell tumor with heterologous element: a case report and a review of the literature

    PubMed Central

    Chen, Longwen; Tunnell, Cairo Dana; Petris, Giovanni De

    2014-01-01

    The patient was a 19-year-old female who presented with a chief complaint of progressive pelvic pain. Preoperative ultrasound of the right ovary revealed an ovarian torsion as the cause of the patients progressive pain. Laparoscopy confirmed the torsion and revealed a right ovary measuring 10 cm in greatest diameter. Intraoperative incision into the ovary revealed a simple ovarian cystic mass measuring 3.0 x 1.5 x 0.8 cm. A solid component within the cyst was identified. Histological sections of the cystic mass demonstrated mononuclear and hyperchromatic Sertoli cells with a trabecular growth pattern. Clusters of medium-sized epithelioid cells with abundant eosinophilic cytoplasm consistent with Leydig cells were also identified between the trabeculae of Sertoli cells. In addition, focal areas of intestinal type mucinous epithelium were identified embedded within the trabeculae of Sertoli cells. Immunohistochemical studies revealed that the Sertoli cells were positive for calretinin (bright) while the Leydig cells were positive for calretinin (dim), inhibin, CAM5.2 and AE1&3. CEA showed positivity mainly of the intraluminal contents of the mucinous type intestinal epithelium. The patient had an uneventful post-operative course and was disease-free for 3 years. PMID:24696734

  20. Morphometry and morphology of nucleus of the Sertoli and interstitial cells of the tambaqui Colossoma macropomum (Cuvier, 1881) (Pisces: Characidae) during the reproductive cycle.

    PubMed

    Nakaghi, L S O; Mitsuiki, D; Santos, H S L; Pacheco, M R; Ganeco, L N

    2003-02-01

    This study allowed the characterization of the tambaqui Colossoma macropomum testes structural organization, emphasizing Sertoli and interstitial cells and analyzing morphometrically the Sertoli cell nucleus diameter and the interstitial tissue area during the reproductive cycle. Fragments of tambaqui testes were collected in the following reproductive cycle stages: immature, resting, maturation I and II, mature, and regression, and were histologically processed. The Sertoli cells were found at the periphery of the cysts of germinative lineage cells and the nuclei were shown to be smaller as these cells developed. The interstitial cells were better observed between the seminiferous lobules next to vessels in the interstitial tissue of maturing testes. PMID:12914420

  1. A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

    PubMed

    Miyamoto, T; Bando, Y; Koh, E; Tsujimura, A; Miyagawa, Y; Iijima, M; Namiki, M; Shiina, M; Ogata, K; Matsumoto, N; Sengoku, K

    2016-01-01

    About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia. PMID:26452337

  2. SERTOLI CELLS IN THE BOAR TESTIS: CHANGES DURING DEVELOPMENT AND COMPENSATORY HYPERTROPHY AFTER HEMICASTRATION AT DIFFERENT AGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changes in Sertoli cell numbers and testicular structure during normal development and during compensatory hypertrophy were assessed in crossbred Meishan x White Composite males. Boars were assigned at birth to unilateral castration at 1, 10, 56 or 112 days, or remained as intact controls through 22...

  3. Modulation of Dhh signaling and altered Sertoli cell function in mice lacking the GPR37-prosaposin receptor.

    PubMed

    La Sala, Gina; Marazziti, Daniela; Di Pietro, Chiara; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

    2015-05-01

    The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P < 0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation. PMID:25609427

  4. Testosterone deficiency induced by progressive stages of diabetes mellitus impairs glucose metabolism and favors glycogenesis in mature rat Sertoli cells.

    PubMed

    Rato, Luís; Alves, Marco G; Duarte, Ana I; Santos, Maria S; Moreira, Paula I; Cavaco, José E; Oliveira, Pedro F

    2015-09-01

    The incidence of type 2 diabetes mellitus and its prodromal stage, pre-diabetes, is rapidly increasing among young men, leading to disturbances in testosterone synthesis. However, the impact of testosterone deficiency induced by these progressive stages of diabetes on the metabolic behavior of Sertoli cells remains unknown. We evaluated the effects of testosterone deficiency associated with pre-diabetes and type 2 diabetes on Sertoli cells metabolism, by measuring (1) the expression and/or activities of glycolysis and glycogen metabolism-related proteins and (2) the metabolite secretion/consumption in Sertoli cells obtained from rat models of different development stages of the disease, to unveil the mechanisms by which testosterone deregulation may affect spermatogenesis. Glucose and pyruvate uptake were decreased in cells exposed to the testosterone concentration found in pre-diabetic rats (600nM), whereas the decreased testosterone concentrations found in type 2 diabetic rats (7nM) reversed this profile. Lactate production was not altered, although the expression and/or activity of lactate dehydrogenase and monocarboxylate transporter 4 were affected by progressive testosterone-deficiency. Sertoli cells exposed to type 2 diabetic conditions exhibited intracellular glycogen accumulation. These results illustrate that gradually reduced levels of testosterone, induced by progressive stages of diabetes mellitus, favor a metabolic reprogramming toward glycogen synthesis. Our data highlights a pivotal role for testosterone in the regulation of spermatogenesis metabolic support by Sertoli cells, particularly in individuals suffering from metabolic diseases. Such alterations may be in the basis of male subfertility/infertility associated with the progression of diabetes mellitus. PMID:26148570

  5. Immunohistochemical expression of SOX9 protein in immature, mature, and neoplastic canine Sertoli cells.

    PubMed

    Banco, Barbara; Palmieri, Chiara; Sironi, Giuseppe; Fantinato, Eleonora; Veronesi, Maria C; Groppetti, Debora; Giudice, Chiara; Martignoni, Benedetta; Grieco, Valeria

    2016-05-01

    Sex-determining region Y box9 gene (SOX9) protein plays a pivotal role in male sexual development. It regulates the transcription of the anti-Müllerian hormone gene promoting development of testis cords, multiplication, and maturation of Sertoli cells (SCs) and maintenance of spermatogenesis in adult testis. The immunohistochemical expression of SOX9 in normal testes has been reported in humans, mice, and rats. The present study aimed to investigate the expression of SOX9 in canine SCs during testicular maturation and neoplastic transformation. Canine testicular samples derived from three fetuses, four newborns, four prepubertal puppies, five adult dogs, 31 Sertoli cell tumors (SCTs) (one metastasizing), and five Leydig cell tumors (LCTs) were selected from departmental archive and tested immunohistochemically with a polyclonal antibody against SOX9 (1:150). All SCs from fetal, neonatal, and adult testes had a strong and exclusively nuclear labeling for SOX9. In SCs from prepubertal testes, SOX9 staining was highly variable with one negative sample (one of four), two samples with exclusively nuclear staining (two of four), and one with both nuclear and cytoplasmic labeling (one of four). Leydig cells (LCs) and LCTs were always negative. All 31 SCTs were positive for SOX9. The expression of SOX9 was nuclear, nuclear and cytoplasmic, and exclusively cytoplasmic in 18 of 31, 11 of 31, and two of 31 SCTs, respectively. This first report on the immunohistochemical expression of SOX9 in canine testes reports that in normal SCs from fetal, neonatal, and adult testes SOX9 labeled the nucleus, as in humans and laboratory animals. The cytoplasmic labeling observed in one prepubertal pairs of testes and in 11 SCTs could reflect SC immaturity or dedifferentiation, paralleling results observed in rat testes. The expression of SOX9 in SCs and SCTs and its absence in LCs and LCTs suggests that SOX9 is a reliable diagnostic marker for both normal and neoplastic SCs. PMID:26777558

  6. Changes in the morphology and protein expression of germ cells and Sertoli cells in plateau pikas testes during non-breeding season

    PubMed Central

    Liu, Ming; Cao, Guangming; Zhang, Yanming; Qu, Jiapeng; Li, Wei; Wan, Xinrong; Li, Yu-xia; Zhang, Zhibin; Wang, Yan-ling; Gao, Fei

    2016-01-01

    Plateau pikas are seasonally breeding small herbivores that inhabit the meadow ecosystem of the Qinghai-Tibetan Plateau. Testis regression in plateau pikas begins in early June, and the male pikas are completely infertile, with a dramatically reduced testis size, in late July. In this study, a decreased germ cell number in the testes was first noted in early June. By late June, only Sertoli cells and a small number of spermatogonia remained. Interestingly, large gonocyte-like germ cells were observed in early July. In late July, the number of gonocyte-like cells per tubule increased significantly, and most of the Sertoli cell nuclei moved to and clustered in the center of the seminiferous tubules. The gonocyte-like germ cells and Sertoli cells began to express AP-2γ and anti-Mullerian hormone (AMH) proteins, which were detected in the germ cells and Sertoli cells of juvenile pikas but not in adult testes. Simultaneously, LC3 puncta dramatically increased in the seminiferous tubules of the pikas’ testes during the non-breeding season. Our study found that spermatogonia and Sertoli cells in non-breeding adult pikas morphologically resembled those in juvenile pikas and expressed specific markers, indicating that de-differentiation-like transitions may occur during this process. PMID:26939551

  7. Changes in the morphology and protein expression of germ cells and Sertoli cells in plateau pikas testes during non-breeding season.

    PubMed

    Liu, Ming; Cao, Guangming; Zhang, Yanming; Qu, Jiapeng; Li, Wei; Wan, Xinrong; Li, Yu-Xia; Zhang, Zhibin; Wang, Yan-Ling; Gao, Fei

    2016-01-01

    Plateau pikas are seasonally breeding small herbivores that inhabit the meadow ecosystem of the Qinghai-Tibetan Plateau. Testis regression in plateau pikas begins in early June, and the male pikas are completely infertile, with a dramatically reduced testis size, in late July. In this study, a decreased germ cell number in the testes was first noted in early June. By late June, only Sertoli cells and a small number of spermatogonia remained. Interestingly, large gonocyte-like germ cells were observed in early July. In late July, the number of gonocyte-like cells per tubule increased significantly, and most of the Sertoli cell nuclei moved to and clustered in the center of the seminiferous tubules. The gonocyte-like germ cells and Sertoli cells began to express AP-2γ and anti-Mullerian hormone (AMH) proteins, which were detected in the germ cells and Sertoli cells of juvenile pikas but not in adult testes. Simultaneously, LC3 puncta dramatically increased in the seminiferous tubules of the pikas' testes during the non-breeding season. Our study found that spermatogonia and Sertoli cells in non-breeding adult pikas morphologically resembled those in juvenile pikas and expressed specific markers, indicating that de-differentiation-like transitions may occur during this process. PMID:26939551

  8. Metallothionein gene expression under different time in testicular Sertoli and spermatogenic cells of rats treated with cadmium.

    PubMed

    Ren, Xu Yi; Zhou, Yong; Zhang, Jian Peng; Feng, Wei Hua; Jiao, Bing Hua

    2003-01-01

    The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. To clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different times in freshly isolated testicular Sertoli and spermatogenic cells and liver of rats treated with Cd. Adult male Sprague-Dawley rats received a s.c. injection of 4.0 micromol Cd/kg and 1, 3, 6, or 24h later and untreated animals (0h) tissue were sampled and testicular Sertoli and spermatogenic cells isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. Testicular lesions were not grossly or histologically observed in rats treated with 4.0 micromol Cd/kg. In the present study, we demonstrated that the rat testis indeed expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3h, followed by a decline, in contrast, the mRNA levels in Sertoli cells peaked at 6h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in Sertoli cells and liver of rats treated with Cd. However, the MT1 mRNA levels of spermatogenic cells decreased 0-3h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0-3h after Cd treatment, followed by a reduction, but induced extents of them are lower than those of Sertoli cells and liver. Cd exposure substantially increased hepatic MT, but did not increase MT translation in Sertoli and spermatogenic cells. These results indicate: (1) that Cd-induced MT mRNA expression is cell- and time-dependent; (2) that the inability to induce the metal-detoxicating MT protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver. PMID:12642155

  9. Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone

    PubMed Central

    Fallarino, Francesca; Luca, Giovanni; Calvitti, Mario; Mancuso, Francesca; Nastruzzi, Claudio; Fioretti, Maria C.; Grohmann, Ursula; Becchetti, Ennio; Burgevin, Anne; Kratzer, Roland; van Endert, Peter; Boon, Louis; Calafiore, Riccardo

    2009-01-01

    Type I diabetes mellitus is caused by autoimmune destruction of pancreatic ? cells, and effective treatment of the disease might require rescuing ? cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional ? cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-?dependent emergence of autoantigen-specific regulatory T cells and recovery of ? cell function in the diabetic recipients. PMID:19822646

  10. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    PubMed Central

    2011-01-01

    Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96) and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%). DOX-96 showed a lower limit of detection (1.25 × 10(4) cells/ml); whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4) cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring. PMID:21575213

  11. Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice.

    PubMed

    Welborn, Joshua P; Davis, Matthew G; Ebers, Steven D; Stodden, Genna R; Hayashi, Kanako; Cheatwood, Joseph L; Rao, Manjeet K; MacLean, James A

    2015-07-01

    The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

  12. Follicle-stimulating hormone induces hyperpolarization of immature rat Sertoli cells in monolayer culture.

    PubMed Central

    Joffre, M; Roche, A

    1988-01-01

    1. The effect of FSH on the membrane potential of Sertoli cells has been investigated by intracellular microelectrode recording from monolayer cultures of cells, isolated from immature rat testes by enzymatic treatment. 2. In standard Earle's solution, the membrane potential of unstimulated cells in a 3-day-old monolayer was -21.6 +/- 0.2 mV (n = 300). The recorded potentials were unchanged on varying the culture period from 3 to 7 days or by removal of chloride or calcium from the media. However, they were decreased in a low-sodium or potassium-rich medium. 3. Ovine FSH caused hyperpolarization of the cell in a dose-dependent manner. Maximal values were obtained with concentrations ranging between 2.9 and 5.9 micrograms/ml (-37.0 +/- 0.2 mV; n = 310). Dibutyryl cyclic AMP (1 mM) produced a similar effect to FSH. Under similar conditions human chorionic gonadotrophin (hCG) had no effect on the membrane potential. 4. The FSH-induced hyperpolarization was unchanged by chloride or bicarbonate replacement. It was decreased by low-sodium media (9 mM) (-29.6 +/- 0.3 mV; n = 110), by removal of calcium (-32.4 +/- 0.6 mV; n = 128) and by an increase in the potassium concentration of the bathing medium. A tenfold increase in potassium concentration depolarized the cell by 29.5 mV against 16 mV for unstimulated cells. 5. FSH-induced hyperpolarization was decreased by application of ouabain (10(-4) M), quinidine (1.4 x 10(-4) M) and cobalt (10(-3) M) (respectively: -28.7 +/- 0.3 mV, n = 124; -26.6 +/- 0.3 mV, n = 52 and -24.8 +/- 0.3 mV, n = 68) but was unchanged by amiloride (10(-4) M) and TTX (3 x 10(-6) M). None of these drugs modified the membrane potential of unstimulated cells. 6. All these results suggest that FSH stimulation of Sertoli cells in monolayer culture involves the modification of their membrane permeabilities. Images Plate 1 PMID:2458458

  13. The protective effects of quercetin on the cytotoxicity of atrazine on rat Sertoli-germ cell co-culture.

    PubMed

    Abarikwu, S O; Pant, A B; Farombi, E O

    2012-08-01

    To evaluate the direct effect of atrazine (ATZ) and the protective effect of quercetin (QT) on testicular cells, we used primary cultures of rat Sertoli-germ cells (SGCs). ATZ (232 ?m) up-regulated the mRNA expression of GATA-4, androgen receptor (AR), androgen-binding protein (ABP), steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (CYP11A1), cyclooxygenase-2 (COX-2) and NF-?appaB (NF-?B) and down-regulated the expression of stem cell factor (SCF) mRNA. There was no change on the mRNA expression of oestrogen receptor-alpha (ER-?). Simultaneous supplementation of QT in the culture normalizes the expression of these genes. The stimulatory action of follicle stimulating hormone (10 ng/mL) on ATZ-induced StAR and CYP11A1 mRNA levels were also prevented by QT. Furthermore, ATZ-stimulatory action on AR mRNA was opposed in a dose-dependent manner in the presence of increasing concentrations of QT (10-50 ?m).The dislodgement of germ cells from the Sertoli cells monolayer and decrease in SGCs viability was prevented by QT. To show whether or not the disrupted interactions of Sertoli and germ cells impaired spermatogenesis, adult male rats exposed in vivo to ATZ (50 mg/kg b.wt) for 1 week had their daily spermatozoa production (DSP) per gram testis lowered by 30%. DSP was significantly increased in the QT(10 mg/kg) + ATZ-treated rats as compared with the ATZ-treated rats. Taken together, ATZ can alter SGCs expression of spermatogenesis- and steroiodogenesis-related genes resulting in a decrease in sperm production in the testis as well as cell viability. QT might block these molecular events-induced by ATZ thereby protecting testicular Sertoli-germ cells from ATZ-induced toxicity. PMID:22372587

  14. Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

    PubMed

    Luaces, Juan Pablo; Rossi, Luis Francisco; Sciurano, Roberta Beatriz; Rebuzzini, Paola; Merico, Valeria; Zuccotti, Maurizio; Merani, Maria Susana; Garagna, Silvia

    2014-03-01

    The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis. PMID:24451984

  15. Hemicastration causes and testosterone prevents enhanced uptake of (/sup 3/H)thymidine by Sertoli cells in testes of immature rats

    SciTech Connect

    Orth, J.M.; Higginbotham, C.A.; Salisbury, R.L.

    1984-02-01

    Rat pups were hemicastrated and uptake of (/sup 3/H)thymidine by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated (/sup 3/H)thymidine was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropins were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats.

  16. Partial Orchiectomy for the Treatment of a Benign Sclerosing Sertoli Cell Tumor: A Report of a Rare Tumor in Association With Testicular Microlithiasis

    PubMed Central

    Ball, Adam James

    2015-01-01

    The diagnosis of a testicular sclerosing Sertoli cell tumor is rare. Approximately 17 cases have been previously described in the literature worldwide. We present a case and review the evaluation and surgical management of a sclerosing Sertoli cell tumor presenting in a 26 year-old African-American adult, in association with testicular microlithiasis and oligospermia. Imaging techniques and the pathological assessment will be reviewed.

  17. New insights on hormones and factors that modulate Sertoli cell metabolism.

    PubMed

    Rato, Luís; Meneses, Maria João; Silva, Branca M; Sousa, Mário; Alves, Marco G; Oliveira, Pedro F

    2016-05-01

    Sertoli cells (SCs) play a key role in spermatogenesis by providing the physical support for developing germ cells and ensuring them the appropriate nutrients, energy sources, hormones, and growth factors. The control of SCs metabolism has been in the spotlight for reproductive biologists, since it may be crucial to determine germ cells' fate. Indeed, the maintenance of spermatogenesis is highly dependent on the metabolic cooperation established between SCs and germ cells, though this event has been overlooked. It depends on the orchestration of various metabolic pathways and an intricate network of signals. Several factors and/or hormones modulate the metabolic activity of SCs, which are major targets for the hormonal signalling that regulates spermatogenesis. Any alteration in the regulation of these cells' metabolic behaviour may compromise the normal development of spermatogenesis and consequently, male fertility. In this context, SC metabolism arises as a key regulation point for spermatogenesis. Herein, we present an up-to-date overview on the impact of hormones and factors that modulate SC metabolism, with special focus on glycolytic metabolism, highlighting their relevance in determining male reproductive potential. PMID:26711246

  18. Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

    PubMed

    Zimmermann, Céline; Stévant, Isabelle; Borel, Christelle; Conne, Béatrice; Pitetti, Jean-Luc; Calvel, Pierre; Kaessmann, Henrik; Jégou, Bernard; Chalmel, Frédéric; Nef, Serge

    2015-04-01

    Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility. PMID:25710594

  19. A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

    PubMed Central

    Yefimova, Marina G.; Messaddeq, Nadia; Harnois, Thomas; Meunier, Annie-Claire; Clarhaut, Jonathan; Noblanc, Anaïs; Weickert, Jean-Luc; Cantereau, Anne; Philippe, Michel; Bourmeyster, Nicolas; Benzakour, Omar

    2013-01-01

    Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis. PMID:23439251

  20. Immunohistochemical evaluation of the expression of anti-Mllerian hormone in mature, immature and neoplastic canine Sertoli cells.

    PubMed

    Banco, B; Veronesi, M C; Giudice, C; Rota, A; Grieco, V

    2012-01-01

    In mammals, the earliest specific protein expressed by Sertoli cells (SCs) is the anti-Mllerian hormone (AMH), which induces the regression of Mllerian ducts and is produced by SCs until the functional maturation of the testes. The aim of this study was to evaluate the expression of AMH by canine SCs during testicular maturation and neoplastic transformation. Testes from two fetuses, 18 newborn puppies, five puppies aged 43-180 days and six adult dogs, and 24 canine Sertoli cell tumours (SCTs) were studied immunohistochemically for expression of AMH. Fifteen of the 24 SCTs were classified as typical, eight as lipid-rich and one was considered malignant based on evidence of lymph node metastasis. SCs from fetuses and neonatal puppies and puppies up to 45 days old expressed AMH, while SCs from older puppies and adults were negative. All SCTs expressed AMH, suggesting that AMH expression is a useful marker of immature and neoplastic canine SCs. PMID:21571300

  1. Testicular dysgenesis does not affect expression of anti-mllerian hormone by Sertoli cells in premeiotic seminiferous tubules.

    PubMed Central

    Rey, R.; al-Attar, L.; Louis, F.; Jaubert, F.; Barbet, P.; Nihoul-Fkt, C.; Chaussain, J. L.; Josso, N.

    1996-01-01

    Anti-Mllerian hormone (AMH) immunoreactivity was studied on paraffin sections obtained from archival testicular biopsies of 29 children with intersex disorders and of 22 controls. Strong AMH immunoreactivity was observed in Sertoli cell cytoplasm from 8 fetal weeks until puberty. During pubertal maturation, in both normal and intersex patients, AMH expression was present in premeiotic seminiferous tubules, but was no longer detected in neighboring tubules with meiotic development. AMH immunostaining was abolished in the testis of one patient with persistent Mllerian ducts due to a mutation of the AMH gene, but was conserved in the testes of two patients with mutations of the AMH receptor gene. Testicular dysgenesis usually results in sexual ambiguity, with low testosterone and AMH serum levels and persistence of Mllerian derivatives. AMH immunoreactivity was conserved in premeiotic seminiferous tubules of dysgenetic testes, and also in sex-cord cells of a gonadoblastoma. In patients with asymmetric gonadal differentiation, the streak gonad was AMH-negative. In conclusion, secretion of AMH is a constitutive feature of the immature Sertoli cell and its expression is altered only by mutations of the AMH gene, but not by gonadal dysgenesis. The degree of regression of Mllerian ducts and serum AMH levels reflect the number, not the functional value, of Sertoli cells present in the immature testis. Images Figure 1 Figure 2 Figure 3 PMID:8623936

  2. Frequent mutation and nuclear localization of ?-catenin in sertoli cell tumors of the testis.

    PubMed

    Perrone, Federica; Bertolotti, Alessia; Montemurro, Gabriella; Paolini, Biagio; Pierotti, Marco A; Colecchia, Maurizio

    2014-01-01

    The Sertoli cell tumor (SCT) of the testis is a sex cord stromal tumor, usually sporadic, rarely associated with genetic syndromes. Much remains unclear about the molecular genetic changes involved in SCT and its histogenesis. Recently, nuclear ?-catenin immunostaining has been reported in a case of bilateral SCT, but the molecular basis of the aberrant nuclear ?-catenin expression remains uncertain. In the present study, ?-catenin immunohistochemical assay and mutational analysis of exon 3 of the CTNNB1 gene by direct sequencing were performed in 14 SCTs, 2 of which had an unfavorable clinical course. Immunohistochemical study showed that ?-catenin was located in the cytoplasm of tumor cells in 4 cases (28.6%) and in both the nuclei and the cytoplasm in the remaining 10 cases (71.4%). ?-Catenin mutations were detected in 10 of the 14 patients (71.4%) under evaluation. Ten of 10 mutation-carrying cases showed strong nuclear and diffuse cytoplasmic ?-catenin immunoreactivity. Seven of the 8 CTNNB1-mutated tumors tested for cyclin D1 displayed diffuse immunoreactivity in the nuclei of tumor cells. We conclude that CTNNB1 exon 3 mutations are likely to be involved in the pathogenesis of male SCT with nuclear accumulation of ?-catenin and affect the expression of cyclin D1. PMID:24061522

  3. Exposure to 2,4-dichlorophenoxyacetic acid alters glucose metabolism in immature rat Sertoli cells.

    PubMed

    Alves, M G; Neuhaus-Oliveira, A; Moreira, P I; Socorro, S; Oliveira, P F

    2013-07-01

    The purpose of this study was to determine the effects of 2,4-D, an herbicide used worldwide also known as endocrine disruptor, in Sertoli cell (SC) metabolism. Immature rat SCs were maintained 50h under basal conditions or exposed to 2,4-D (100nM, 10?M and 1mM). SCs exposed to 10?M and 1mM of 2,4-D presented lower intracellular glucose and lactate content. Exposure to 10?M of 2,4-D induced a significant decrease in glucose transporter-3 mRNA levels and phosphofructokinase-1 mRNA levels decreased in cells exposed to 100nM and 10?M of 2,4-D. Exposure to 100nM and 10?M also induced a decrease in lactate dehydrogenase (LDH) mRNA levels while the LDH protein levels were only decreased in cells exposed to 1mM of 2,4-D. Exposure to 2,4-D altered glucose uptake and metabolization in SCs, as well as lactate metabolism and export that may result in impaired spermatogenesis. PMID:23538319

  4. An Integrative Omics Strategy to Assess the Germ Cell Secretome and to Decipher Sertoli-Germ Cell Crosstalk in the Mammalian Testis

    PubMed Central

    Lavigne, Rgis; Hernio, Nolwen; Teixeira-Gomes, Ana-Paula; Dacheux, Jean-Louis; Pineau, Charles

    2014-01-01

    Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an integrative omics strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this integrative omics strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture. PMID:25111155

  5. Immunohistochemical expression of markers of immaturity in sertoli and seminal cells in canine testicular atrophy.

    PubMed

    Giudice, C; Banco, B; Veronesi, M C; Ferrari, A; Di Nardo, A; Grieco, V

    2014-01-01

    During maturation from fetal to adult testis, both Sertoli cells (SCs) and germ cells (GCs) switch from an immature to a mature immunophenotype. Immature canine SCs express cytokeratins (CKs), desmin (DES), vimentin (VIM), anti-Mllerian hormone (AMH) and inhibin (INH)-?, while mature SCs retain only expression of VIM. Immature GCs express placental alkaline phosphatase (PLAP), which is lost in spermatocytes. Re-expression of markers of immaturity has been observed in human atrophic testes and in human and canine testicular tumours. In human medicine, testicular atrophy is considered a risk factor for testicular cancer. In the present study 13 canine atrophic testes were examined immunohistochemically. VIM was expressed in the SCs of all cases, while CK, DES, INH-? and AMH were expressed in a variable percentage of SCs in two, five, five and eight cases, respectively. PLAP was expressed by single GCs in one case. Markers of immaturity are therefore expressed by SCs and GCs in canine atrophic testes. Similar results were reported previously in canine testicular neoplasia, suggesting that testicular atrophy may represent a risk factor for tumour development in the dog. PMID:24064049

  6. Intraperitoneal injection of microencapsulated Sertoli cells restores muscle morphology and performance in dystrophic mice.

    PubMed

    Chiappalupi, Sara; Luca, Giovanni; Mancuso, Francesca; Madaro, Luca; Fallarino, Francesca; Nicoletti, Carmine; Calvitti, Mario; Arato, Iva; Falabella, Giulia; Salvadori, Laura; Di Meo, Antonio; Bufalari, Antonello; Giovagnoli, Stefano; Calafiore, Riccardo; Donato, Rosario; Sorci, Guglielmo

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-?1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases. PMID:26523508

  7. Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice

    PubMed Central

    Chiappalupi, Sara; Luca, Giovanni; Mancuso, Francesca; Madaro, Luca; Fallarino, Francesca; Nicoletti, Carmine; Calvitti, Mario; Arato, Iva; Falabella, Giulia; Salvadori, Laura; Di Meo, Antonio; Bufalari, Antonello; Giovagnoli, Stefano; Calafiore, Riccardo; Donato, Rosario; Sorci, Guglielmo

    2015-01-01

    We report data about the effects of intraperitoneal (i.p.) injection of specific pathogen-free (SPF) porcine Sertoli cells (SeC) encapsulated into clinical grade alginate-based microcapsules (SeC-MC) on muscles of chronic and presymptomatic dystrophic, mdx mice. Mdx mouse is the best characterized animal model of Duchenne muscular dystrophy (DMD), an X-linked lethal myopathy due to mutation in the gene of dystrophin, which is crucial for myofiber integrity during muscle contraction. Our data show that three weeks after i.p. injection of SeC-MC significantly reduced adipose and fibrous tissue deposition, reduced macrophage infiltrate, and reduced numbers of damaged myofibers are found in muscles of 12-month-old mdx mice, which reproduce chronic DMD conditions. Compared with muscles of mock-treated mdx mice muscles of SeC-MC-treated mice show upregulation of the dystrophin paralogue, utrophin which is localized to the periphery of myofibers. Moreover, our data show that i.p. injection of SeC-MC into presymptomatic, 2-week-old mdx mice, although not fully preventing myofiber degeneration, results in protection against myofiber necrosis and muscle inflammation. Extensive discussion of these data can be found in Ref. [1]. PMID:26759818

  8. Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice.

    PubMed

    Chiappalupi, Sara; Luca, Giovanni; Mancuso, Francesca; Madaro, Luca; Fallarino, Francesca; Nicoletti, Carmine; Calvitti, Mario; Arato, Iva; Falabella, Giulia; Salvadori, Laura; Di Meo, Antonio; Bufalari, Antonello; Giovagnoli, Stefano; Calafiore, Riccardo; Donato, Rosario; Sorci, Guglielmo

    2015-12-01

    We report data about the effects of intraperitoneal (i.p.) injection of specific pathogen-free (SPF) porcine Sertoli cells (SeC) encapsulated into clinical grade alginate-based microcapsules (SeC-MC) on muscles of chronic and presymptomatic dystrophic, mdx mice. Mdx mouse is the best characterized animal model of Duchenne muscular dystrophy (DMD), an X-linked lethal myopathy due to mutation in the gene of dystrophin, which is crucial for myofiber integrity during muscle contraction. Our data show that three weeks after i.p. injection of SeC-MC significantly reduced adipose and fibrous tissue deposition, reduced macrophage infiltrate, and reduced numbers of damaged myofibers are found in muscles of 12-month-old mdx mice, which reproduce chronic DMD conditions. Compared with muscles of mock-treated mdx mice muscles of SeC-MC-treated mice show upregulation of the dystrophin paralogue, utrophin which is localized to the periphery of myofibers. Moreover, our data show that i.p. injection of SeC-MC into presymptomatic, 2-week-old mdx mice, although not fully preventing myofiber degeneration, results in protection against myofiber necrosis and muscle inflammation. Extensive discussion of these data can be found in Ref. [1]. PMID:26759818

  9. Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene

    SciTech Connect

    Turner, B.; Vordermark, J.S.; Fechner, P.Y.

    1995-07-03

    We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and muellerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y-bearing cells. The subjects in this report demonstrate that there is a continuum in the extent of the testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development. 20 refs.

  10. Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis

    PubMed Central

    Rebourcet, Diane; O'Shaughnessy, Peter J.; Pitetti, Jean-Luc; Monteiro, Ana; O'Hara, Laura; Milne, Laura; Tsai, Yi Ting; Cruickshanks, Lyndsey; Riethmacher, Dieter; Guillou, Florian; Mitchell, Rod T.; van t Hof, Rob; Freeman, Tom C.; Nef, Serge; Smith, Lee B.

    2014-01-01

    Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:24803659

  11. Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells.

    PubMed

    de Liz Oliveira Cavalli, Vera Lcia; Cattani, Daiane; Heinz Rieg, Carla Elise; Pierozan, Paula; Zanatta, Leila; Benedetti Parisotto, Eduardo; Wilhelm Filho, Danilo; Mena Barreto Silva, Ftima Regina; Pessoa-Pureur, Regina; Zamoner, Ariane

    2013-12-01

    Glyphosate is the primary active constituent of the commercial pesticide Roundup. The present results show that acute Roundup exposure at low doses (36 ppm, 0.036 g/L) for 30 min induces oxidative stress and activates multiple stress-response pathways leading to Sertoli cell death in prepubertal rat testis. The pesticide increased intracellular Ca(2+) concentration by opening L-type voltage-dependent Ca(2+) channels as well as endoplasmic reticulum IP3 and ryanodine receptors, leading to Ca(2+) overload within the cells, which set off oxidative stress and necrotic cell death. Similarly, 30 min incubation of testis with glyphosate alone (36 ppm) also increased (45)Ca(2+) uptake. These events were prevented by the antioxidants Trolox and ascorbic acid. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK play a role in eliciting Ca(2+) influx and cell death. Roundup decreased the levels of reduced glutathione (GSH) and increased the amounts of thiobarbituric acid-reactive species (TBARS) and protein carbonyls. Also, exposure to glyphosate-Roundup stimulated the activity of glutathione peroxidase, glutathione reductase, glutathione S-transferase, ?-glutamyltransferase, catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, supporting downregulated GSH levels. Glyphosate has been described as an endocrine disruptor affecting the male reproductive system; however, the molecular basis of its toxicity remains to be clarified. We propose that Roundup toxicity, implicated in Ca(2+) overload, cell signaling misregulation, stress response of the endoplasmic reticulum, and/or depleted antioxidant defenses, could contribute to Sertoli cell disruption in spermatogenesis that could have an impact on male fertility. PMID:23820267

  12. Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review

    PubMed Central

    Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Flix, Ana

    2014-01-01

    Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker. PMID:25926909

  13. Blood-testis barrier and Sertoli cell function: lessons from SCCx43KO mice.

    PubMed

    Gerber, Jonathan; Heinrich, Julia; Brehm, Ralph

    2016-02-01

    The gap junction protein connexin43 (CX43) plays a vital role in mammalian spermatogenesis by allowing for direct cytoplasmic communication between neighbouring testicular cells. In addition, different publications suggest that CX43 in Sertoli cells (SC) might be important for blood-testis barrier (BTB) formation and BTB homeostasis. Thus, through the use of the Cre-LoxP recombination system, a transgenic mouse line was developed in which only SC are deficient of the gap junction protein, alpha 1 (Gja1) gene. Gja1 codes for the protein CX43. This transgenic mouse line has been commonly defined as the SC specific CX43 knockout (SCCx43KO) mouse line. Within the seminiferous tubule, SC aid in spermatogenesis by nurturing germ cells and help them to proliferate and mature. Owing to the absence of CX43 within the SC, homozygous KO mice are infertile, have reduced testis size, and mainly exhibit spermatogenesis arrest at the level of spermatogonia, seminiferous tubules containing only SC (SC-only syndrome) and intratubular SC-clusters. Although the SC specific KO of CX43 does not seem to have an adverse effect on BTB integrity, CX43 influences BTB composition as the expression pattern of different BTB proteins (like OCCLUDIN, ?-CATENIN, N-CADHERIN, and CLAUDIN11) is altered in mutant males. The supposed roles of CX43 in dynamic BTB regulation, BTB assembly and/or disassembly and its possible interaction with other junctional proteins composing this unique barrier are discussed. Data collectively indicate that CX43 might represent an important regulator of dynamic BTB formation, composition and function. PMID:26556893

  14. NC1 domain of collagen ?3(IV) derived from the basement membrane regulates Sertoli cell blood-testis barrier dynamics

    PubMed Central

    Wong, Elissa W.P.; Cheng, C. Yan

    2013-01-01

    The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen ?3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Col?3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNF?. While recombinant Col?3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Col?3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Col?3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, ?-catenin, ?-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis. PMID:23885308

  15. Activation of innate immune system in response to lipopolysaccharide in chicken Sertoli cells.

    PubMed

    Michailidis, Georgios; Anastasiadou, Maria; Guibert, Edith; Froment, Pascal

    2014-09-01

    Sertoli cells (SCs) play an important physiological role in the testis, as they support, nourish, and protect the germ cells. As protection of the developing spermatozoa is an emerging aspect of reproductive physiology, this study examined the expression pattern of innate immune-related genes, including avian β-defensins (AvBDs), Toll-like receptors (TLRs), and cytokines, and investigated the time course of an inflammatory response in rooster SCs triggered by exposure to the bacterial endotoxin lipopolysaccharide (LPS). SCs were isolated from 6-week-old chicken, cultured in vitro, and stimulated with 1 μg/ml LPS at different time courses (0, 6, 12, 24, and 48  h). Data on expression analysis revealed that all ten members of the chicken TLR family, nine members of the AvBD family, as well as eight cytokine genes were expressed in SCs. Quantitative real-time PCR analysis revealed that LPS treatment resulted in significant induction of the expression levels of six TLRs, six AvBDs, and four cytokine genes, while two cytokine genes were downregulated and two other genes were unchanged. The increasing interleukin 1β (IL1β) production was confirmed in the conditioned medium. Furthermore, the phagocytosis of SCs was increased after LPS treatment. In conclusion, these findings provide evidence that SCs express innate immune-related genes and respond directly to bacterial ligands. These genes represent an important component of the immune system, which could be integrated into semen, and present a distinctive constituent of the protective repertoire of the testis against ascending infections. PMID:24920664

  16. Asynchronic steroid activity of Leydig and Sertoli cells related to spermatogenic and testosterone cycle in Phymaturus antofagastensis.

    PubMed

    Boretto, J M; Ibargengoyta, N R; Jahn, G A; Acosta, J C; Vincenti, A E; Forns, M W

    2010-05-01

    The severe environments where Phymaturus lizards inhabit in the Andes highlands and in Patagonia, Argentina, impose restrictions on their reproduction, offering a framework for the development of life history strategies to overcome hard weather conditions. Among them, prolonged female cycles, asynchrony between sexes in receptivity, and sperm storage in males, were described. Asynchrony in the reproductive timing between males and females is a consequence of different energy requirements for gametogenesis, and often imply the existence of cellular mechanisms to enhance fertilization, such as the asynchronic steroid synthesis between testicular compartments, allowing gametogenesis independently of mating. In the present study ultrastructural and hormone assays were combined for the first time in liolaemids. Specifically, morphological features of steroid activity in Leydig and Sertoli cells, and serum testosterone concentrations have been studied in the lizard Phymaturus antofagastensis. Leydig and Sertoli cells presented morphological features characteristic of steroid synthesis during the spermatogenesis, and evident asynchronic steroid production between testicular compartments. Active Sertoli cells and inactive Leydig cells were observed in spring and autumn, while in mid-summer their steroid activity was synchronic in coincidence with maximal abundance of spermatozoa in epididymis. Serum testosterone concentration was at its maximum in mid-summer (126-230 ng ml(-1)), and minimum in late spring (4-24 ng ml(-1)) and early autumn (2-17 ng ml(-1)). In view of these results, P. antofagastensis males show an original approach to adjust their reproductive activity to physiological and environmental constraints at high latitudes and altitudes in the Andean highlands of Argentina. PMID:20152839

  17. Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride

    SciTech Connect

    Quirk, S.M.; Reichert, L.E. Jr.

    1988-07-01

    Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-(2-3H) inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total (3H)inositol phosphates ((3H)inositol mono-, di-, and triphosphates (IP, IP2, and IP3)) in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%.

  18. Nuclear Localization of ?-Catenin in Sertoli Cell Tumors and Other Sex Cord-Stromal Tumors of the Testis: An Immunohistochemical Study of 87 Cases.

    PubMed

    Zhang, Chen; Ulbright, Thomas M

    2015-10-01

    The diagnosis and subclassification of Sertoli cell tumors (SCT) of the testis are often challenging to general surgical pathologists because of the rarity of the tumors. Immunohistochemical study to date has limited diagnostic value. Nuclear localization of ?-catenin, which correlated closely with CTNNB1 gene mutation, was recently reported in SCTs. We investigated the utility of ?-catenin nuclear localization in diagnosing SCTs and differentiating them from other testicular sex cord-stromal tumors. Immunohistochemical staining for ?-catenin was evaluated in 87 cases of testicular sex cord-stromal tumor: 33 SCTs, not otherwise specified (SCT-NOS) (15 with benign and 18 with malignant features), 10 sclerosing SCTs (SSCT), 5 large cell calcifying SCTs (LCCSCT), 6 Sertoli-stromal cell tumors, 10 Leydig cell tumors, 7 juvenile granulosa cell tumors, 4 adult granulosa cell tumors, and 12 sex cord-stromal tumors, unclassified. Twenty-one of 33 (64%) SCT-NOS, 6 of 10 (60%) SSCTs, and 4 of 6 (67%) Sertoli-stromal cell tumors showed strong, diffuse ?-catenin nuclear staining. Nuclear ?-catenin positivity was more frequent in SCTs-NOS with benign features than in those with malignant features (93% and 39%, respectively, P=0.13) and, in the Sertoli-stromal cell tumors, occurred only in the Sertoli component. All 5 LCCSCTs and all other types of sex cord-stromal tumor were negative for ?-catenin nuclear staining. In conclusion, SCT-NOS and SSCT frequently show ?-catenin nuclear localization. Positive nuclear staining of ?-catenin is specific for SCT-NOS, SSCT, and Sertoli-stromal cell tumor among testicular sex cord-stromal tumors but has limited sensitivity (63%) in this group. The similar reactivity of SCT-NOS and SSCT provides additional support that these 2 variants are not distinct entities. PMID:26034868

  19. Perfluorooctanesulfonate (PFOS) perturbs male rat Sertoli cell blood-testis barrier function by affecting F-actin organization via p-FAK-Tyr(407): an in vitro study.

    PubMed

    Wan, Hin-Ting; Mruk, Dolores D; Wong, Chris K C; Cheng, C Yan

    2014-01-01

    Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10-20 ?M (?5-10 ?g/mL) was found to be more potent than bisphenol A (100 ?M) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr(407) was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr(407) was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr(407) and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

  20. Formation and structure of transplantable tissue constructs generated in simulated microgravity from Sertoli cells and neuron precursors.

    PubMed

    Cameron, Don F; Hushen, J J; Colina, L; Mallery, J; Willing, A; Sanberg, P R; Saporta, Samuel

    2004-01-01

    Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD. PMID:15690977

  1. A new role for follicle-stimulating hormone in the regulation of calcium flux in Sertoli cells: Inhibition of Na+/Ca++ exchange

    SciTech Connect

    Grasso, P.; Joseph, M.P.; Reichert, L.E. Jr. )

    1991-01-01

    Elucidation of mechanisms regulating intracellular calcium levels in steroidogenic tissues is important for understanding control of cellular function. We have previously described FSH receptor-mediated flux of 45Ca++ into cultured rat Sertoli cells and receptor-enriched proteoliposomes via voltage-sensitive and voltage-independent calcium channels. In the present study, we report heretofore unrecognized inhibitory effects of FSH on Na+/Ca++ exchange in these two systems. An outwardly directed Na+ gradient, developed by preincubating Sertoli cell monolayers in buffer made hypertonic with NaCl, resulted in uptake of 45Ca++ that was unaffected by calcium channel blocking agents, ruthenium red or methoxyverapamil, but was enhanced by ouabain, a specific inhibitor of Na+/K(+)-ATPase. Sodium-dependent 45Ca++ flux into Sertoli cells was inhibited in a concentration-related manner by increased extracellular Na+ (up to 135 mM). FSH consistently and reproducibly (28.9 +/- 3.8%, 10 separate assays) reduced sodium-dependent 45Ca++ influx in the absence or presence of ouabain. A lesser effect on Na+/Ca++ exchange was seen when Li+ replaced Na+ in the preincubation buffer, and a marked reduction occurred when Sertoli cells were incubated in buffer containing KCl, presumably due to membrane depolarization. FSH-sensitive Na+/45Ca++ exchange was also observed when using FSH receptor-enriched proteoliposomes. Our earlier calcium channel studies indicated that FSH affects Ca++ entry into Sertoli cells via a receptor-mediated process. The results reported here demonstrate that the interaction of FSH with its receptor is associated with changes in Na+/Ca++ exchange as well, and suggest that this activity may also be involved in regulating intracellular free Ca++ levels in the Sertoli cell.

  2. Bilateral Laparoscopic Gonadectomy in a Patient With Complete Androgen Insensitivity Syndrome and Bilateral Sertoli-Leydig Cell Tumor: A Case Report and Brief Review of the Literature

    PubMed Central

    Asl Zare, Mohammad; Kalantari, Mahmood Reza; Asadpour, Amir Abbas; Kamalati, Ali

    2014-01-01

    Introduction: Complete androgen insensitivity syndrome (previously called testicular feminization) is specified by a 46 XY karyotype and negative sex chromatin, bilateral undescended testes, female genitalia appearance, and lack of mullerian derivatives. Case Presentation: A 28-year-old woman with complete (severe) androgen resistance underwent prophylactic laparoscopic bilateral gonadectomy because of the eventually increased risk of gonadal malignancy. Although the gonads appeared grossly normal, microscopic examination revealed bilateral well differentiated sertolileydig cell tumor (SLCT). Discussion: Our Medline search revealed that this is the first reported case of bilateral sertolileydig cell tumor (SLCT) in androgen insensitivity syndrome. PMID:25032133

  3. The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone.

    PubMed

    Dimitriadis, Fotios; Tsiampali, Chara; Chaliasos, Nikolaos; Tsounapi, Panagiota; Takenaka, Atsushi; Sofikitis, Nikolaos

    2015-10-01

    Spermatogenesis is contingent upon hormones and growth factors acting through endocrine and paracrine pathways either in vivo or in vitro. Sertoli cells (SCs) furnish essential factors for the successful advancement of spermatogenesis and spermiogenesis. Moreover, receptors for follicle stimulating hormone (FSH) and testosterone, which are the main hormonal regulators of spermatogenesis, are identified on SCs. Testosterone, FSH and luteinizing hormone are known to determine the destiny of germ cells and in their absence germ cells undergo apoptosis. Bcl-2 family proteins determine one signaling pathway which seems to be crucial for the homeostasis of male gametes. In addition to paracrine signals, germ cell development also relies on signals generated by SCs via direct membrane contact. The regulatory peptide somatostatin has an important role in the regulation of the proliferation of the male germ cells. Activin A, follistatin and FSH control germ cell development. In vitro culture systems have provided initial evidence supporting the achievement of the completion of the first and second male meiotic division in vitro. This review article provides an overview of the literature regarding the hormonal pathways governing spermatogenesis and spermiogenesis. PMID:26732153

  4. The Oncogenic Roles of DICER1 RNase IIIb Domain Mutations in Ovarian Sertoli-Leydig Cell Tumors12

    PubMed Central

    Wang, Yemin; Chen, Jiamin; Yang, Winnie; Mo, Fan; Senz, Janine; Yap, Damian; Anglesio, Michael S.; Gilks, Blake; Morin, Gregg B.; Huntsman, David G.

    2015-01-01

    DICER1, an endoribonuclease required for microRNA (miRNA) biogenesis, is essential for embryogenesis and the development of many organs including ovaries. We have recently identified somatic hotspot mutations in RNase IIIb domain of DICER1 in half of ovarian Sertoli-Leydig cell tumors, a rare class of sex-cord stromal cell tumors in young women. These hotspot mutations lost IIIb cleavage activity of DICER1 in vitro and failed to produce 5p-derived miRNAs in mouse Dicer1-null ES cells. However, the oncogenic potential of these hotspot DICER1 mutations has not been studied. Here, we further revealed that the global expression of 5p-derived miRNAs was dramatically reduced in ovarian Sertoli-Leydig cell tumors carrying DICER1 hotspot mutations compared with those without DICER1 hotspot mutation. The miRNA production defect was associated with the deregulation of genes controlling cell proliferation and the cell fate. Using an immortalized human granulosa cell line, SVOG3e, we determined that the D1709N-DICER1 hotspot mutation failed to produce 5p-derived miRNAs, deregulated the expression of several genes that control gonadal differentiation and cell proliferation, and promoted cell growth. Re-expression of let-7 significantly inhibited the growth of D1709N-DICER1 SVOG3e cells, accompanied by the suppression of key regulators of cell cycle control and ovarian gonad differentiation. Taken together, our data revealed that DICER1 hotspot mutations cause systemic loss of 5p-miRNAs that can both drive pseudodifferentiation of testicular elements and cause oncogenic transformation in the ovary. PMID:26408257

  5. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis.

    PubMed Central

    Eisel, U; Reynolds, K; Riddick, M; Zimmer, A; Niemann, H; Zimmer, A

    1993-01-01

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light chain in the seminiferous epithelium of transgenic mice. Spermatogenesis was severely impaired and mature spermatozoa were completely absent. Late spermatids exhibited pleomorphic shapes and acrosomal distortions. The number of Leydig cells was greatly increased. In situ hybridization analysis revealed that the toxin acts on Sertoli cells. Affected cells exhibited an aberrant distribution of actin filaments and many cells contained large vacuoles. Our results demonstrate that tetanus toxin is active in non-neuronal cells and suggest an important function for members of the synaptobrevin gene family during the late stages of spermatogenesis. Images PMID:8253064

  6. Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

    PubMed

    Egbowon, Biola F; Harris, Wayne; Arnott, Gordon; Mills, Chris Lloyd; Hargreaves, Alan J

    2016-04-01

    The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1?M CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12?M CdCl2 for 4h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12?M CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12?M Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure. PMID:26724415

  7. The Dynamic of the Apical Ectoplasmic Specialization between Spermatids and Sertoli Cells: The Case of the Small GTPase Rap1

    PubMed Central

    2014-01-01

    Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception. PMID:24719879

  8. Isolated Large Cell Calcifying Sertoli Cell Tumor in a Young Boy, not Associated with Peutz-Jeghers Syndrome or Carney Complex

    PubMed Central

    Lai, Jin-Ping; Lee, Chyi-Chia; Crocker, Melissa; Najmuddin, Mufaddal; Lange, Eileen; Merino, Maria; Stratakis, Constantine A

    2015-01-01

    Background Large cell calcifying sertoli cell tumor (LCCSCT) is an exceedingly rare lesion of the testicle. It is most often seen in patients with Carney complex (CNC) or Peutz-Jeghers syndrome (PJS). We now report the first pediatric patient with what appears to be bilateral LCCSCT and no other conditions or a genetic syndrome, such as PJS or CNC, have been associated with it. Methods A 10-year-old boy was found to have a right testicular mass during a routine pediatric examination; he underwent right orchiectomy. He was then evaluated clinically for PJS or CNC and underwent genetic testing. His tumor was studied by immunohistochemistry for the expression of calretinin, NY-ESO-1, inhibin, CD99, S100, PLAP, AE1/AE3, Bcl-2, p53, and Mib1. Results Patient did not have clinical features or genetic abnormalities of CNC and PJS. Microscopic features showed large, round or cubical intratubular and aggregated tumor cells with prominent nuclear atypia, large and prominent nucleoli and extensive calcification. In the Immunohistochemical studies, calretinin and inhibin alpha were up regulated in LCCSCT as compared to the adjacent benign Sertoli cells. Meanwhile, NY-ESO-1 and CD99 were down-regulated in LCCSCT. Focally and weakly positive S100 was found in the tumor tissue, but no S100 expression was present in the adjacent Sertoli cells. There was no expression of PLAP, P53, Bcl-2, Mib1 and AE1/AE3 in LCCSCT and adjacent Sertoli cells. Micro-calcifications were found in the other gonad by ultrasonography, suggesting LCCSCT. Conclusion LCCSCT is a rare testicular neoplasm, and may present in isolated rather than in more typical association with syndromes such as CNC and PJS. PMID:26587565

  9. Sertoli cells in culture secrete paracrine factor(s) that inhibit peritubular myoid cell proliferation: identification of heparinoids as likely candidates

    SciTech Connect

    Tung, P.S.; Fritz, I.B. )

    1991-06-01

    Conditioned medium from Sertoli cells, prepared from testes of 20-day-old rats, contains component(s) that inhibit the incorporation of (3H)-thymidine into DNA of peritubular myoid cells (PMC) and inhibit the proliferation of PMC. These components are trypsin-resistant, heat-stable compounds having a molecular weight less than 30,000. The active inhibitory components in Sertoli cell conditioned medium are inactivated by treatment with heparinase, but not by treatment with hyaluronidase or chondroitin sulfate lyases. Addition of heparin or heparan sulfate results in inhibition of DNA synthesis by PMC in a dose-dependent manner, whereas other glycosaminoglycans (GAGs) examined (hyaluronic acid, keratan sulfate, and chondroitin sulfate) have no detectable effects. Heparin and heparan sulfate are unique among GAGs tested in inhibiting the characteristic multilayer growth pattern of PMC following the attainment of confluence in serum-rich medium. On the basis of these and other data presented, it is concluded that heparin and other heparin-like GAGs synthesized by Sertoli cells are implicated in the modulation of growth of PMC in vitro during co-culture. It is postulated that heparin may play a similar role in maintaining the quiescent peritubular myoid cell phenotype in vivo.

  10. Androgen receptor binding to an androgen-responsive element in the promoter of the Srsf4 gene inhibits its expression in mouse Sertoli cells.

    PubMed

    Guo, Huan; Li, Yuchi; Luo, Manling; Lin, Shouren; Chen, Jianbo; Ma, Qian; Gu, Yanli; Jiang, Zhimao; Gui, Yaoting

    2015-12-01

    The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y) ) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells. Mol. Reprod. Dev. 82: 976-985, 2015. 2015 Wiley Periodicals, Inc. PMID:26308373

  11. Follicle-stimulating hormone amplifies insulin-like growth factor I-mediated activation of AKT/protein kinase B signaling in immature rat Sertoli cells.

    PubMed

    Khan, Shafiq A; Ndjountche, Lilianne; Pratchard, Lauren; Spicer, L J; Davis, John S

    2002-06-01

    FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 microM), (Bu)(2)cAMP (40 microM), or forskolin (10 microM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10-20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1-4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 microM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells. PMID:12021190

  12. Testicular juvenile granulosa cell and Sertoli cell tumours: a clinicopathological study of 29 cases from the Kiel Paediatric Tumour Registry.

    PubMed

    Harms, D; Kock, L R

    1997-04-01

    Testicular Sertoli cell tumours (SCT) and juvenile granulosa cell tumours (JGCT) are rare in childhood. This study was designed to investigate the clinical picture, morphology and disease course in a comparatively large series of cases (total number = 29). Of 198 cases of childhood testicular tumour documented in the Kiel Paediatric Tumour Registry 18 were cases of infantile SCT (9.1%) and 11 of JGCT (5.6%). The average age at the time of diagnosis was 4.2 months for infantile SCT and 0.4 months for IGCT. SCT and JGCT often showed infiltrative growth into adjacent testicular tissue, dense cellularity and considerable proliferation activity. Immunohistochemically all cases expressed vimentin intermediate filaments in both tumour types. Next in frequency of expression were cytokeratins (SCT: 7/16; JGCT: 7/10) and smooth-muscle actin (SCT: 9/15; JGCT: 4/10). Follow-up studies (24/29) showed that in cases of tumour manifestation in infancy and after complete tumour removal (usually orchiectomy) no local recurrences and no metastases occurred. The most important conclusion for diagnosis and therapy is that despite infiltrative growth, incomplete differentiation, dense cellularity and considerable proliferation activity, after surgical excision infantile SCT and JGCT have a good prognosis. Adjuvant chemotherapy or more extensive operations with lymphadenectomy are thus not indicated. PMID:9134041

  13. Ultrastructural modifications in the mitochondrion of mouse Sertoli cells after inhalation of lead, cadmium or lead-cadmium mixture.

    PubMed

    Bizarro, Patricia; Acevedo, Sandra; Nio-Cabrera, Geraldine; Mussali-Galante, Patricia; Pasos, Francisco; Avila-Costa, Maria Rosa; Fortoul, Teresa I

    2003-01-01

    CD-1 mice inhaled 0.01 M lead acetate, 0.006 M cadmium chloride or Pb-Cd mixture during 1h twice a week during 4 weeks. Testes were processed for transmission electron microscopic analysis. The percentage of damaged mitochondria was related to exposure time and the type of metal inhaled, noticing more damage when the mixture was administered. A dose-time relationship was found. Cadmium chloride caused the most severe mitochondrial alteration compared to lead acetate, whereas the mixture was more aggressive compared with each metal alone. Our results suggest that the changes in Sertoli cell could lead to a transformation process that may interfere with spermatogenesis. PMID:14555194

  14. Use of Aromatase Inhibitors in Large Cell Calcifying Sertoli Cell Tumors: Effects on Gynecomastia, Growth Velocity, and Bone Age

    PubMed Central

    Crocker, Melissa K.; Gourgari, Evgenia; Stratakis, Constantine A.

    2014-01-01

    Context: Large cell calcifying Sertoli cell tumors (LCCSCT) present in isolation or, especially in children, in association with Carney Complex (CNC) or Peutz-Jeghers Syndrome (PJS). These tumors overexpress aromatase (CYP19A1), which leads to increased conversion of delta-4-androstenedione to estrone and testosterone to estradiol. Prepubertal boys may present with growth acceleration, advanced bone age, and gynecomastia. Objective: To investigate the outcomes of aromatase inhibitor therapy (AIT) in prepubertal boys with LCCSCTs. Design: Case series of a very rare tumor and chart review of cases treated at other institutions. Setting: Tertiary care and referral center. Patients: Six boys, five with PJS and one with CNC, were referred to the National Institutes of Health for treatment of LCCSCT. All patients had gynecomastia, testicular enlargement, and advanced bone ages, and were being treated by their referring physicians with AIT. Interventions: Patients were treated for a total of 660 months on AIT. Main Outcome Measures: Height, breast tissue mass, and testicular size were all followed; physical examination, scrotal ultrasounds, and bone ages were obtained, and hormonal concentrations and tumor markers were measured. Results: Tumor markers were negative. All patients had decreases in breast tissue while on therapy. Height percentiles declined, and predicted adult height moved closer to midparental height as bone age advancement slowed. Testicular enlargement stabilized until entry into central puberty. Only one patient required unilateral orchiectomy. Conclusions: Patients with LCCSCT benefit from AIT with reduction and/or elimination of gynecomastia and slowing of linear growth and bone age advancement. Further study of long-term outcomes and safety monitoring are needed but these preliminary data suggest that mammoplasty and/or orchiectomy may be foregone in light of the availability of medical therapy. PMID:25226294

  15. Early Postnatal Exposure to a Low Dose of Decabromodiphenyl Ether Affects Expression of Androgen and Thyroid Hormone Receptor-Alpha and Its Splicing Variants in Mouse Sertoli Cells

    PubMed Central

    Miyaso, Hidenobu; Nakamura, Noriko; Naito, Munekazu; Hirai, Shuichi; Matsuno, Yoshiharu; Itoh, Masahiro; Mori, Chisato

    2014-01-01

    Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (Thra) transcripts and its splicing variants and androgen receptor (Ar) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the Ar, Thra and its splicing variants, and Thra splicing factors (Hnrnpa1, Srsf1, and Hnrnph1) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding Ar, Thra, and its variant, Thra1, declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or Thra2, Hnrnph1, or Srsf1 transcript levels were observed between control and decaBDE-exposed mice. However, the Thra1:Thra2 and Hnrnpa1:Srsf1 ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of Ar and Thra transcripts in Sertoli cells, accompanied by an imbalance in the ratios of Thra splicing variants, resulting in smaller testicular size and impaired spermatogenesis. PMID:25479311

  16. Identification of genetic networks involved in the cell injury accompanying endoplasmic reticulum stress induced by bisphenol A in testicular Sertoli cells

    SciTech Connect

    Tabuchi, Yoshiaki . E-mail: ytabu@cts.u-toyama.ac.jp; Takasaki, Ichiro; Kondo, Takashi

    2006-07-07

    To identify detailed mechanisms by which bisphenol A (BPA), an endocrine-disrupting chemical, induces cell injury in mouse testicular Sertoli TTE3 cells, we performed genome-wide microarray and computational gene network analyses. BPA (200 {mu}M) significantly decreased cell viability and simultaneously induced an increase in mRNA levels of HSPA5 and DDIT3, endoplasmic reticulum (ER) stress marker genes. Of the 22,690 probe sets analyzed, BPA down-regulated 661 probe sets and up-regulated 604 probe sets by >2.0-fold. Hierarchical cluster analysis demonstrated nine gene clusters. In decreased gene clusters, two significant genetic networks were associated with cell growth and proliferation and the cell cycle. In increased gene clusters, two significant genetic networks including many basic-region leucine zipper transcription factors were associated with cell death and DNA replication, recombination, and repair. The present results will provide additional novel insights into the detailed molecular mechanisms of cell injury accompanying ER stress induced by BPA in Sertoli cells.

  17. Implication of actin microfilaments in maintenance of intercellular bridges and the Sertoli cell barrier in the rat seminiferous epithelium

    SciTech Connect

    Weber, J.E.

    1987-01-01

    Within the seminiferous epithelium, germ cells are connected to one another by intercellular bridges. Additionally, young germ cells are separated from more advanced germ cells by the Sertoli cell barrier, the occluding junctions of which are associated with actin microfilaments. To examine how microfilaments influence these structures in the rat the actin-disrupting agent cytochalasin D (CD) was injected intratesticularly (i.t.). In preliminary studies the optical injection volume was found to be 50 {mu}l and, by using the dye trypan blue, the injected solution was shown to enter the lymphatic system and rapidly spread throughout the testis. A 50% clearance of {sup 3}H-insulin from the testis was achieved at 3 hr and 95% by 24 hr. Vehicles with varying solubility properties did not cause testicular damage. Intercellular bridges were found to be dynamic structures. As spermatogenesis progressed, the bridge diameter gradually increased. The formation and degradation of bridge partitioning complexes within pre-existing bridges of dividing cells were described.

  18. Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

    PubMed Central

    Papadopoulos, Dimitrios; Dietze, Raimund; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-01-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility. PMID:26938869

  19. Mono-(2-ethylhexyl) phthalate (MEHP) affects intercellular junctions of Sertoli cell: A potential role of oxidative stress.

    PubMed

    Sobarzo, Cristian M; Rosana, Nogueira de Morais; Livia, Lustig; Berta, Denduchis; Schteingart, Helena F

    2015-12-01

    We analyzed the potential role of oxidative stress induced by mono (2-ethylhexyl) phthalate (MEHP) in adherent cell junction protein expression of prepubertal rat Sertoli cells (SC) in vitro. Five-day SC cultures were treated with MEHP (200?M) for 24h and compared to cells in basal conditions. Western blot and immunofluorescent (IF) analyses showed that MEHP induced increase of N-cadherin and catenin expression, modifying its distribution. Concomitantly, Cx-43 expression decreased significantly and delocalization of the IF signal for tight junction proteins (occludin, claudin-11 and ZO-1) occurred. Indicative of oxidative stress, MEHP induced in SC an increase of lipoperoxides, a decrease in glutathione (GSH) levels and a concomitant increase in Glutathione S-Transferases (GST) activity. Antioxidant N-acetyl-cysteine (1mM) treatment prevented GSH decrease and N-cadherin and ?-catenin up-regulation induced by MEHP. Our data suggest that oxidative stress signaling is a mechanism involved in adherent cell junctions disruption induced by MEHP in SC cultures. PMID:26498383

  20. Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids, triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells.

    PubMed

    Boulogne, Barbara; Habert, Ren; Levacher, Christine

    2003-06-01

    The regulation of early fetal germ cell growth has not been studied in cell culture, probably due to the poor survival of these cells. However, cell culture is the only system in which the control of cell growth can be studied independently of the influence of secreted testicular factors, which are diluted in the medium. We successfully cultured dispersed testicular cells from 16.5-day-old rat fetuses in defined medium and compared the growth of these cells with that of cells from 3-day-old neonates. In this system, fetal gonocytes displayed low levels of mitotic activity and their numbers remained stable. In contrast, neonatal gonocytes displayed high levels of mitotic activity and increased in number, these characteristics resembling those observed in vivo. We found that retinoic acid had deleterious effects on the number of gonocytes but did not affect Sertoli cell proliferation in fetal and neonatal cell cultures. Moreover, in fetal cell cultures, the decrease in the number of gonocytes resulted from a decrease in mitotic activity, probably due to a direct effect of retinoids on fetal gonocytes. Among the selective agonists for the retinoic acid receptor (RARalpha agonist, RARbeta agonist, and RARgamma agonist) and the retinoic X receptor (pan-RXR agonist) tested, only the RARalpha agonist reproduced the effects of retinoic acid at concentrations lower than its Kd value in both fetal and neonatal cell cultures. As both RARalpha and RXRalpha are present in fetal and neonatal gonocytes, we suggest that retinoic acid exerts its effects on gonocytes via a RARalpha-RXRalpha heterodimer, with RARalpha functioning as an active partner and RXRalpha as a passive partner. In this culture system, we show for the first time that triiodothyronine (T3) inhibits testicular fetal Sertoli cell and germ cell growth. We also tested intracellular signaling factors and found that a cAMP analog increased Sertoli cell proliferation and germ cell survival in both fetal and neonatal cells whereas phorbol esters (PMA) strongly inhibited the proliferation of fetal but not of neonatal gonocytes. None of the tested factors (T3, dbcAMP, and PMA) seemed to interact with the all-trans retinoic acid pathway. Thus, fetal gonocytes and neonatal gonocytes differ in intrinsic properties, and their growth is not regulated in the same manner. Despite their low level of mitotic activity, fetal gonocytes were more sensitive to various factors than neonatal gonocytes. PMID:12704731

  1. Sustained Expression of Insulin by a Genetically Engineered Sertoli Cell Line after Allotransplantation in Diabetic BALB/c Mice1

    PubMed Central

    Kaur, Gurvinder; Thompson, Lea Ann; Pasham, Mithun; Tessanne, Kim; Long, Charles R.; Dufour, Jannette M.

    2014-01-01

    ABSTRACT Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression. PMID:24695630

  2. Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

    SciTech Connect

    Tabuchi, Yoshiaki . E-mail: ytabu@ms.toyama-mpu.ac.jp; Kondo, Takashi; Suzuki, Yoshihisa; Obinata, Masuo

    2005-04-15

    Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 deg C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 deg C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21{sup waf1}, milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.

  3. Smad2/3 Upregulates the Expression of Vimentin and Affects Its Distribution in DBP-Exposed Sertoli Cells

    PubMed Central

    Zhang, Xi; Wang, Xiaogang; Liu, Taixiu; Mo, Min; Ao, Lin; Liu, Jinyi; Cao, Jia; Cui, Zhihong

    2015-01-01

    Sertoli cells (SCs) in the testes provide physical and nutritional support to germ cells. The vimentin cytoskeleton in SCs is disrupted by dibutyl phthalate (DBP), which leads to SCs dysfunction. In a previous study, we found that peroxisome proliferator-activated receptor alpha (PPARα) influenced the distribution of vimentin by affecting its phosphorylation in DBP-exposed SCs. In the present study, we investigated the role of Smad2/3 in regulating the expression of vimentin in DBP-exposed SCs. We hypothesized that Smad2/3 affects the distribution of vimentin by regulating its expression and that there is cross talk between Smad2/3 and PPARα. The real-time PCR and ChIP-qPCR results showed that SB431542 (an inhibitor of Smad2/3) could significantly attenuate the expression of vimentin induced by DBP in SCs. Phosphorylated and soluble vimentin were both downregulated by SB431542 pretreatment. WY14643 (an agonist of PPARα) pretreatment stimulated, while GW6471 (an antagonist of PPARα) inhibited, the activity of Smad2/3; SB431542 pretreatment also inhibited the activity of PPARα, but it did not rescue the DBP-induced collapse in vimentin. Our results suggest that, in addition to promoting the phosphorylation of vimentin, DBP also stimulates the expression of vimentin by activating Smad2/3 in SCs and thereby induces irregular vimentin distribution. PMID:26819576

  4. Toxicogenomic Screening of Replacements for Di(2-Ethylhexyl) Phthalate (DEHP) Using the Immortalized TM4 Sertoli Cell Line.

    PubMed

    Nardelli, Thomas C; Erythropel, Hanno C; Robaire, Bernard

    2015-01-01

    Phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) are being phased out of many consumer products because of their endocrine disrupting properties and their ubiquitous presence in the environment. The concerns raised from the use of phthalates have prompted consumers, government, and industry to find alternative plasticizers that are safe, biodegradable, and have the versatility for multiple commercial applications. We examined the toxicogenomic profile of mono(2-ethylhexyl) phthalate (MEHP, the active metabolite of DEHP), the commercial plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH), and three recently proposed plasticizers: 1,4-butanediol dibenzoate (BDB), dioctyl succinate (DOS), and dioctyl maleate (DOM), using the immortalized TM4 Sertoli cell line. Results of gene expression studies revealed that DOS and BDB clustered with control samples while MEHP, DINCH and DOM were distributed far away from the control-DOS-BDB cluster, as determined by principle component analysis. While no significant changes in gene expression were found after treatment with BDB and DOS, treatment with MEHP, DINCH and DOM resulted in many differentially expressed genes. MEHP upregulated genes downstream of PPAR and targeted pathways of cholesterol biosynthesis without modulating the expression of PPAR's themselves. DOM upregulated genes involved in glutathione stress response, DNA repair, and cholesterol biosynthesis. Treatment with DINCH resulted in altered expression of a large number of genes involved in major signal transduction pathways including ERK/MAPK and Rho signalling. These data suggest DOS and BDB may be safer alternatives to DEHP/MEHP than DOM or the commercial alternative DINCH. PMID:26445464

  5. Sperm retrieval and live birth rates in presumed Sertoli-cell-only syndrome in testis biopsy: a single centre experience.

    PubMed

    Gul, U; Turunc, T; Haydardedeoglu, B; Yaycioglu, O; Kuzgunbay, B; Ozkardes, H

    2013-01-01

    We aimed to investigate sperm retrieval rates (SRR) by testicular sperm extraction (TESE), factors affecting SRR, and fertilization rate (FR), implantation rate (IR), clinical pregnancy rate (CPR) and live birth rate (LBR) in patients with presumed Sertoli-cell-only syndrome in testis biopsy (SCOS). We retrospectively evaluated files of 134 patients with SCOS who underwent TESE. Group I were patients in whom spermatozoa were retrieved and Group II were patients in whom no spermatozoa could be retrieved. SRR, Follicle stimulating hormone (FSH), Luteinizing hormone (LH), and testosterone levels, and the volume of testicles were compared between groups. In addition, FR, IR, CPR and LBR were determined. Sperm retrieval was achieved in 37 (27.6%) patients (Group I), and the remaining 97 (72.4%) patients made Group II. There were no significant differences in age, infertility time, testicular volume, serum FSH, LH and testosterone levels between Groups I and II (p>0.05). Intracytoplasmic sperm injection (ICSI) was performed in 36 patients. FR, IR, and CPR were 60.8623.03, 36.5341.78 and 51.3% respectively. Cycle and patient based LBRs were 37.8 and 45.1% respectively. SRR in SCOS is lower than patients with non-obstructive azoospermia (NOA) in general. No parameters to predict spermatozoa retrieval were determined. In patients with SCOS, ICSI achieves similar live birth rate to other patients with NOA. PMID:23258629

  6. Melatonin regulates the development and function of bovine Sertoli cells via its receptors MT1 and MT2.

    PubMed

    Yang, Wu-Cai; Tang, Ke-Qiong; Fu, Chang-Zhen; Riaz, Hasan; Zhang, Qiong; Zan, Lin-Sen

    2014-06-10

    Melatonin and its receptors are found in the testis of many species, where they mediate testicular functions. The present study aimed to investigate the expression of melatonin receptors (MT1 and MT2) in bovine Sertoli cells (SCs), using reverse transcription polymerase chain reaction (RT-PCR) and western blot. In addition, we assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after treatment with various concentrations (0, 80, 160, and 320 pg/mL) of melatonin at different time points (24, 48, or 72 h). We found that bovine SCs express MT1 and MT2 receptors, which were regulated by melatonin in time- and dose-dependent manners after treatment with melatonin. Exogenous melatonin up-regulated the expression of spermatogenesis-related genes, including Cyclin D1, Cyclin E, Pdgfa, Dhh, Occludin, and Claudin, and decreased the mRNA levels of P21 and Kit1 in a time or dose-dependent manner. Meanwhile, melatonin supplementation significantly affected Inhba, Inhbb and Inha mRNA expression. These findings were consistent with inhibin B levels detected in the culture medium. In conclusion, exogenous melatonin acts via its receptors and appears to play regulatory roles in the development and function of bovine SCs. PMID:24768045

  7. Characterization of a new superfusion, two-compartment culture system for Sertoli cells: influence of extracellular matrix on the cell permeability and dynamics of transferrin secretion.

    PubMed

    Guillou, F

    1990-01-01

    A new superfusion two-compartment culture system was developed in the laboratory of the author and was used to investigate the influence of testicular extracellular matrix on barrier formation by Sertoli cells in culture, and the acute dynamic changes in the bidirectional secretion of transferrin. Only Sertoli cells growing on extracellular matrix formed a monolayer that was specifically impermeable to inulin, peroxidase, and FSH, but did not affect the passage of testosterone. Moreover, in these conditions, they were highly polarized morphologically. The bidirectional secretion (basal/apical ratio) of transferrin was affected by the duration of the stationary culture preceding the superfusion. After 2 days of culture, the amount of transferrin secreted during the subsequent 20 h superfusion was higher in the basal chamber than in the apical chamber. In contrast, after 5 days of culture, the amount of secreted transferrin was higher in the apical compartment. The author compared his data with those previously reported by other authors, using stationary two-compartment chambers. PMID:2324005

  8. Effect of transglutaminase substrates and polyamines on the cellular sequestration and processing of follicle-stimulating hormone by rat Sertoli cells

    SciTech Connect

    Dias, J.A.

    1986-08-01

    Transglutaminase (TGase) substrates monodansyl cadaverine (MDC, monodansyl-1,5 diaminopentane) and methylamine (MA) and polyamines (PA) were tested for their effects on the cellular processing of radioiodinated human follicle-stimulating hormone (/sup 125/I-hFSH). Specifically bound /sup 125/I-hFSH that could be released from cells during 10-min incubation period with acidified (pH 3.9) Hanks balanced-salt solution was considered membrane-bound unsequestered hormone. The rate at which cells sequestered /sup 125/I-hFSH into cellular compartments resistant to acid dissociation depended on the length of time in which cells were incubated with hormone. Cells incubated with /sup 125/I-hFSH for 15, 60, and 120 min had half-lives of sequestration of 26, 55 and 67 min respectively. One hundred-micromolar MDC inhibited degradation of /sup 125/I-hFSH as measured by the presence of radioactivity in the medium that was soluble in trichloroacetic acid. The rate of sequestration was never slower than that of controls, indicating that MDC did not decrease the ability of Sertoli cells to sequester /sup 125/I-hFSH. Despite these two observations, radioactivity associated with cells (acid-resistant radioactivity) was lower in cells treated with MDC than in controls. No effect of MDC on specific binding of 125I-hFSH was observed. Similar results were observed with MA, albeit at higher levels (0.0025-0.0425 M), consistent with their relative potency to inhibit TGase activity. Polyamines, spermine, and putrescine also decreased cell-associated radioactivity despite decreasing degradation of hFSH. TGase substrates (MDC, MA, PA) prevented entry of sequestered 125I-hFSH into the degradative pathways of Sertoli cells. These data suggest that transglutamination may influence the fate of sequestered FSH in Sertoli cells but not the rate at which sequestration occurs.

  9. Novel Role for p110? PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

    PubMed Central

    Guillermet-Guibert, Julie; Smith, Lee B.; Halet, Guillaume; Whitehead, Maria A.; Pearce, Wayne; Rebourcet, Diane; Len, Kelly; Crpieux, Pascale; Nock, Gemma; Strmstedt, Maria; Enerback, Malin; Chelala, Claude; Graupera, Mariona; Carroll, John; Cosulich, Sabina; Saunders, Philippa T. K.; Huhtaniemi, Ilpo; Vanhaesebroeck, Bart

    2015-01-01

    The organismal roles of the ubiquitously expressed class I PI3K isoform p110? remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110?, we document that full inactivation of p110? leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110? kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110? results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110? was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110? also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110? inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110? inactivation. In line with a crucial role for p110? in SCs, selective inactivation of p110? in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110? and AR have previously been reported to functionally interact. PMID:26132308

  10. Sertoli-cell-specific knockout of connexin 43 leads to multiple alterations in testicular gene expression in prepubertal mice

    PubMed Central

    Giese, Sarah; Hossain, Hamid; Markmann, Melanie; Chakraborty, Trinad; Tchatalbachev, Svetlin; Guillou, Florian; Bergmann, Martin; Failing, Klaus; Weider, Karola; Brehm, Ralph

    2012-01-01

    SUMMARY A significant decline in human male reproductive function has been reported for the past 20 years but the molecular mechanisms remain poorly understood. However, recent studies showed that the gap junction protein connexin-43 (CX43; also known as GJA1) might be involved. CX43 is the predominant testicular connexin (CX) in most species, including in humans. Alterations of its expression are associated with different forms of spermatogenic disorders and infertility. Men with impaired spermatogenesis often exhibit a reduction or loss of CX43 expression in germ cells (GCs) and Sertoli cells (SCs). Adult male transgenic mice with a conditional knockout (KO) of the Gja1 gene [referred to here as connexin-43 (Cx43)] in SCs (SCCx43KO) show a comparable testicular phenotype to humans and are infertile. To detect possible signaling pathways and molecular mechanisms leading to the testicular phenotype in adult SCCx43KO mice and to their failure to initiate spermatogenesis, the testicular gene expression of 8-day-old SCCx43KO and wild-type (WT) mice was compared. Microarray analysis revealed that 658 genes were significantly regulated in testes of SCCx43KO mice. Of these genes, 135 were upregulated, whereas 523 genes were downregulated. For selected genes the results of the microarray analysis were confirmed using quantitative real-time PCR and immunostaining. The majority of the downregulated genes are GC-specific and are essential for mitotic and meiotic progression of spermatogenesis, including Stra8, Dazl and members of the DM (dsx and map-3) gene family. Other altered genes can be associated with transcription, metabolism, cell migration and cytoskeleton organization. Our data show that deletion of Cx43 in SCs leads to multiple alterations of gene expression in prepubertal mice and primarily affects GCs. The candidate genes could represent helpful markers for investigators exploring human testicular biopsies from patients showing corresponding spermatogenic deficiencies and for studying the molecular mechanisms of human male sterility. PMID:22699423

  11. Effects of 4-nonylphenol isomers on cell receptors and mitogen-activated protein kinase pathway in mouse Sertoli TM4 cells.

    PubMed

    Liu, Xiaozhen; Nie, Shaoping; Chen, Yangjie; Huang, Danfei; Xie, Mingyong

    2014-12-01

    In the present study, experiments were performed to investigate the effects of nonylphenol (NP) isomers (4-[1,2, 4-trimethylhexyl]-phenol (NP41), 4-[1,2, 5-trimethylhexyl]-phenol (NP42)) on Sertoli TM4 cells. NP41 decreased mRNA expression levels of androgen receptor and toll-like receptor (TLR)-4 in 20-40?M (P<0.05), and increased mRNA levels of estrogen receptor (ER)-? and progesterone receptor in 1-40?M (P<0.05). NP42 treatment only evoked significant decrease in mRNA expression levels of ER-? in 20-40?M (P<0.05). Similarly, NP41 (1-40?M) drastically increased the protein expression of ER-?, which was significantly decreased in 20-40?M NP42 groups (P<0.01). Both NP41 and NP42 showed no effect on the expression of ER-?. Protein levels of follicle stimulating hormone receptor were increased significantly in high concentrations of NP41 (40?M) and NP42 (10-40?M) challenged cells. Furthermore, NP41 and NP42 showed various effects on the expression of junction-associated molecules and inhibin B secretion in TM4 cells. Additionally, activation of JNK1/2 pathway was induced by NP41 and NP42. However, ERK1/2 and p38 pathways were inhibited in TM4 cells exposed to low concentrations of NP41 (0.1-20?M) and NP42 (0.1-1?M), and high concentrations of NP41 (40?M) and NP42 (10-40?M) resulted in a return of p-ERK1/2 and p-p38 to control levels. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signaling pathways, and the effects may be related to the structure of NP isomer. PMID:25242005

  12. Leptin modulates human Sertoli cells acetate production and glycolytic profile: a novel mechanism of obesity-induced male infertility?

    PubMed

    Martins, Ana D; Moreira, Ana C; Sá, Rosália; Monteiro, Mariana P; Sousa, Mário; Carvalho, Rui A; Silva, Branca M; Oliveira, Pedro F; Alves, Marco G

    2015-09-01

    Human feeding behavior and lifestyle are gradually being altered, favoring the development of metabolic diseases, particularly type 2 diabetes and obesity. Leptin is produced by the adipose tissue acting as a satiety signal. Its levels have been positively correlated with fat mass and hyperleptinemia has been proposed to negatively affect male reproductive function. Nevertheless, the molecular mechanisms by which this hormone affects male fertility remain unknown. Herein, we hypothesize that leptin acts on human Sertoli cells (hSCs), the "nurse cells" of spermatogenesis, altering their metabolism. To test our hypothesis, hSCs were cultured without or with leptin (5, 25 and 50ng/mL). Leptin receptor was identified by qPCR and Western blot. Protein levels of glucose transporters (GLUT1, GLUT2 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by Western Blot. LDH activity was assessed and metabolite production/consumption determined by proton nuclear magnetic resonance. Oxidative damage was evaluated by assessing lipid peroxidation, protein carbonilation and nitration. Our data shows that leptin receptor is expressed in hSCs. The concentration of leptin found in lean, healthy patients, upregulated GLUT2 protein levels and concentrations of leptin found in lean and obese patients increased LDH activity. Of note, all leptin concentrations decreased hSCs acetate production illustrating a novel mechanism for this hormone action. Moreover, our data shows that leptin does not induce or protect hSCs from oxidative damage. We report that this hormone modulates the nutritional support of spermatogenesis, illustrating a novel mechanism that may be linked to obesity-induced male infertility. PMID:26071642

  13. Pituitary binding of 3H-labeled Sertoli cell factor in vitro: a potential radioreceptor assay for inhibin.

    PubMed

    Seethalakshmi, L; Steinberger, A; Steinberger, E

    1984-10-01

    We have previously demonstrated that binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The present study further characterized the binding of [3H]SCF to rat anterior pituitary in vitro and explored its potential use as a radioreceptor assay for SCF and other inhibin preparations. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 mu Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of approximately 20,000 dpm/micrograms protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing one pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37 C; 30 min) or heat (100 C; 10 min). Storage of the pituitary glands at -20 C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high mol wt fraction of ram testis fluid (mol wt, greater than 10,000), low mol wt fraction of ram testis fluid (mol wt, less than 5,000), ovine testicular lymph, and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low mol wt fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). In contrast, various noninhibin substances tested [rat androgen-binding protein, bovine LH, BSA, native GnRH, or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-EA and D-Ala6-des-Gly10-GnRH-N-EA] did not significantly compete for the [3H]SCF binding. The binding ability correlated well with inhibin biological activity in vitro. These results provide additional evidence for the presence of SCF-binding sites in the rat anterior pituitary which interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. Our results also indicate that the pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations. PMID:6090098

  14. Toxicogenomic Screening of Replacements for Di(2-Ethylhexyl) Phthalate (DEHP) Using the Immortalized TM4 Sertoli Cell Line

    PubMed Central

    Nardelli, Thomas C.; Erythropel, Hanno C.; Robaire, Bernard

    2015-01-01

    Phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) are being phased out of many consumer products because of their endocrine disrupting properties and their ubiquitous presence in the environment. The concerns raised from the use of phthalates have prompted consumers, government, and industry to find alternative plasticizers that are safe, biodegradable, and have the versatility for multiple commercial applications. We examined the toxicogenomic profile of mono(2-ethylhexyl) phthalate (MEHP, the active metabolite of DEHP), the commercial plasticizer diisononyl cyclohexane-1,2-dicarboxylate (DINCH), and three recently proposed plasticizers: 1,4-butanediol dibenzoate (BDB), dioctyl succinate (DOS), and dioctyl maleate (DOM), using the immortalized TM4 Sertoli cell line. Results of gene expression studies revealed that DOS and BDB clustered with control samples while MEHP, DINCH and DOM were distributed far away from the control-DOS-BDB cluster, as determined by principle component analysis. While no significant changes in gene expression were found after treatment with BDB and DOS, treatment with MEHP, DINCH and DOM resulted in many differentially expressed genes. MEHP upregulated genes downstream of PPAR and targeted pathways of cholesterol biosynthesis without modulating the expression of PPAR’s themselves. DOM upregulated genes involved in glutathione stress response, DNA repair, and cholesterol biosynthesis. Treatment with DINCH resulted in altered expression of a large number of genes involved in major signal transduction pathways including ERK/MAPK and Rho signalling. These data suggest DOS and BDB may be safer alternatives to DEHP/MEHP than DOM or the commercial alternative DINCH. PMID:26445464

  15. Dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone in male reproductive health: Implications of differential regulation of human Sertoli cells metabolic profile.

    PubMed

    Dias, Tânia R; Alves, Marco G; Almeida, Susana P; Silva, Joaquina; Barros, Alberto; Sousa, Mário; Silva, Branca M; Silvestre, Samuel M; Oliveira, Pedro F

    2015-11-01

    Dehydroepiandrosterone (DHEA) is a precursor of androgen synthesis whose action is partially exerted through its metabolites. 7-Oxo-dehydroepiandrosterone (7-oxo-DHEA) is a common DHEA metabolite, non-convertible to androgens, which constitutes a promising therapeutic strategy for multiple conditions. Sertoli cells (SCs) are responsible for the support of spermatogenesis, having unique metabolic characteristics strongly modulated by androgens. Consequently, disruptions in androgen synthesis compromise SCs function and hence male fertility. We aimed to evaluate the effects of DHEA and 7-oxo-DHEA in human SCs (hSCs) metabolism and oxidative profile. To do so, hSCs were exposed to increasing concentrations of DHEA and 7-oxo-DHEA (0.025, 1 and 50 μM) that revealed to be non-cytotoxic in these experimental conditions. We measured hSCs metabolites consumption/production by (1)H NMR, the protein expression levels of key players of the glycolytic pathway by Western blot as well as the levels of carbonyl groups, nitration and lipid peroxidation by Slot blot. The obtained data demonstrated that 7-oxo-DHEA is a more potent metabolic modulator than DHEA since it increased hSCs glycolytic flux. DHEA seem to redirect hSCs metabolism to the Krebs cycle, while 7-oxo-DHEA has some inhibitory effect in this path. The highest 7-oxo-DHEA concentrations (1 and 50 μM) also increased lactate production, which is of extreme relevance for the successful progression of spermatogenesis in vivo. None of these steroids altered the intracellular oxidative profile of hSCs, illustrating that, at the concentrations used they do not have pro- nor antioxidant actions in hSCs. Our study represents a further step in the establishment of safe doses of DHEA and 7-oxo-DHEA to hSCs, supporting its possible use in hormonal and non-hormonal therapies against male reproductive problems. PMID:26134425

  16. Glucocorticoid receptors in murine erythroleukaemic cells

    SciTech Connect

    Hammond, K.D.; Torrance, J.M.; DiDomenico, M.

    1987-01-01

    Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation. Specific binding of dexamethasone was measured. A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified. Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 +/- 8.2 pmol/g protein) than in untreated controls (87.9 +/- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM). Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.

  17. Listeriolysin O, but not Murine E-cadherin, is Involved in Invasion of Listeria monocytogenes into Murine Liver Parenchymal Cells

    PubMed Central

    Kanayama, Yu-ju; Kaneko, Masakazu; Emoto, Yoshiko; Emoto, Masashi

    2015-01-01

    Human E-cadherin and listeriolysin O (LLO) are involved in invasion of Listeria monocytogenes into human liver parenchymal cells (LPC). Yet, it remains to be determined whether murine E-cadherin and LLO participate in invasion of L. monocytogenes into murine LPC. In the present study, involvement of murine E-cadherin and LLO in invasion of L. monocytogenes into murine LPC was investigated. Murine E-cadherin was expressed on murine LPC, but the expression became undetectable by insertion of transgene of Simian virus 40 large T antigen. Although invasion of L. monocytogenes into murine LPC was found regardless of murine E-cadherin expression, infection rate of L. monocytogenes being unable to secrete LLO was lower than that of L. monocytogenes being capable of secreting LLO. Our RESULTS verify that invasion of L. monocytogenes into murine LPC occurs independently of murine E-cadherin and indicate that LLO participates in invasion of L. monocytogenes into murine LPC. PMID:26668665

  18. Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

    PubMed Central

    Monaco, L; Foulkes, N S; Sassone-Corsi, P

    1995-01-01

    Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479863

  19. Isolation of murine adipose-derived stem cells.

    PubMed

    Yu, Gang; Wu, Xiying; Kilroy, Gail; Halvorsen, Yuan-Di C; Gimble, Jeffrey M; Floyd, Z Elizabeth

    2011-01-01

    Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Primary adipocytes grown in culture and derived from murine adipose tissue are essential for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stem cells along with a protocol for inducing adipogenesis in this cell population. PMID:21082392

  20. [The ultrastructural manifestations of the regenerative processes in the Sertoli cells under the action of low-intensity electromagnetic radiation in the rats subjected to stress].

    PubMed

    Korolev, Yu N; Geniatulina, M S; Nikulina, L A; Mikhailik, L V

    2015-01-01

    The experiments on the outbred female rats using the electron microscopic technique have demonstrated that the application of ultrahigh frequency low-intensity electromagnetic radiation (LIEMR) with a flux density below 1 mCW/Cm2 and a frequency of approximately 1,000 MHz in the regime of primary prophylaxis and therapeutic-preventive action suppressed the development of the post-stress pathological ultrastructural changes and increased the activity of the regenerative processes in the Sertoli cells. It was shown that the developing adaptive and compensatory changes in the Sertoli cells most frequently involve the energy-producing structures (mitochondria) that undergo the enlargement of their average and total dimensions. Simultaneously, the amount of granular endoplasmic reticulum and the number of ribosomes increased while the intracellular links between the organelles strengthened and the reserve potential of the cells improved. It is concluded that the observed effects may be due to the action of both local and systemic regulation mechanisms. PMID:26285333

  1. Very high alpha-fetoprotein in a young man due to concomitant presentation of hepatocellular carcinoma and Sertoli cell testis tumor

    PubMed Central

    Ersoy, Ozdal

    2005-01-01

    Studies reported that there is a close relationship between hepatocellular carcinoma (HCC) and testis carcinoma. Both tumors can be presented as synchronal tumors, or as testicular metastases of HCC or as hepatic metastases of testicular tumor[7]. HCC is one of the most common malignancies worldwide and the incidence of HCC increases with age[8]. The relationship between hepatitis B incidence and HCC rates is also well recognized. Alpha fetoprotein (AFP) is produced by 70% of HCC. Though a level of AFP >400 ng/mL is diagnostic for HCC, in the presence of active hepatitis B infection, the cut-off level should be considered to be at least 1 000-4 000 ng/mL. Like HCC, germ cell tumors of the testis also release AFP; but it is shown that some of Sertoli cell tumors of testis can also release AFP[10]. Herein we have reported about the first case of HCC in the literature which is presented concomitantly with Sertoli-Leydig tumor of testis, leading to extremely high level of AFP in a 21-year-old man. PMID:16437617

  2. Assessment of testicular function after acute and chronic irradiation: Further evidence for an influence of late spermatids on Sertoli cell function in the adult rat

    SciTech Connect

    Pineau, C.; Velez de la Calle, J.F.; Pinon-Lataillade, G.; Jegou, B.

    1989-06-01

    To study cell to cell communications within the testis of adult Sprague-Dawley rats, we used acute whole body neutron plus gamma-irradiation over 7-121 days postirradiation and chronic whole body gamma-irradiation over 14-84 days of irradiation and 7-86 days postirradiation. Neither irradiation protocol had an effect on the body weight of the animals. Neutron plus gamma-rays induced dramatic damages to spermatogonia, preleptotene spermatocytes, spermatozoa, and, to a lesser extent, pachytene spermatocytes. In contrast, gamma-rays induced a selective destruction of spermatogonia. Subsequently, in both experiments a maturation-depletion process led to a marked decrease in all germ cell types. A complete or near complete recovery of the different germ cell types and spermatozoa took place during the two postirradiation periods. Under both irradiation protocols Sertoli cells number was unchanged. Androgen-binding protein and FSH levels were normal in spite of the disappearance of most germ cells from spermatogonia to early spermatids. However, the decline of androgen-binding protein as well as the rise of FSH and their subsequent recovery were highly correlated to the number of late spermatids and spermatozoa. Moreover, it appeared that spermatocytes may also interfere with the production of inhibin (Exp B). With neither irradiation was Leydig cell function altered, except in Exp B in which elevated LH levels were temporarily observed. Correlation analysis suggested a relationship between preleptotene spermatocytes and Leydig cell function. In conclusion, this study establishes that chronic gamma-irradiation is particularly useful in the study of intratesticular paracrine regulation in vivo and provides further support to the concept that late spermatids play a major role in controlling some aspects of Sertoli cell function in the adult rat.

  3. Isolation of Murine Valve Endothelial Cells

    PubMed Central

    Miller, Lindsey J.; Lincoln, Joy

    2014-01-01

    Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms. PMID:25177896

  4. Intratubular Large Cell Hyalinizing Sertoli Cell Tumor of the Testes in a 4-Year-Old Male With Peutz-Jeghers Syndrome.

    PubMed

    Armijo, Beeling; Bocklage, Theresa; Heideman, Richard

    2015-04-01

    Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disorder that typically displays familial inheritance. Gastrointestinal polyposis and cutaneous pigmentation is a classic presentation of this syndrome. The reported lifetime cumulative cancer risk in PJS patients is >76% when compared with the general public with females affected more often than males. The prepubertal testicular tumor registry found Sertoli cell tumors (SCTs) to compose approximately 1% of all pediatric solid tumors. Prepubertal testicular masses are relatively rare. Only a small number of SCT cases have been reported in the first decade of life. The concurrence of PJS and feminizing SCTs of the testes is an increasingly recognized cause of prepubertal gynecomastia. The testicular lesions observed in patients with PJS primarily represent multifocal intratubular large cell hyalinizing SCTs with a distinct morphology that differs from large cell calcifying SCTs and sex cord tumors with annular tubules. Here, we describe the diagnosis and treatment course of a 4-year-old male with a SCT of the testes and diagnosis of PJS. PMID:25171448

  5. Treatment of High Risk SertoliLeydig Cell Tumors of the Ovary Using a Gonadotropin Releasing Hormone (GnRH) Analog

    PubMed Central

    Lashkari, Harsha Prasada; Nash, Ruth; Albanese, Assunta; Okoye, Bruce; Millar, Robert; Pritchard-Jones, Kathy

    2013-01-01

    SertoliLeydig cell tumors are rare ovarian neoplasms. We report two unusual cases with bilateral SLCTs suggesting evidence of genetic predisposition and at high risk of recurrence. To reduce this risk, we exploited the use of GnRH analog to lower gondadotropin and potentially directly inhibit the tumors through expressed GnRH receptors. We used it as maintenance antitumor therapy for 2 years after completion of chemotherapy, to cover the period of risk for recurrence. Both patients remain in complete remission at >2 years after completing leuprorelin therapy. Of note, both patients carry DICER1 mutations, frequently found in pleuropulmonary blastoma syndrome. Pediatr Blood Cancer 2013; 60: E16E18. 2012 Wiley Periodicals, Inc. PMID:23193086

  6. Xenograft of microencapsulated Sertoli cells for the cell therapy of type 2 diabetes mellitus in spontaneously diabetic nonhuman primates: preliminary data.

    PubMed

    Luca, G; Cameron, D F; Arato, I; Mancuso, F; Linden, E H; Calvitti, M; Falabella, G; Szekeres, K; Bodo, M; Ricci, G; Hansen, B C; Calafiore, R

    2014-01-01

    Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans. PMID:25131093

  7. Redefining Myeloid Cell Subsets in Murine Spleen

    PubMed Central

    Hey, Ying-Ying; Tan, Jonathan K. H.; O’Neill, Helen C.

    2016-01-01

    Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed “L-DC” in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11bhiCD11cloMHCII−Ly6C−Ly6G− subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−Ly6C−Ly6G− cells, which are CD43+, Siglec-F− and CD115−. Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types. PMID:26793192

  8. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  9. Telomere sister chromatid exchange in telomerase deficient murine cells

    SciTech Connect

    Wang, Yisong; Giannone, Richard J; Liu, Yie

    2005-01-01

    We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

  10. The Wilms Tumor Gene, Wt1, Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

    PubMed Central

    Wang, Ya Qing; Chen, Min; Zhang, Jun; Hao, Jian Xiu; Wang, Yan Bo; Sha, Ri Na; Huang, Yi; Liu, Xiao; Hu, Jing Chu; Sun, Guang Qing; Li, Hong Gang; Xiong, Cheng Liang; Xie, Jun; Jiang, Zhi Mao; Cai, Zhi Ming; Wang, Jun; Wang, Jian; Huff, Vicki; Gui, Yao Ting; Gao, Fei

    2013-01-01

    Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA), indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1flox and Cre-ERTM mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the bloodtestis barrier (BTB) was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin) and Wnt signaling genes (Wnt4, Wnt11) were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans. PMID:23935527

  11. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    SciTech Connect

    Carette, Diane; Perrard, Marie-Hélène; Prisant, Nadia; Gilleron, Jérome; Pointis, Georges; Segretain, Dominique; Durand, Philippe

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  12. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  13. Improving in vitro Sertoli cell/Gonocyte co-culture model for assessing male reproductive toxicity: lessons learned from comparisons of cytotoxicity versus genomic responses to phthalates

    PubMed Central

    Yu, Xiaozhong; Hong, SungWoo; Moreira, Estefania G; Faustman, Elaine M

    2009-01-01

    Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/Gonocyte coculture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE) on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally nontoxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants. PMID:19560483

  14. Improving in vitro Sertoli cell/gonocyte co-culture model for assessing male reproductive toxicity: Lessons learned from comparisons of cytotoxicity versus genomic responses to phthalates

    SciTech Connect

    Yu Xiaozhong; Hong, Sung Woo; Moreira, Estefania G.; Faustman, Elaine M.

    2009-09-15

    Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/gonocyte co-culture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE) on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally non-toxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, the gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in the gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants.

  15. Cytotoxicity of crotoxin on murine erythroleukemia cells in vitro.

    PubMed

    Corin, R E; Viskatis, L J; Vidal, J C; Etcheverry, M A

    1993-02-01

    The cytotoxic effect of crotoxin, a heterodimeric phospholipase A2 from the venom of Crotalus durissus terrificus, was examined on murine erythroleukemia cells in vitro. Crotoxin cytocidal effect on cell growth had an EC50 of approximately 0.1-0.2 microM (3.0-5.0 micrograms/ml) in serum-free medium. Cytotoxicity was independent of cell growth since both quiescent and proliferating cells had similar sensitivities to the toxin. Dissociation of the crotoxin complex and phospholipase A2 activity of its subunit B are required for cytotoxicity, since the covalently linked crotoxin complex or the specific alkylation of the active site on the subunit B abolish the cytotoxic activity on murine erythroleukemia cells. Specific interaction between crotoxin and murine erythroleukemia cells appears to be required since the homologous phospholipase A2 from Crotalus atrox venom, with a higher phospholipase A2 specific activity than crotoxin, was 86-fold less potent than crotoxin. The data in this report show that the cytotoxic effect of crotoxin on murine erythroleukemia cells is consistent with the specific binding of the toxin resulting in cytocidal action mediated by the phospholipase A2 activity of crotoxin subunit B. PMID:8349431

  16. Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

    PubMed

    Le Gac, F; Loir, M; le Bail, P Y; Ollitrault, M

    1996-05-01

    Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II > insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish. PMID:8722689

  17. Sertoli-Leydig cell tumors: hormonal profile after dynamic test with GnRH analogue: triptorelin represents a useful tool to evaluate tumoral hyperandrogenism.

    PubMed

    Turra, J; Granzotto, M; Gallea, M; Faggian, D; Conte, L; Litta, P; Vettor, R; Mioni, R

    2015-01-01

    We report the case of a 15-year-old woman with signs of hyperandrogenism affected by a Sertoli-Leydig cell tumor (SLCT). In our patient, blood analysis showed a high testosterone (T) level (T: 8.53?nmol/L; nv?

  18. SYNCHRONIZATION OF RAPID GLOBIN EXPRESSION IN MURINE ERYTHROLEUKEMIC CELLS

    EPA Science Inventory

    The addition of butyric acid (BA) to murine erythroleukemia cells (MELC) produces the expression of primarily A and E2 hemoglobins while DMSO incubation produces the expression of primarily A hemoglobin. Preincubation of MELC with DMSO followed by BA induction accelerates the exp...

  19. Procedures for the isolation and culture of Sertoli cells from the testes of infant, juvenile, and adult rhesus monkeys (Macaca mulatta).

    PubMed

    Majumdar, S S; Winters, S J; Plant, T M

    1998-03-01

    The purpose of the present study was to establish culture conditions for the in vitro study of the rhesus monkey Sertoli cell (Sc) at three major stages of development, namely infancy, adulthood, and the intervening prepubertal period. Conditions for the culture of Sc from juveniles were first established using collagenase and pancreatin digestion of seminiferous tubules. The addition of 1% fetal bovine serum for the first 24 h of culture was necessary for attachment of Sc clusters. Confluency of Sc from juveniles was reached as early as 4 days of culture. Histochemical and ultrastructural observations confirmed that the cultures were enriched with Sc and that contamination by peritubular cells was minimal (2%). Although application of similar culture conditions was successful in establishing cultures of Sc from infants, significant modification of the procedure was required before Sc from adults could be cultured. Specifically, adult testicular tissue required two sequential collagenase digestions at elevated temperature. The yield of adult Sc, however, remained low. Cultures of juvenile Sc produced substantial quantities of 31-kDa inhibin, which was bioactive as reflected by its ability to suppress FSH secretion from rat pituitary cells in vitro. Although aromatase activity in juvenile Sc cultures was stimulated by FSH, inhibin synthesis, as reflected by immunoactive inhibin production and steady-state levels of alpha inhibin mRNA, was not increased by FSH. The establishment of conditions for the culture of infant, juvenile, and adult Sc from the rhesus monkey will provide a model for study of the postnatal ontogeny of Sc function in higher primates. PMID:9510950

  20. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes

    PubMed Central

    KANG, Hoin; PARK, Jong Im; ROH, Sangho

    2015-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  1. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-02-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  2. Cadmium-induced Activation of Stress Signaling Pathways, Disruption of Ubiquitin-dependent Protein Degradation and Apoptosis in Primary Rat Sertoli Cell-Gonocyte Cocultures

    PubMed Central

    Yu, Xiaozhong; Hong, Sungwoo; Faustman, Elaine M.

    2008-01-01

    Cadmium (Cd) is a ubiquitous environmental pollutant that has been associated with male reproductive toxicity in both humans and animal models. The underlying mechanism of this response, however, is still uncharacterized. To address this issue, we employed a recently developed and optimized three-dimensional primary Sertoli cell-gonocyte coculture system and examined the time- and dose-dependent effects of Cd on morphological alterations, cell viability, activation of stress signaling pathway proteins, and the disruption of the ubiquitin proteasome system (UPS). Our results demonstrated that Cd exposure lead to time- and dose-dependent morphological changes that are associated with the induction of apoptosis. In response to Cd, we also saw a disruption of the UPS as evaluated through the accumulation of high–molecular weight polyubiquitinated proteins (HMW-polyUb) as well as alterations in proteasome activity. Robust activation of cellular stress response, measured through the increased phosphorylation of stress-activated protein kinase/c-jun N-terminal kinase and p38, paralleled the accumulation of HMW-polyUb. In addition, p53, a key regulatory protein, was upregulated and underwent increased ubiquitination in response to Cd. To further characterize the role of the UPS in Cd cellular response, we compared the above changes with two classic proteasomal inhibitors, lactacystin, and MG132. The stress response and the accumulation of HWM-polyUb induced by Cd were consistent with the response seen with MG132 but not with lactacystin. In addition, Cd treatment resulted in a dose- and time-dependent effect on proteasome activity, but the overall Cd-induced proteasomal inhibition was unique as compared to MG132 and lactacystin. Taken together, our studies further characterize Cd-induced in vitro testicular toxicity and highlight the potential role of the UPS in this response. PMID:18463101

  3. 4-Nonylphenol induces apoptosis, autophagy and necrosis in Sertoli cells: Involvement of ROS-mediated AMPK/AKT-mTOR and JNK pathways.

    PubMed

    Duan, Peng; Hu, Chunhui; Quan, Chao; Yu, Tingting; Zhou, Wei; Yuan, Meng; Shi, Yuqin; Yang, Kedi

    2016-02-01

    The xenoestrogen 4-nonylphenol (NP) induces reproductive dysfunction of male rats, but the fundamental mechanism of this phenomenon is largely unexplored. Sertoli cells (SCs) are pivotal for spermatogenesis and male fertility. The involvement of autophagy in NP-induced apoptotic and necrotic death of SCs was investigated. In this study, 24-h exposure of SCs to 20-30?M NP decreased cell viability, caused G2/M arrest, triggered ??m loss, increased ROS production and induced caspase-dependent apoptsis, necrosis as well as autophagosome formation. NP-induced autophagy was confirmed by monodansylcadaverine-staining and LC3-I/LC3-II conversion. Furthermore, NP up-regulated the (Thr172)p-AMPK/AMPK and (Thr183/185)p-JNK/JNK ratios. This was followed by the down-regulation of (Ser473)p-Akt/Akt, (Thr1462)p-TSC2/TSC2, (Ser2448)p-mTOR/mTOR, (Thr389)p-p70S6K/p70S6K and (Thr37/45)p-4EBP1/4EBP1. Intriguingly, NP-induced apoptosis, autophagy and necrosis could be inhibited through blocking ROS generation by N-acetylcysteine. Autophagy inhibitor 3-MA enhanced NP-induced apoptosis and necrosis. Moreover, The activation of AMPK/mTOR/p70s6k/4EBP1 and JNK signalling pathways induced by NP could be efficiently reversed by pretreatment of N-acetylcysteine or 3-MA. Collectively, our findings provide the first evidence that NP promotes apoptosis, autophagy and necrosis simultaneously in SCs and that this process may involve ROS-dependent JNK- and Akt/AMPK/mTOR pathways. Modulation of autophagy induced by NP may serve as a survival mechanism against apoptosis and necrosis. PMID:26804764

  4. Dickkopf Homolog 3 (DKK3) Plays a Crucial Role Upstream of WNT/?-CATENIN Signaling for Sertoli Cell Mediated Regulation of Spermatogenesis

    PubMed Central

    Kunj, Neetu; Sarda, Kanchan; Pradhan, Bhola Shankar; Majumdar, Subeer S.

    2013-01-01

    Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (46 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/?-CATENIN activity. Elevated ?-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/?-CATENIN modulation. PMID:23667645

  5. Human pontine glioma cells can induce murine tumors.

    PubMed

    Caretti, Viola; Sewing, A Charlotte P; Lagerweij, Tonny; Schellen, Pepijn; Bugiani, Marianna; Jansen, Marc H A; van Vuurden, Dannis G; Navis, Anna C; Horsman, Ilona; Vandertop, W Peter; Noske, David P; Wesseling, Pieter; Kaspers, Gertjan J L; Nazarian, Javad; Vogel, Hannes; Hulleman, Esther; Monje, Michelle; Wurdinger, Thomas

    2014-01-01

    Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy (1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or (2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprising human cells. Of note, direct injection of cells obtained postmortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question. PMID:24777482

  6. Human Pontine Glioma Cells Can Induce Murine Tumors

    PubMed Central

    Caretti, Viola; Sewing, A. Charlotte P.; Lagerweij, Tonny; Schellen, Pepijn; Bugiani, Marianna; Jansen, Marc H. A.; van Vuurden, Dannis G.; Navis, Anna C.; Horsman, Ilona; Vandertop, W. Peter; Noske, David P.; Wesseling, Pieter; Kaspers, Gertjan J.L.; Nazarian, Javad; Vogel, Hannes; Hulleman, Esther; Monje, Michelle; Wurdinger, Thomas

    2014-01-01

    Diffuse intrinsic pontine glioma (DIPG), with a median survival of only nine months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy 1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or 2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprised of human cells. Of note, direct injection of cells obtained post mortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question. PMID:24777482

  7. Pancreatic Differentiation from Murine Embryonic Stem Cells.

    PubMed

    Sakano, Daisuke; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Pluripotent stem cells are considered as a cell source for replacement therapies for pancreatic beta cells and other organs.We identified tetrabenazine (TBZ), vesicular monoamine transporter 2 (VMAT2) inhibitor as a promoter of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Ngn3-positive endocrine progenitor cells. A cell-permeable cAMP analog, dBu-cAMP promotes beta cell maturation in late stage of differentiation. The induced beta cells can secrete insulin in a glucose-dependent manner.Our protocol consists of a three -step differentiation process. ES cell recapitulate embryonic developmental processes in vitro. Therefore, the ES cell differentiation system is a useful model for the understanding of molecular mechanism of beta-cell differentiation and are useful for application for future regenerative medicine. PMID:25762295

  8. Ex vivo expansion of murine and human hematopoietic stem cells.

    PubMed

    Doan, Phuong L; Chute, John P

    2014-01-01

    Hematopoietic stem cells have the capacity to self-renew and give rise to the entirety of the mature blood and immune system throughout the lifespan of an organism. Here, we describe methods to isolate and culture murine bone marrow (BM) CD34(-)ckit(+)Sca1(+)Lineage(-) (CD34(-)KSL) hematopoietic stem cells (HSCs). We also describe a method to measure functional HSC content via the competitive repopulation assay. Furthermore, we summarize methods to isolate and culture human CD34(+)CD38(-)Lineage(-) cells which are enriched for human hematopoietic stem and progenitor cells. PMID:25062631

  9. Flow Cytometric Analysis of Immune Cells Within Murine Aorta.

    PubMed

    Gjurich, Breanne N; Taghavie-Moghadam, Parsa L; Galkina, Elena V

    2015-01-01

    The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis. PMID:26445788

  10. Flow Cytometric Analysis of Immune Cells Within Murine Aorta

    PubMed Central

    Gjurich, Breanne N.; Taghavie-Moghadam, Parsa L.; Galkina, Elena V.

    2015-01-01

    The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis. PMID:26445788

  11. Intramyocardial Cell Delivery: Observations in Murine Hearts

    PubMed Central

    Poggioli, Tommaso; Sarathchandra, Padmini; Rosenthal, Nadia; Santini, Maria P.

    2014-01-01

    Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells. Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe. Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load. PMID:24513973

  12. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  13. Hyperimmune human ABO blood-typing sera: reactivity with murine laminin and cytotoxicity for metastatic murine tumour cells.

    PubMed

    McCoy, J P; Schrier, D; Lovett, E J; Judd, W J; Varani, J

    1983-01-01

    Commercially prepared ABO blood-typing antisera have been tested for their ability to bind to murine laminin and their cytotoxic effects upon high and low metastatic variants of a murine fibrosarcoma. Previous studies have shown that alpha-D-galactopyranosyl end-groups comprise the major antigenic determinants on type B erythrocytes and that these same end-groups are present on murine laminin purified from the EHS sarcoma and on a laminin-like glycoprotein on the surface of the high, but not low, metastatic fibrosarcoma cells. In the present study we found that all sera containing anti-B activity were cytotoxic to the high, but not the low, metastatic cells and that all of these sera reacted strongly against immobilized murine laminin in an enzyme-linked immunosorbent assay. Sera lacking anti-B activity, i.e. anti-A antisera, were much less cytotoxic to either cell line and three of the four anti-A sera did not bind to murine laminin. The laminin reactivity and cytotoxic effect of the anti-B sera were specifically abrogated by preincubation of the sera with water-soluble blood group B substance or with murine laminin but not with water-soluble blood group A substance. PMID:6863409

  14. Nanoelectroablation therapy for murine basal cell carcinoma

    SciTech Connect

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S.; Epstein, Ervin H.; Tang, Jean Y.; Stanford University, Stanford, CA 94305

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  15. XC cell fusion by murine leukemia viruses: fusion from without.

    PubMed

    Ogura, H

    1976-12-01

    Concentrated murine leukemia virus (MuLV) or MuLV producing cells induce XC cell fusion within an hour leading to syncytia formation. While MuLV inactivated by UV irradiation, beta-propiolactone or hydroxylamine treatment still caused cell fusion, Bromelin- or trypsin treated MuLV was no longer able to fuse XC cells. Though sonicated MuLV induced no XC cell fusion, it interfered with cell fusion as caused by untreated MuLV. XC cells infected by diluted MuLV of a titer lower than 1 X 10(5) PFU/ml formed no syncytia although they produced MuLV. The cell fusion mechanism is discussed. PMID:187916

  16. Bioengineering murine mastocytoma cells to produce anticoagulant heparin.

    PubMed

    Gasimli, Leyla; Glass, Charles A; Datta, Payel; Yang, Bo; Li, Guoyun; Gemmill, Trent R; Baik, Jong Youn; Sharfstein, Susan T; Esko, Jeffrey D; Linhardt, Robert J

    2014-03-01

    Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities. PMID:24326668

  17. Antagonistic Effects of a Mixture of Low-Dose Nonylphenol and Di-N-Butyl Phthalate (Monobutyl Phthalate) on the Sertoli Cells and Serum Reproductive Hormones in Prepubertal Male Rats In Vitro and In Vivo

    PubMed Central

    Xiang, Zou; Qian, Weiping; Han, Xiaodong; Li, Dongmei

    2014-01-01

    The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EDCs) which at high doses in some species of laboratory animals, such as mice and rats, have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EDCs in the environment, their combined effects warrant clarification. In this study, we attempted to determine the mixture effects of NP and DBP on the testicular Sertoli cells and reproductive endocrine hormones in serum in male rats based on quantitative data analysis by a mathematical model. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from 9-day-old Sprague-Dawley rats followed by treatment with NP and MBP, singly or combined. Cell viability, apoptosis, necrosis, membrane integrity and inhibin-B concentration were tested. In the in vivo experiment, rats were gavaged on postnatal days 2335 with a single or combined NP and DBP treatment. Serum reproductive hormone levels were recorded. Next, Bliss Independence model was employed to analyze the quantitative data obtained from the in vitro and in vivo investigation. Antagonism was identified as the mixture effects of NP and DBP (MBP). In this study, we demonstrate the potential of Bliss Independence model for the prediction of interactions between estrogenic and antiandrogenic agents. PMID:24676355

  18. In vitro stimulation of murine lymphoid cell cultures by levamisole.

    PubMed Central

    Merluzzi, V J; Badger, A M; Kaiser, C W; Cooperband, S R

    1975-01-01

    Levamisole has been reported to act as an immunological adjuvant. Experiments reported here on the effect of this agent on a variety of murine lymphoid culture systems were designed to gain an insight into its mechanism of action. We have found levamisole to be a weak mitogen for mouse spleen cells producing a dose related response which peaks at 48 hr in culture. The drug acted to augment the response of spleen cells to sub-optimal concentrations of concanavalin A, but had no unusual effect on the lipopolysaccharide stimulation of B-cell DNA synthesis in vitro. Levamisole was directly stimulatory on enriched T-cell populations and was found to have two actions: (1) to stimulate a subpopulation of T cells and (2) to augment the response of suboptimal mitogen concentrations of concanavalin A. In addition, we have found that murine thymocytes stimulated by concanavalin A were greatly potentiated in the presence of levamisole, but this population of cells could not be stimulated directly by the drug. PMID:1083786

  19. Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development

    PubMed Central

    Frodermann, Vanessa; van Duijn, Janine; van Pel, Melissa; van Santbrink, Peter J.; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C. A.

    2015-01-01

    Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies. PMID:26490642

  20. Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development.

    PubMed

    Frodermann, Vanessa; van Duijn, Janine; van Pel, Melissa; van Santbrink, Peter J; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C A

    2015-01-01

    Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies. PMID:26490642

  1. Flow cytometric quantification of radiation responses of murine peritoneal cells

    SciTech Connect

    Tokita, N.; Raju, M.R.

    1982-01-01

    Methods have been developed to distinguish subpopulations of murine peritoneal cells, and these were applied to the measurement of early changes in peritoneal cells after irradiation. The ratio of the two major subpopulations in the peritoneal fluid, lymphocytes and macrophages, was measured rapidly by means of cell volume distribution analysis as well as by hypotonic propidium iodide (PI) staining. After irradiation, dose and time dependent changes were noted in the cell volume distributions: a rapid loss of peritoneal lymphocytes, and an increase in the mean cell volume of macrophages. The hypotonic PI staining characteristics of the peritoneal cells showed two or three distinctive G/sub 1/ peaks. The ratio of the areas of these peaks was also found to be dependent of the radiation dose and the time after irradiation. These results demonstrate that these two parameters may be used to monitor changes induced by irradiation (biological dosimetry), and to sort different peritoneal subpopulations.

  2. Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

    PubMed Central

    Schollaert, Kaila L.; Stephens, Michael R.; Gray, Jerilyn K.; Fulkerson, Patricia C.

    2014-01-01

    Eosinophils are produced in the bone marrow from CD34+ eosinophil lineagecommitted progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineagecommitted progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineagecommitted progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineagecommitted progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineagecommitted progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5. PMID:25551463

  3. Cyclosporine inhibition of a murine B cell lymphoma.

    PubMed

    Pisetsky, D S; Haughton, G

    1986-03-01

    The effect of cyclosporine (CsA) on the CH12 murine B cell lymphoma was investigated to determine whether sensitivity to this agent is retained by malignant B cells. This tumour produces an antibody to bromelain-treated red blood cells and may represent transformation of a B cell with certain activation properties associated with early resting B cells. In in vitro cultures, the growth and proliferation of CH12 were inhibited by CsA in concentrations of 0.1-1.0 microgram/ml; these levels were ineffective against non-lymphoid tumours, although some non-specific cell toxicity was noted at higher levels. IgM antibody production, as measured by enzyme-linked immunosorbent assay (ELISA), was inhibited over the same range. CH12 cells stimulated by lipopolysaccharide, however, were less sensitive to CsA than untreated cells. These studies indicate that malignant B cells may be sensitive to CsA, perhaps reflecting their derivation from a functionally distinct B cell population with enhanced drug sensitivity. PMID:3486733

  4. Cyclosporine inhibition of a murine B cell lymphoma.

    PubMed Central

    Pisetsky, D S; Haughton, G

    1986-01-01

    The effect of cyclosporine (CsA) on the CH12 murine B cell lymphoma was investigated to determine whether sensitivity to this agent is retained by malignant B cells. This tumour produces an antibody to bromelain-treated red blood cells and may represent transformation of a B cell with certain activation properties associated with early resting B cells. In in vitro cultures, the growth and proliferation of CH12 were inhibited by CsA in concentrations of 0.1-1.0 microgram/ml; these levels were ineffective against non-lymphoid tumours, although some non-specific cell toxicity was noted at higher levels. IgM antibody production, as measured by enzyme-linked immunosorbent assay (ELISA), was inhibited over the same range. CH12 cells stimulated by lipopolysaccharide, however, were less sensitive to CsA than untreated cells. These studies indicate that malignant B cells may be sensitive to CsA, perhaps reflecting their derivation from a functionally distinct B cell population with enhanced drug sensitivity. PMID:3486733

  5. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

  6. Murine norovirus transcytosis across an in vitro polarized murine intestinal epithelial monolayer is mediated by M-like cells.

    PubMed

    Gonzalez-Hernandez, Mariam B; Liu, Thomas; Blanco, Luz P; Auble, Heather; Payne, Hilary C; Wobus, Christiane E

    2013-12-01

    Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host. PMID:24049163

  7. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  8. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  9. Migration of Dendritic Cells from Murine Skeletal Muscle

    PubMed Central

    Wang, Lei; Eghtesad, Saman; Clemens, Paula R.

    2010-01-01

    To better understand the role of dendritic cells (DCs) in skeletal muscle, we investigated the migration of DCs from murine skeletal muscle and compared that to previously studied footpad (FP) DC trafficking. We adoptively transferred carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled mature DCs to syngeneic mice and followed them in various lymphatic tissues at different time points. Injection of DCs into the tibialis anterior muscle resulted in the peak number of CFSE+ DCs recovered in spleen at 12h, not at 24h, when the largest number of these cells appeared in the draining lymph nodes. Interestingly, this result for adoptive transfer of DCs to skeletal muscle differs with what is previously reported for adoptive transfer to the FP, a result that we also confirmed in parallel studies. These findings could have a significant impact on 1) understanding muscle diseases with immunological complications such as muscular dystrophies and 2) the immunologic effects of treatments for muscle diseases. PMID:20580121

  10. Control of Murine Cytomegalovirus Infection by ?? T Cells

    PubMed Central

    Sell, Sabrina; Dietz, Monika; Schneider, Andrea; Holtappels, Rafaela; Mach, Michael; Winkler, Thomas H.

    2015-01-01

    Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of ?? T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of ?? T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 ??-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1-/- mice lacking any T- and B-cells. ?? T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1-/- mice after adoptive transfer. ?? T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the ?? T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused V?1 and V?2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add ?? T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that ?? T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by ?? T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described. PMID:25658831

  11. FLOW CYTOMETRIC COMPARISON OF THE EFFECTS OF TRIALKYTING ON THE MURINE ERYTHROLEUKEMIC CELL

    EPA Science Inventory

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) ...

  12. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1989-12-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-(3-thiotriphosphate)) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

  13. A Novel Cell Line from Spontaneously Immortalized Murine Microglia

    PubMed Central

    Kulas, Joshua; Combs, Colin K.

    2014-01-01

    Background Purified microglia cultures are useful tools to study microglial behavior in vitro. Microglial cell lines serve as an attractive alternative to primary microglia culture, circumventing the costly and lengthy preparation of the latter. However, immortalization by genetic or pharmacologic manipulations may show altered physiology from primary microglia. New Method A novel microglial cell line was isolated from a primary glial culture of postnatal murine cerebral cortices. The culture contained a population of spontaneously transformed microglia that continued to divide without genetic or pharmacological manipulations. After several clones were isolated, one particular clone, SIM-A9, was analyzed for its microglial characteristics. Results SIM-A9 cells expressed macrophage/microglia-specific proteins, CD68 and Iba1. SIM-A9 cells were responsive to exogenous inflammatory stimulation with lipopolysaccharide and ?-amyloid, triggering tyrosine kinase-based and NF?B signaling cascades as well as TNF? secretion. SIM-A9 cells also exhibited phagocytic uptake of fluorescent labeled ?-amyloid and bacterial bioparticles. Furthermore, lipopolysaccharide increased the levels of inducible nitric oxide synthase and cyclooxygenase-2, whereas IL-4 stimulation increased arginase-1 levels demonstrating that SIM-A9 cells are capable of switching their profiles to pro- or anti-inflammatory phenotypes, respectively. Comparison with Existing Methods The use of SIM-A9 cells avoids expensive and lengthy procedures required for the preparation of primary microglia. Spontaneously immortalized SIM-A9 cells are expected to behave more comparably to primary microglia than virally transformed or pharmacologically induced microglial cell lines. Conclusions SIM-A9 cells exhibit key characteristics of cultured primary microglia and may serve as a valuable model system for the investigation of microglial behavior in vitro. PMID:24975292

  14. The murine Fhit locus: isolation, characterization, and expression in normal and tumor cells.

    PubMed

    Pekarsky, Y; Druck, T; Cotticelli, M G; Ohta, M; Shou, J; Mendrola, J; Montgomery, J C; Buchberg, A M; Siracusa, L D; Manenti, G; Fong, L Y; Dragani, T A; Croce, C M; Huebner, K

    1998-08-01

    The murine Fhit locus maps near the centromere nu proximal Ptprg locus on mouse chromosome 14. The cDNA sequence and structure are similar to those of the human gene, with exons 5-9 encoding the protein. The predominant mRNA in the tissues and cell lines tested was an alternatively spliced form missing exon 3. Most murine cell lines tested, including lines established from normal mouse embryos and tumors, expressed very low or undetectable levels of Fhit mRNA. Most normal mouse tissues expressed wild-type Fhit mRNA, whereas approximately 40% of murine lung carcinomas expressed wild-type and aberrant Fhit RT-PCR products that lacked various exons. Several tumorigenic mouse cell lines exhibited homozygous deletions of Fhit exons. We conclude that the murine Fhit gene, like its human counterpart, is a target of alterations involved in murine carcinogenesis. PMID:9699672

  15. The kin17 Protein in Murine Melanoma Cells.

    PubMed

    Ramos, Anelise C; Gaspar, Vanessa P; Kelmer, Sabrina M G; Sellani, Tarciso A; Batista, Ana G U; De Lima Neto, Quirino A; Rodrigues, Elaine G; Fernandez, Maria A

    2015-01-01

    kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma. PMID:26610484

  16. The kin17 Protein in Murine Melanoma Cells

    PubMed Central

    Ramos, Anelise C.; Gaspar, Vanessa P.; Kelmer, Sabrina M. G.; Sellani, Tarciso A.; Batista, Ana G. U.; De Lima Neto, Quirino A.; Rodrigues, Elaine G.; Fernandez, Maria A.

    2015-01-01

    kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma. PMID:26610484

  17. Murine coagulation factor VIII is synthesized in endothelial cells

    PubMed Central

    Everett, Lesley A.; Cleuren, Audrey C. A.; Khoriaty, Rami N.

    2014-01-01

    The primary cellular source of factor VIII (FVIII) biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. LMAN1 is a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and FVIII to the plasma. Lman1 mutations result in combined deficiency of FV and FVIII, with levels of both factors reduced to ?10% to 15% of normal in human patients. We generated Lman1 conditional knockout mice to characterize the FVIII secretion profiles of endothelial cells and hepatocytes. We demonstrate that endothelial cells are the primary biosynthetic source of murine FVIII and that hepatocytes make no significant contribution to the plasma FVIII pool. Utilizing RiboTag mice and polyribosome immunoprecipitation, we performed endothelial cellspecific messenger RNA isolation and quantitative polymerase chain reaction analyses to confirm that endothelial cells highly express F8 and to explore the heterogeneity of F8 expression in different vascular beds. We demonstrate that endothelial cells from multiple, but not all, tissues contribute to the plasma FVIII pool in the mouse. PMID:24719406

  18. Complementation analysis of the murine scid cell line

    SciTech Connect

    Zdzienicka, M.Z. |; Priestly, A.; Jeggo, P.A.

    1995-09-01

    It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

  19. Induction of murine interleukin 1: stimuli and responsive primary cells.

    PubMed Central

    Koide, S; Steinman, R M

    1987-01-01

    An interleukin 1 alpha (IL-1 alpha) cDNA probe and an IL-1 responsive T-cell clone (D10.G4; half-maximal stimulation, 0.1-1 pM) have been used to study the production of IL-1 by primary murine cell populations, particularly macrophages and dendritic cells. Spleen and peritoneal macrophages produced IL-1 mRNA and released biologically active IL-1 when challenged with lipopolysaccharide (LPS). Induction of IL-1 was evident over a dose range of 0.01-10 micrograms of LPS per ml, and maximal mRNA levels were maintained from 4 to 20 hr. Several other stimuli did not induce IL-1 in cultured macrophages, including phorbol 12-myristate 13-acetate, gamma-interferon, Con A, macrophage colony-stimulating factor, IL-3, cachectin, and activated T cells. Activated T cells could markedly reduce the response of peritoneal macrophages to LPS. When other cell types were compared with macrophages, keratinocytes had high levels of IL-1 mRNA, apparently in response to endogenous LPS. However B and T lymphocytes did not yield detectable IL-1 during proliferative responses to LPS and Con A, respectively, while dendritic cells produced little or no IL-1 when challenged with a battery of stimuli. Therefore, IL-1 may not be required for the potent accessory function of dendritic cells in lymphocyte mitogenesis. The results indicate that macrophages and dendritic cells have different secretory capacities. The macrophage is the principal leukocyte that synthesizes IL-1, and select stimuli increase and decrease the levels of macrophage IL-1 mRNA. Images PMID:3495797

  20. High-dimensional analysis of the murine myeloid cell system.

    PubMed

    Becher, Burkhard; Schlitzer, Andreas; Chen, Jinmiao; Mair, Florian; Sumatoh, Hermi R; Teng, Karen Wei Weng; Low, Donovan; Ruedl, Christiane; Riccardi-Castagnoli, Paola; Poidinger, Michael; Greter, Melanie; Ginhoux, Florent; Newell, Evan W

    2014-12-01

    Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system. PMID:25306126

  1. Glycomics of Proteoglycan Biosynthesis in Murine Embryonic Stem Cell Differentiation

    PubMed Central

    Nairn, Alison V.; Kinoshita-Toyoda, Akiko; Toyoda, Hidenao; Xie, Jin; Harris, Kyle; Dalton, Stephen; Kulik, Michael; Pierce, J. Michael; Toida, Toshihiko; Moremen, Kelley W.; Linhardt, Robert J.

    2014-01-01

    Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13- and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4- and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5- and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells. PMID:17915907

  2. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  3. Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

    SciTech Connect

    Savion, N.; Disatnik, M.H.; Nevo, Z.

    1987-01-01

    Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells attach, invade, and penetrate confluent vascular endothelial cell monolayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the (/sup 35/S)O/sub 4//sup -/-labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The degradation of (/sup 35/S)O/sub 4//sup -/-labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10..mu..g/ml), arteparon (10..mu..g/ml), and heparin at a concentration of 3 ..mu..g/ml. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage haparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.

  4. B cell lymphoma and myeloma in murine Gaucher's disease.

    PubMed

    Pavlova, E V; Wang, S Z; Archer, J; Dekker, N; Aerts, J M F G; Karlsson, S; Cox, T M

    2013-09-01

    Multiple myeloma and B cell lymphoma are leading causes of death in Gaucher's disease but the nature of the stimulus driving the often noted clonal expansion of immunoglobulin-secreting B cells and cognate lymphoid malignancy is unknown. We investigated the long-term development of B cell malignancies in an authentic model of non-neuronopathic Gaucher's disease in mice: selective deficiency of β-glucocerebrosidase in haematopoietic cells [Gba(tm1Karl/tm1Karl)Tg(Mx1-cre)1Cgn/0, with excision of exons 9-11 of the murine GBA1 gene, is induced by poly[I:C]. Mice with Gaucher's disease showed visceral storage of β-glucosylceramide and greatly elevated plasma β-glucosylsphingosine [median 57.9 (range 19.8-159) nm; n = 39] compared with control mice from the same strain [median 0.56 (range 0.04-1.38) nm; n = 29] (p < 0.0001). Sporadic fatal B cell lymphomas developed in 11 of 21 GD mice (6-24 months) but only two of eight control animals developed tumours by age 24 months. Unexpectedly, most mice with overt lymphoma had absent or few Gaucher cells but local inflammatory macrophages were present. Eleven of 39 of Gaucher mice developed monoclonal gammopathy, but in the control group only one animal of 25 had clonal immunoglobulin abnormalities. Seven of 10 of the B cell lymphomas were found to secrete a monoclonal paraprotein and the lymphomas stained intensely for pan-B cell markers; reactive T lymphocytes were also present in tumour tissue. In the Gaucher mouse strain, it was notable that, as in patients with this disease, CD138(+) plasma cells frequently surrounded splenic macrophages engorged with glycosphingolipid. Our strain of mice, with inducible deficiency of β-glucocerebrosidase in haematopoietic cells and a high frequency of sporadic lethal B cell malignancies, faithfully recapitulates human Gaucher's disease: it serves as a tractable model to investigate the putative role of bioactive sphingolipids in the control of B cell proliferation and the pathogenesis of myelomatosis-the most prevalent human cancer associated with this disorder. PMID:23775597

  5. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB. PMID:10865941

  6. Echinacea pupurea extracts modulate murine dendritic cell fate and function

    PubMed Central

    Benson, Jenna M.; Pokorny, Amanda J.; Rhule, Ava; Wenner, Cynthia A.; Kandhi, Vamsikrishna; Cech, Nadja B.; Shepherd, David M.

    2010-01-01

    Echinacea is a top-selling herbal remedy that purportedly acts as an immunostimulant. However, the specific immunomodulatory effects of Echinacea remain to be elucidated. We focused on defining the effects of Echinacea purpurea extracts in dendritic cells (DCs), which generate innate and adaptive immune responses. We hypothesized that E. purpurea extracts would enhance murine bone marrow-derived DC (BMDC) activation leading to increased immune responses. The fate and function of DCs from C57Bl/6 mice was evaluated following 48 h exposure to E. purpurea root and leaf extracts. Flow cytometry revealed that the polysaccharide-rich root extract increased the expression of MHC class II, CD86, and CD54 surface biomarkers whereas the alkylamide-rich leaf extract inhibited expression of these molecules. Production of IL-6 and TNF-? increased in a concentration-dependent manner with exposure to the root, but not leaf, extract. In contrast, the leaf but not root extract inhibited the enzymatic activity of cyclooxygenase-2. While both extracts decreased the uptake of ovalbumin by BMDCs, the leaf but not root extract inhibited the antigen-specific activation of nave CD4+ T cells from OT II/Thy1.1 mice. Collectively, these results suggest that E. purpurea can be immunostimulatory, immunosuppressive, and/or anti-inflammatory depending on the portion of the plant and extraction method. PMID:20149833

  7. Echinacea purpurea extracts modulate murine dendritic cell fate and function.

    PubMed

    Benson, Jenna M; Pokorny, Amanda J; Rhule, Ava; Wenner, Cynthia A; Kandhi, Vamsikrishna; Cech, Nadja B; Shepherd, David M

    2010-05-01

    Echinacea is a top-selling herbal remedy that purportedly acts as an immunostimulant. However, the specific immunomodulatory effects of Echinacea remain to be elucidated. We focused on defining the effects of Echinacea purpurea extracts in dendritic cells (DCs), which generate innate and adaptive immune responses. We hypothesized that E. purpurea extracts would enhance murine bone marrow-derived DC (BMDC) activation leading to increased immune responses. The fate and function of DCs from C57Bl/6 mice was evaluated following 48h exposure to E. purpurea root and leaf extracts. Flow cytometry revealed that the polysaccharide-rich root extract increased the expression of MHC class II, CD86, and CD54 surface biomarkers whereas the alkylamide-rich leaf extract inhibited expression of these molecules. Production of IL-6 and TNF-alpha increased in a concentration-dependent manner with exposure to the root, but not leaf, extract. In contrast, the leaf but not root extract inhibited the enzymatic activity of cyclooxygenase-2. While both extracts decreased the uptake of ovalbumin by BMDCs, the leaf but not root extract inhibited the antigen-specific activation of nave CD4(+) T cells from OT II/Thy1.1 mice. Collectively, these results suggest that E. purpurea can be immunostimulatory, immunosuppressive, and/or anti-inflammatory depending on the portion of the plant and extraction method. PMID:20149833

  8. Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

    PubMed Central

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S.; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS. PMID:23675538

  9. Replication of enterotropic and polytropic murine coronaviruses in cultured cell lines of mouse origin.

    PubMed

    Kyuwa, S; Ohsawa, K; Sato, H; Urano, T

    2000-10-01

    To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively. PMID:11109550

  10. Intravenous anesthetic propofol suppresses leukotriene production in murine dendritic cells.

    PubMed

    Inada, Takefumi; Ueshima, Hironobu; Shingu, Koh

    2013-01-01

    Leukotrienes, divided into cysteinyl leukotrienes (CysLTs), which are important mediators of asthmatic responses, and leukotriene B4 (LTB4), a chemotactic and chemokinetic agent for leukocytes, are potent lipid mediators generated from arachidonic acid by 5-lipoxygenase (5-LO). Leukotrienes are also considered to have immunoregulatory and pro-inflammatory actions. Propofol is an intravenous anesthetic widely used for anesthesia and sedation that is alleged to possess anti-inflammatory properties. The present study examined the effect of propofol on leukotriene production by dendritic cells (DC). In murine bone marrow-derived DC, propofol significantly suppressed CysLT and LTB4 production after short-term stimulation with zymosan. The protein levels of cytosolic phospholipase A2 and 5-LO, or arachidonic acid release from plasma membranes, were not affected by the presence of propofol. Although zymosan treatment induced or enhanced the phosphorylation of ERK1/2, p-38 MAPK, and JNK, which presumably up-regulates the activity of 5-LO, the presence of propofol had no additional effect on the phosphorylation status of any of these MAPKs. Similarly, zymosan significantly increased the concentration of intracellular calcium, which is the most crucial activator of 5-LO, but no additional concentration changes were observed with the addition of propofol. Lastly, in an in-vitro cell-free ferrous oxidation-xylenol orange assay, propofol significantly inhibited the 5-LO activity of purified human recombinant 5-LO enzyme with an IC50 of ~7.5 µM. Thus, propofol's inhibition of 5-LO is not likely restricted to the circumstances surrounding the production of leukotrienes from DC, but applicable to other types of immune and non-immune cells that produce leukotrienes. The 5-LO-inhibiting activity of propofol may, at least in part, contribute to the well-known anti-inflammatory activity of propofol. PMID:22953970

  11. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    ERIC Educational Resources Information Center

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine

  12. Anti-leukemic properties of deferasirox via apoptosis in murine leukemia cell lines

    PubMed Central

    Jeon, Sol-Rim; Lee, Jae-Wook; Jang, Pil-Sang; Cho, Bin; Jeong, Dae-Chul

    2015-01-01

    Background Although deferasirox (DFX) is reported to have anti-tumor effects, its anti-leukemic activity remains unclear. We evaluated the effect of DFX treatment on two murine lymphoid leukemia cell lines, and clarified the mechanisms underlying its potential anti-leukemic activity. Methods L1210 and A20 murine lymphoid leukemia cell lines were treated with DFX. Cell viability and apoptosis were evaluated by the 3-(4,5-dimethylthaizol-2-yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and fluorescence-activated cell sorting (FACS) analysis, respectively. Immunoblotting was performed to detect the expression of key apoptotic proteins. Results In dose- and time-dependent manner, DFX decreased viability and increased apoptosis of murine leukemic cells. Fas expression was significantly higher in A20 cells than in L1210 cells at all DFX concentrations tested. Although both cell lines exhibited high caspase 3 and caspase 9 expression, a critical component of the intrinsic mitochondrial apoptotic pathway, expression was greater in L1210 cells. In contrast, caspase 8, a key factor in the extrinsic apoptotic pathway, showed greater expression in A20 cells. Cytochrome c expression was significantly higher in L1210 cells. In both cell lines, co-treatment with ferric chloride and DFX diminished the expression of these intracellular proteins, as compared to DFX treatment alone. Conclusion Treatment with DFX increased caspase-dependent apoptosis in two murine lymphoid leukemia cell lines, with differing apoptotic mechanisms in each cell line. PMID:25830128

  13. Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells

    PubMed Central

    Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, Denis

    2015-01-01

    Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used. PMID:26498381

  14. Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line

    PubMed Central

    Cox, Courtney; Cao, Shengbo; Lu, Yuanan

    2009-01-01

    Background Human Noroviruses are the predominant cause of non-bacterial gastroenteritis worldwide. To facilitate prevention and control, a norovirus isolated from mice can provide a model to understand human noroviruses. To establish optimal viral infectivity conditions for murine noroviruses, several cell lines of hematopoietic lineage, including murine BV-2, RAW 264.7, and TIB, as well as human CHME-5, were tested comparatively for their sensitivity to murine norovirus-1. Results Except for CHME-5, all three murine-derived cell lines were susceptible to MNV infection. Viral infection of these cells was confirmed by RT-PCR. Using both viral plaque and replication assays, BV-2 and RAW 264.7 cells were determined to have comparable sensitivities to MNV-1 infection. Comparisons of cell growth characteristics, general laboratory handling and potential in-field applications suggest the use of BV-2 to be more advantageous. Conclusion Results obtained from these studies demonstrate that an immortalized microglial cell line can support MNV-1 replication and provides a more efficient method to detect and study murine noroviruses, facilitating future investigations using MNV-1 as a model to study, detect, and control Human Norovirus. PMID:19903359

  15. Primary amenorrhea in a young Polish woman with complete androgen insensitivity syndrome and Sertoli-Leydig cell tumor: identification of a new androgen receptor gene mutation and evidence of aromatase hyperactivity and apoptosis dysregulation within the tumor.

    PubMed

    Jarzabek, Katarzyna; Philibert, Pascal; Koda, Mariusz; Sulkowski, Stanislaw; Kotula-Balak, Malgorzata; Bilinska, Barbara; Kottler, Marie-Laure; Wolczynski, Slawomir; Sultan, Charles

    2007-09-01

    Primary amenorrhea in 46,XY females can be due to complete androgen insensitivity syndrome (CAIS), pure gonadal dysgenesis, 17-hydroxysteroid dehydrogenase deficiency, or mixed gonadal dysgenesis. The present paper describes a new de novo non-sense mutation in exon 1 (K141Z) of the androgen receptor gene (AR) and the expression in CAIS testis of aromatase, estrogen receptors, as well as proliferation- and apoptosis-associated proteins. CAIS is a rare disease characterized by absent virilization in 46,XY individuals and the development of a female phenotype despite normal or even elevated androgen levels. CAIS is usually caused by a mutation in AR, which leads to organ resistance to androgens. Testicular tumors such as Sertoli-Leydig cell tumor often develop in patients with CAIS. The immunohistochemical findings in the testes of our CAIS patient suggest that the high expression of aromatase and other molecular changes in the testis may be responsible for pubertal breast development and the increased risk of testicular tumor. PMID:17852420

  16. Characterization of the expression and gene promoter of CD22 in murine B cells.

    PubMed

    Andersson, K B; Draves, K E; Magaletti, D M; Fujioka, S; Holmes, K L; Law, C L; Clark, E A

    1996-12-01

    CD22 is a B cell-restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSA(lo) memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD- B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22- murine pro-B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12-myristate 13-acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5' of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP-1, AP-2, C/EBP and SP-1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3-kb promoter fragment 5' of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp-1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo. PMID:8977319

  17. Involvement of a chromatin modifier in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury: Probably an indirect action via the regulation of NFκB/FasL circuitry

    SciTech Connect

    Chen, Shiwei; Dong, Yushu; Xu, Chun; Jiang, Liming; Chen, Yongjie; Jiang, Cheng; Hou, Wugang; Li, Wei

    2013-11-01

    Highlights: •MTA1 expression is upregulated in SCs upon MEHP treatment. •Knockdown of MTA1 in SCs impairs the MEHP-induced NFκB signaling activation. •Knockdown of MTA1 inhibits recruitment of NFκB onto FasL promoter in MEHP-treated SCs. -- Abstract: The Fas/FasL signaling pathway, controlled by nuclear factor-κB (NFκB) at the transcriptional level, is critical for triggering germ cell apoptosis in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell (SC) injury, but the exact regulation mechanism remain unknown. Here, we discovered that expression level of Metastasis associated protein 1 (MTA1), a component of the Mi-2/nucleosome remodeling and deacetylase complex, was upregulated in SCs during the early recovery after MEHP exposure. This expression change was in line with the dynamic changes in germ cell apoptosis in response to MEHP treatment. Furthermore, a knockdown of MTA1 by RNAi in SCs was found to impair the MEHP-induced early activation of NFκB pathway and abolish the recruitment of NFκB onto FasL promoter, which consequently diminished the MEHP-triggered FasL induction. Considering that Fas/FasL is a well characterized apoptosis initiating signaling during SCs injury, our results point to a potential “switch on” effect of MTA1, which may govern the activation of NFκB/FasL cascade in MEHP-insulted SCs. Overall, the MTA1/NFκB/FasL circuit may serve as an important defensive/repairing mechanism to help to control the germ cell quality after SCs injury.

  18. Murine trophoblast cells are not killed by tumor necrosis factor-alpha.

    PubMed

    Drake, B L; Head, J R

    1990-03-01

    Purified midgestation murine trophoblast cannot be killed by a variety of cell-mediated effector mechanisms, with the exception of highly lytic effectors such as lymphokine-activated killer cells. We now report that this trophoblast population is also resistant to tumor necrosis factor (TNF)-alpha. PMID:2109800

  19. Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen

    PubMed Central

    Xu, Zhengzhong; Meng, Chuang; Qiang, Bin; Gu, Hongyan; Sun, Lin; Yin, Yuelan; Pan, Zhiming; Chen, Xiang; Jiao, Xinan

    2015-01-01

    Macrophages (M?) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and M?s in immunity against Mycobacterium bovis Bacillus Calmette-Gurin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic M?s compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in M?s were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic M?s were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic M?s participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation. PMID:26473844

  20. Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

    PubMed Central

    JUN, Jin-Gon; MAEDA, Seishi; KUWAHARA-OTANI, Sachi; TANAKA, Koichi; HAYAKAWA, Tetsu; SEKI, Makoto

    2014-01-01

    ABSTRACT Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems. PMID:25069412

  1. Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages.

    PubMed

    Flao, E; Husain, S M; Sample, J T; Woodland, D L; Blackman, M A

    2000-07-15

    Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells. PMID:10878386

  2. Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

    PubMed Central

    Uphoff, Cord C.; Lange, Sandra; Denkmann, Sabine A.; Garritsen, Henk S. P.; Drexler, Hans G.

    2015-01-01

    Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%. PMID:25927683

  3. Phenotype and Stability of Neural Differentiation of Androgenetic Murine ES Cell-Derived Neural Progenitor Cells

    PubMed Central

    Wolber, Wanja; Ahmad, Ruhel; Choi, Soon Won; Eckardt, Sigrid; McLaughlin, K. John; Schmitt, Jessica; Geis, Christian; Heckmann, Manfred; Sirn, Anna-Leena; Mller, Albrecht M.

    2013-01-01

    Uniparental zygotes with two paternal (androgenetic, AG) or two maternal genomes (gynogenetic, GG) cannot develop into viable offsprings but form blastocysts from which pluripotent embryonic stem (ES) cells can be derived. For most organs, it is unclear whether uniparental ES cells can give rise to stably expandable somatic stem cells that can repair injured tissues. Even if previous reports indicated that the capacity of AG ES cells to differentiate in vitro into pan-neural progenitor cells (pNPCs) and into cells expressing neural markers is similar to biparental [normal fertilized (N)] ES cells, their potential for functional neurogenesis is not known. Here we show that murine AG pNPCs give rise to neuron-like cells, which then generate sodium-driven action potentials while maintaining fidelity of imprinted gene expression. Neural engraftment after intracerebral transplantation was achieved only by late (22 days) AG and N pNPCs with in vitro low colony-forming cell (CFC) capacity. However, persisting CFC formation seen, in particular, in early (13 or 16 days) differentiation cultures of N and AG pNPCs correlated with a high incidence of trigerm layer teratomas. As AG ES cells display functional neurogenesis and in vivo stability similar to N ES cells, they represent a unique model system to study the roles of paternal and maternal genomes on neural development and on the development of imprinting-associated brain diseases.

  4. Neonatal testicular cell transplantation restores murine spermatogenesis damaged in the course of herpes simplex virus-induced orchitis.

    PubMed

    Malolina, Ekaterina A; Kulibin, Andrey Yu; Kushch, Alla A

    2014-11-17

    Genital tract infection and inflammation may affect male fertility, causing germ and Sertoli cell loss. We determined if testicular cell transplantation is effective at repairing testicular injury induced by herpes simplex virus (HSV) orchitis. ROSA26 mice were used as donors and the recipients were C57BL/6 mice after HSV testicular inoculation; some of the recipients were treated with the antiviral drug acyclovir (ACV). ACV reduced the amount of HSV antigen in testes on Day 3 after transplantation and enhanced the efficacy of transplantation at Day 30. In recipient testes, donor Sertoli cells formed new seminiferous tubules; significantly more new tubules were observed in the testes of ACV-treated mice compared with mice not treated with ACV (17.8% vs 3.6%). Over half (50.4%) of new tubules in ACV-treated testes contained germ cells and round spermatids were detected in 14.2% of new tubules compared with 15.9% and 5.3% in testes not treated with ACV, respectively. At Day 150 the seminiferous epithelium was completely recovered in some donor tubules and elongated spermatids were observed inside it. Thus, our findings reveal the effectiveness of the combination of antiviral therapy with neonatal testis-cell transplantation for the restoration of spermatogenesis damaged by viral infection. PMID:25399480

  5. Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells.

    PubMed Central

    Hilton, D J; Nicola, N A; Metcalf, D

    1988-01-01

    Leukemia inhibitory factor (LIF), a glycoprotein capable of suppressing the clonogenicity and inducing the differentiation of the murine myeloid leukemia cell line M1, was radioiodinated to a high specific radioactivity with retention of full biological activity. Binding of 125I-labeled LIF to M1 cells reached a steady state at 37 degrees C after approximately equal to 40 min and was in competition with unlabeled LIF but not granulocyte colony-stimulating factor or a range of other cytokines or differentiation-inducing agents. Specific binding was demonstrable to cells from a range of murine hemopoietic tissues including the bone marrow, the spleen, and the peritoneal cavity. Autoradiography revealed macrophages, monocytes, and their precursors to be the major cell types responsible for 125I-labeled LIF binding within these tissues. Receptors on M1 cells were of high affinity (apparent Kd, 100-200 pM) and few in number (300-500 per cell). Images PMID:3137563

  6. METHODOLOGICAL CONSIDERATIONS FOR THE MURINE HEPATOMA CELL BIOASSAY-DIRECTED ISOLATION OF CANCER CHEMOPREVENTIVE AGENTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of a murine hepatoma (Hepa 1c1c7) cell line over a decade ago for the isolation of phase II enzyme inducing agents by a group from Johns Hopkins University led to the identification of sulforaphane as a major cancer chemopreventive agent in broccoli. We have found that choices among...

  7. COMPARATIVE TOXICITY OF DIFFERENT EMISSION PARTICLES IN MURINE PULMONARY EPITHELIAL CELLS AND MACROPHAGES

    EPA Science Inventory

    Comparative Toxicity of Different Emission Particles in Murine Pulmonary Epithelial Cells and Macrophages. T Stevens1, M Daniels2, P Singh2, M I Gilmour2. 1 UNC, Chapel Hill 27599 2Experimental Toxicology Division, NHEERL, RTP, NC 27711

    Epidemiological studies have shown ...

  8. De novo infection of B cells during murine gammaherpesvirus 68 latency.

    PubMed

    Freeman, Michael L; Burkum, Claire E; Yager, Eric J; Woodland, David L; Blackman, Marcia A

    2011-10-01

    The mechanisms by which gammaherpesviruses maintain latency are unclear. Here we used a murine gammaherpesvirus model to show that previously uninfected B cells in immunocompetent mice can acquire virus during latency. In vivo depletion of T cells allowed viral reactivation, as measured by increased viral loads, but not enhanced transfer of virus to new cells. In the absence of both immune T cells and antibody following the transfer of latently infected cells into nave animals, there was robust infection of new B cells. These data confirm that both T cells and antibody contribute to the control of gammaherpesvirus latency, reactivation, and spread. PMID:21849446

  9. Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research

    PubMed Central

    Fuertes Marraco, Silvia A.; Grosjean, Frédéric; Duval, Anaïs; Rosa, Muriel; Lavanchy, Christine; Ashok, Devika; Haller, Sergio; Otten, Luc A.; Steiner, Quynh-Giao; Descombes, Patrick; Luber, Christian A.; Meissner, Felix; Mann, Matthias; Szeles, Lajos; Reith, Walter; Acha-Orbea, Hans

    2012-01-01

    Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research. PMID:23162549

  10. Comparative study of artificial chromosome centromeres in human and murine cells

    PubMed Central

    Moralli, Daniela; Jefferson, Andrew; Valeria Volpi, Emanuela; Larin Monaco, Zoia

    2013-01-01

    Human artificial chromosomes (HAC) are a valuable tool in the analysis of complex chromatin structures such as the human centromere because of their small size and relative simplicity compared with normal human chromosomes. This report includes a comprehensive study of the centromere and chromatin composition of HAC, expressing human genes, generated in human cells and transferred to murine cells. The analysis involved chromatin immuno-precipitation and immuno-FISH on metaphase chromosomes and chromatin fibres. In both the cell types, the HAC consisted of alphoid and non-alphoid DNA and were mainly euchromatic in composition, although a pericentromeric heterochromatic region was present on all the HAC. Fibre-FISH and chromatin immuno-precipitation data indicated that the position of the centromere differed between HAC in human cells and in murine cells. Our work highlights the importance and utilisation of HAC for understanding the epigenetic aspects of chromosome biology. PMID:23403904

  11. Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt-dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells.

    PubMed

    Chojnacka, Katarzyna; Zarzycka, Marta; Hejmej, Anna; Mruk, Dolores D; Gorowska, Ewelina; Kotula-Balak, Malgorzata; Klimek, Monika; Bilinska, Barbara

    2016-03-01

    We investigated the effects of 2-hydroxyflutamide (HF), an active metabolite of the anti-androgen flutamide, on the activation of the phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) in rat Sertoli cells. Sertoli cells, isolated from 20-day-old rat testes, were cultured in vitro and treated with HF, testosterone, or HF+testosterone. Studies by western blotting demonstrated that HF inhibited the testosterone-mediated increase in c-Src activity (p<0.05). In contrast, Akt phosphorylation was augmented within 5min after HF treatment (p<0.01). This effect was accompanied by a rapid upregulation in PTEN phosphorylation (p<0.001). Despite no changes in Raf-1 phosphorylation, HF increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (p<0.001), indicating that the effect of the anti-androgen on ERK1/2 was independent of PI3K/Akt-pathway activation at this level. Since HF inhibited the testosterone-mediated increase in c-Src activity, it is likely that activation of both Akt and ERK1/2 occurred in a p-Src independent manner. Activation of PI3K/Akt-pathway by HF resulted in the reduced level of Sertoli cell functional marker, connexin 43 (p<0.01). Collectively, these data provide evidence that HF rapidly and transiently affects the protein kinase-dependent signaling pathways, acting both as an antagonist and agonist. Moreover, using testes of flutamide-treated rats for 7days, we demonstrated that the anti-androgen can modulate the protein kinase-dependent pathways in long term by enhancing Akt and ERK1/2 protein expression (p<0.05). PMID:26437446

  12. Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells

    PubMed Central

    Hendrickson, Jeanne E; Hod, Eldad A.; Hudson, Krystalyn E.; Spitalnik, Steven L.; Zimring, James C.

    2011-01-01

    Background Although a subset of recent studies have suggested red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh plus stored RBCs. Study Design and Methods Fresh, 14-day stored, or fresh plus 14-day stored leukoreduced RBCs from HOD transgenic donors (with RBC specific expression of hen egg lysozyme, ovalbumin, and human Duffyb) were transfused into nave C57BL/6 recipients. Serum cytokines and anti-HOD alloimmunization were evaluated following transfusion. Results In 6 of 6 experiments (n=90 mice total), a pro-inflammatory serum cytokine storm of interleukin-6, keratinocyte-derived chemokine/CXCL1, and monocyte chemoattractant protein-1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti-HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p<0.05). Conclusions These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform old blood groups may be needed in future clinical studies to most accurately investigate the biological effects of older RBC units. PMID:21645005

  13. Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.

    PubMed

    Tomasi, S; Lohzic-Le Dvhat, F; Sauleau, P; Bzivin, C; Boustie, J

    2004-04-01

    Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 < or = 20 microg/ml) against at least one of the two murine cancer cell lines (Ganoderma lucidum, Meripilus giganteus, Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before. PMID:15125575

  14. Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells.

    PubMed

    Mezzapelle, Rosanna; Rrapaj, Eltjona; Gatti, Elena; Ceriotti, Chiara; Marchis, Francesco De; Preti, Alessandro; Spinelli, Antonello E; Perani, Laura; Venturini, Massimo; Valtorta, Silvia; Moresco, Rosa Maria; Pecciarini, Lorenza; Doglioni, Claudio; Frenquelli, Michela; Crippa, Luca; Recordati, Camilla; Scanziani, Eugenio; de Vries, Hilda; Berns, Anton; Frapolli, Roberta; Boldorini, Renzo; D'Incalci, Maurizio; Bianchi, Marco E; Crippa, Massimo P

    2016-01-01

    Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment. PMID:26961782

  15. Human malignant mesothelioma is recapitulated in immunocompetent BALB/c mice injected with murine AB cells

    PubMed Central

    Mezzapelle, Rosanna; Rrapaj, Eltjona; Gatti, Elena; Ceriotti, Chiara; Marchis, Francesco De; Preti, Alessandro; Spinelli, Antonello E.; Perani, Laura; Venturini, Massimo; Valtorta, Silvia; Moresco, Rosa Maria; Pecciarini, Lorenza; Doglioni, Claudio; Frenquelli, Michela; Crippa, Luca; Recordati, Camilla; Scanziani, Eugenio; de Vries, Hilda; Berns, Anton; Frapolli, Roberta; Boldorini, Renzo; D’Incalci, Maurizio; Bianchi, Marco E.; Crippa, Massimo P.

    2016-01-01

    Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment. PMID:26961782

  16. Effects of some endocrine disruptors on cell cycle progression and murine dendritic cell differentiation.

    PubMed

    Pisapia, L; Del Pozzo, G; Barba, P; Caputo, L; Mita, L; Viggiano, E; Russo, G L; Nicolucci, C; Rossi, S; Bencivenga, U; Mita, D G; Diano, N

    2012-08-01

    Endocrine disruptor chemicals (EDCs), which are predominantly present in the environment, are able to mimic or antagonise the biological activity of hormones primarily through the interaction with specific receptors. The main consequences are adverse effects on the growth and development of reproductive organs, the induction of cancer and effects on neuronal differentiation. In this study, we investigated the ability of certain EDCs, Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF), 4-n Nonylphenol (NP) and Octylphenol (OP), belonging to a homogeneous group of phenol origin, to interfere with specific cellular processes, namely, proliferation, by using MCF-7 breast carcinoma cells, and differentiation, by using murine bone marrow dendritic cells. We correlated the data on cell growth with the stimulation of cell cycle progression, which could become a step in the development of cancer, and we established a proliferation ranking between the tested EDCs: NP>BPA>OP>BPB>BPF. In addition, we investigated the ability of NP, BPA and OP to induce the differentiation of dendritic cells, the powerful antigen-presenting cells of the immune system. The differentiation and activation of these cells could affect a well-regulated immune response and determine an allergic sensitisation. We found that BPA and NP were active in determining differentiation. PMID:22531466

  17. Primate gammaretroviruses require an ancillary factor not required for murine gammaretroviruses to infect BHK cells.

    PubMed

    Xu, Wenqin; Eiden, Maribeth V

    2011-04-01

    BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice. PMID:21270153

  18. Primate Gammaretroviruses Require an Ancillary Factor Not Required for Murine Gammaretroviruses To Infect BHK Cells?

    PubMed Central

    Xu, Wenqin; Eiden, Maribeth V.

    2011-01-01

    BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice. PMID:21270153

  19. Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration

    PubMed Central

    de la Garza-Rodea, Anabel S; Boersma, Hester; Dambrot, Cheryl; de Vries, Antoine AF; van Bekkum, Dirk W; Knan-Shanzer, Shoshan

    2015-01-01

    AIM: To study regeneration of damaged human and murine muscle implants and the contribution of added xenogeneic mesenchymal stem cells (MSCs). METHODS: Minced human or mouse skeletal muscle tissues were implanted together with human or mouse MSCs subcutaneously on the back of non-obese diabetic/severe combined immunodeficient mice. The muscle tissues (both human and murine) were minced with scalpels into small pieces (< 1 mm3) and aliquoted in portions of 200 mm3. These portions were either cryopreserved in 10% dimethylsulfoxide or freshly implanted. Syngeneic or xenogeneic MSCs were added to the minced muscles directly before implantation. Implants were collected at 7, 14, 30 or 45 d after transplantation and processed for (immuno)histological analysis. The progression of muscle regeneration was assessed using a standard histological staining (hematoxylin-phloxin-saffron). Antibodies recognizing Pax7 and von Willebrand factor were used to detect the presence of satellite cells and blood vessels, respectively. To enable detection of the bone marrow-derived MSCs or their derivatives we used MSCs previously transduced with lentiviral vectors expressing a cytoplasmic LacZ gene. X-gal staining of the fixed tissues was used to detect ?-galactosidase-positive cells and myofibers. RESULTS: Myoregeneration in implants of fresh murine muscle was evident as early as day 7, and progressed with time to occupy 50% to 70% of the implants. Regeneration of fresh human muscle was slower. These observations of fresh muscle implants were in contrast to the regeneration of cryopreserved murine muscle that proceeded similarly to that of fresh tissue except for day 45 (P < 0.05). Cryopreserved human muscle showed minimal regeneration, suggesting that the freezing procedure was detrimental to human satellite cells. In fresh and cryopreserved mouse muscle supplemented with LacZ-tagged mouse MSCs, ?-galactosidase-positive myofibers were identified early after grafting at the well-vascularized periphery of the implants. The contribution of human MSCs to murine myofiber formation was, however, restricted to the cryopreserved mouse muscle implants. This suggests that fresh murine muscle tissue provides a suboptimal environment for maintenance of human MSCs. A detailed analysis of the histological sections of the various muscle implants revealed the presence of cellular structures with a deviating morphology. Additional stainings with alizarin red and alcian blue showed myofiber calcification in 50 of 66 human muscle implants, and encapsulated cartilage in 10 of 81 of murine muscle implants, respectively. CONCLUSION: In mouse models the engagement of human MSCs in myoregeneration might be underestimated. Furthermore, our model permits the dissection of species-specific factors in the microenvironment. PMID:25992329

  20. CD4+ T Helper 1 Cells Facilitate Regression of Murine Lyme Carditis

    PubMed Central

    Bockenstedt, Linda K.; Kang, Insoo; Chang, Christopher; Persing, David; Hayday, Adrian; Barthold, Stephen W.

    2001-01-01

    Murine Lyme borreliosis, caused by infection with the spirochete Borrelia burgdorferi, results in acute arthritis and carditis that regress as a result of B. burgdorferi-specific immune responses. B. burgdorferi-specific antibodies can attenuate arthritis in mice deficient in both B cells and T cells but have no effect on carditis. Because macrophages comprise the principal immune cell in carditis, T-cell responses that augment cell-mediated immunity may be important for carditis regression. To investigate this hypothesis, we examined the course of Lyme carditis in mice selectively deficient in B cells or ?? T cells. Our results show that carditis regresses in B-cell-deficient B10.Ak mice but not in ?? T-cell-deficient mice, independently of the mouse strain background. Despite prominent macrophage infiltrates, hearts from B. burgdorferi-infected ?? T-cell-deficient mice had less mRNA for tumor necrosis factor alpha as measured by reverse transcription-PCR compared to infected control mice. Anti-inflammatory cytokine mRNA levels were equivalent. Adoptive transfer of gamma interferon-secreting CD4+ T cells into infected ?? T-cell-deficient mice promoted carditis resolution. These results show that ?? T cells can promote resolution of murine Lyme carditis and are the first demonstration of a beneficial role for CD4+ T helper 1 cells in this disease. PMID:11500394

  1. RANKL-mediated osteoclast formation from murine RAW 264.7 cells.

    PubMed

    Collin-Osdoby, Patricia; Osdoby, Philip

    2012-01-01

    Extensive research efforts over the years have provided us with great insights into how bone-resorbing osteoclasts (OCs) develop and function and, based on such work, valuable antiresorptive therapies have been developed to help combat the excessive bone loss that occurs in numerous skeletal disorders. The RAW 264.7 murine cell line has proven to be an important tool for in vitro studies of OC formation and function, having particular advantages over the use of OCs generated from primary bone marrow cell populations or directly isolated from murine bones. These include their ready access and availability, simple culture for this pure macrophage/pre-OC population, sensitive and rapid development into highly bone-resorptive OCs expressing hallmark OC characteristics following their RANKL stimulation, abundance of RAW cell-derived OCs that can be generated to provide large amounts of study material, relative ease of transfection for genetic and regulatory manipulation, and close correlation in characteristics, gene expression, signaling, and developmental or functional processes between RAW cell-derived OCs and OCs either directly isolated from murine bones or formed in vitro from primary bone marrow precursor cells. Here, we describe methods for the culture and RANKL-mediated differentiation of RAW cells into bone-resorptive OCs as well as procedures for their enrichment, characterization, and general use in diverse analytical assays. PMID:22130930

  2. Diabetes, insulin-mediated glucose metabolism and Sertoli/blood-testis barrier function

    PubMed Central

    Alves, Marco G.; Martins, Ana D.; Cavaco, José E.; Socorro, Sílvia; Oliveira, Pedro F.

    2013-01-01

    Blood testis barrier (BTB) is one of the tightest blood-barriers controlling the entry of substances into the intratubular fluid. Diabetes Mellitus (DM) is an epidemic metabolic disease concurrent with falling fertility rates, which provokes severe detrimental BTB alterations. It induces testicular alterations, disrupting the metabolic cooperation between the cellular constituents of BTB, with dramatic consequences on sperm quality and fertility. As Sertoli cells are involved in the regulation of spermatogenesis, providing nutritional support for germ cells, any metabolic alteration in these cells derived from DM may be responsible for spermatogenesis disruption, playing a crucial role in fertility/subfertility associated with this pathology. These cells have a glucose sensing machinery that reacts to hormonal fluctuations and several mechanisms to counteract hyper/hypoglycemic events. The role of DM on Sertoli/BTB glucose metabolism dynamics and the metabolic molecular mechanisms through which DM and insulin deregulation alter its functioning, affecting male reproductive potential will be discussed. PMID:24665384

  3. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

    NASA Technical Reports Server (NTRS)

    Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

    1986-01-01

    Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

  4. Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells.

    PubMed

    Van Den Heuvel, R L; Leppens, H; Schoeters, G E

    1999-04-01

    In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations. PMID:10408357

  5. Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones

    PubMed Central

    Koyama, Sachiko; Soini, Helena A.; Foley, John; Novotny, Milos V.; Lai, Cary

    2013-01-01

    Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner. PMID:23964214

  6. Inhibition of Murine Leukemia Virus Production in Chronically Infected AKR Cells: A Novel Effect of Interferon

    PubMed Central

    Friedman, Robert M.; Ramseur, Janet M.

    1974-01-01

    Treatment of AKR cells that had spontaneously become procedures of a murine leukemia virus with a partially purified mouse interferon (> 5 × 107 international mouse reference units per mg of protein) inhibited endogenous virus production. This inhibitory effect decreased over a 72-hr period in a manner similar to interferon-induced antiviral activity directed against vesicular stomatitis virus in AKR cells. Despite the inhibitory effect of interferon on infectious murine leukemia virus and viral reverse transcriptase (RNA-dependent DNA polymerase) titers in the culture fluids, intracellular levels of viral groups-specific antigens were significantly increased. These results suggest that interferon treatment in AKR cells inhibited the assembly or release of the virus. PMID:4139716

  7. Kinetics of Murine Gammaherpesvirus 68 Gene Expression following Infection of Murine Cells in Culture and in Mice

    PubMed Central

    Rochford, Rosemary; Lutzke, Mary L.; Alfinito, Rosiane S.; Clavo, Anaira; Cardin, Rhonda D.

    2001-01-01

    A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection. PMID:11333874

  8. Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice.

    PubMed

    Rochford, R; Lutzke, M L; Alfinito, R S; Clavo, A; Cardin, R D

    2001-06-01

    A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection. PMID:11333874

  9. Mammospheres from murine mammary stem cell-enriched basal cells: clonal characteristics and repopulating potential.

    PubMed

    Dong, Qiaoxiang; Wang, Danhan; Bandyopadhyay, Abhik; Gao, Hui; Gorena, Karla M; Hildreth, Kim; Rebel, Vivienne I; Walter, Christi A; Huang, Changjiang; Sun, Lu-Zhe

    2013-05-01

    Identification of murine mammary stem cells (MaSCs) has been attempted with various in vitro and in vivo assays. While, the in vivo repopulation assay remains as the most definitive assay for MaSC detection, it is expensive, time-consuming, and technically challenging. The in vitro mammosphere assay was considered unreliable because of major concerns about its clonal origin. In the current study, co-culture experiments with mammary cells from fluorescent protein transgenic mice and time-lapse video microscopy revealed that >90% mammospheres formed from sorted basal epithelial-enriched cells were of clonal origin in terms of stem cell. These basal-cell derived mammospheres were further distinguished morphologically in a 3-dimensional extracellular matrix culture and functionally in the in vivo repopulation assay. Transplant of single mammospheres or the resultant 3-dimensional solid structures into gland-free mammary fat pads yielded a 70% success rate of multilineage mammary gland reconstitution. Thus, this in vitro sphere formation and differentiation assay is a reliable alternative to the in vivo repopulation assay for the study of MaSCs. PMID:23466563

  10. Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

    PubMed Central

    Vella, Joseph B.; Thompson, Seth D.; Bucsek, Mark J.; Song, Minjung; Huard, Johnny

    2011-01-01

    Background Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity. Methodology/Principal Findings Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs. Conclusions The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy. PMID:22195027

  11. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    PubMed Central

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective, controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine vocal fold epithelium. Method Twelve mice were administered daily intraperitoneal injections of a nucleotide dye, bromodeoxyuridine (BrdU), over seven consecutive days. Under this pulse-chase paradigm, slow-cycling cells retain the dye (label-retaining cells; LRCs) while more rapidly cycling cells lose dye to dilution during multiple cell divisions. The percentage of label-retaining cells (%LRCs) was calculated following a chase period of two, four, and eight weeks post-injections. Results The %LRCs decreased significantly from 9.4% at two weeks to 3.1% at eight weeks following injections (p<.05). No statistically significant differences in the quantity of BrdU-positive cells were measured between the anterior, mid-membranous, or cartilaginous regions of the vocal fold (p>.05). Conclusions These findings are consistent with the presence and first report of a small population of putative stem cells along the length of murine vocal fold epithelium. PMID:21330647

  12. RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

    PubMed Central

    KISHIMOTO, MAYUKO; MATSUDA, TAKENORI; YANASE, SHOUGO; KATSUMI, AKIRA; SUZUKI, NOBUAKI; IKEJIRI, MAKOTO; TAKAGI, AKIRA; IKAWA, MASAHITO; KOJIMA, TETSUHITO; KUNISHIMA, SHINJI; KIYOI, HITOSHI; NAOE, TOMOKI; MATSUSHITA, TADASHI; MARUYAMA, MITSUO

    2014-01-01

    ABSTRACT RhoF is a member of the Rho GTPase family that has been implicated in various cell functions including long filopodia formation, adhesion, and migration of cells. Although RhoF is expressed in lymphoid tissues, the roles of RhoF in B cell development remain largely unclear. On the other hand, other members of the Rho GTPase family, such as Cdc42, RhoA, and Rac, have been intensively studied and are known to be required for B cell development in the bone marrow and spleen. We hypothesized that RhoF is also involved in B cell development. To examine our hypothesis, we analyzed B cell development in RhoF knockout (KO) mice and found a significant reduction in marginal zone (MZ) B cells in the spleen, although T cell development in the thymus and spleen was not affected. Consistent with these results, the width of the MZ B cell region in the spleen was significantly reduced in the RhoF KO mice. However, the antigen-specific antibody titer of IgM and IgG3 after MZ B cell-specific antigen (T cell-independent antigen, type I) stimulation was not affected by RhoF deletion. Furthermore, we demonstrated that RhoF was dispensable for stromal cell-derived factor-1α- and B lymphocyte chemoattractant-induced B cell migration. These results suggest that RhoF promotes MZ B cell development in the spleen. PMID:25741038

  13. Neonatal CD71+ Erythroid Cells Do Not Modify Murine Sepsis Mortality.

    PubMed

    Wynn, James L; Scumpia, Philip O; Stocks, Blair T; Romano-Keeler, Joann; Alrifai, Mhd Wael; Liu, Jin-Hua; Kim, Annette S; Alford, Catherine E; Matta, Pranathi; Weitkamp, Jrn-Hendrik; Moore, Daniel J

    2015-08-01

    Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization. PMID:26101326

  14. Murine trophoblast can be killed by lymphokine-activated killer cells.

    PubMed

    Drake, B L; Head, J R

    1989-07-01

    The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface. PMID:2499634

  15. Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

    PubMed Central

    Huang, Cheng; Walker, Aida G.; Grant, Ashley M.; Kolokoltsova, Olga A.; Yun, Nadezhda E.; Seregin, Alexey V.; Paessler, Slobodan

    2014-01-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice. PMID:24901990

  16. In vitro Assay for Screening of Optimal Targets for Antigen-Delivery to Murine Dendritic Cells.

    PubMed

    Pugholm, L H; Varming, K; Agger, R

    2015-12-01

    Targeting of antigen to dendritic cells (DCs) increase the efficiency of immunization procedures and may facilitate the development of more effective vaccines. Several surface molecules on DCs have shown to be useful for antigen targeting, but many more deserves investigation for their efficacy in this respect. With this end in mind, a simple invitro assay for screening of optimal targets for antigen-delivery to murine DCs was established. Splenocytes from mice immunized with rat IgG were targeted invitro with a panel of different rat monoclonal antibodies (mAbs) directed against surface markers on murine DCs. The resulting T-cell activation was analysed by determining the number of IFN-? and IL-4 secreting cells by ELISPOT. A positive effect of targeting was evident with several of the mAbs. Thus, mAbs against CD11c, CD36, CD205 and Clec7A all induced IFN-? responses that were significantly higher than those induced by non-targeting control mAbs. Anti-CD36 also induced IL-4 responses that were significantly higher than the control. The assay described here allows simultaneous analysis of a large number of potential target structures and facilitates direct comparison between the different targets regarding the strength of the T-cell responses induced by the targeted DCs. The assay could be useful as a first-line screening of potential target structures on murine DCs. PMID:26331836

  17. ?? T Cells Confer Protection against Murine Cytomegalovirus (MCMV)

    PubMed Central

    Villacreces, Arnaud; Juzan, Marina; Rousseau, Benot; Dulanto, Sara; Giese, Alban; Costet, Pierre; Praloran, Vincent; Moreau, Jean-Franois; Dubus, Pierre; Vermijlen, David

    2015-01-01

    Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. ?? T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 ?? and/or ?? T cell-deficient mice, we here show that ?? T cells are as competent as ?? T cells to protect mice from CMV-induced death. ?? T cell-mediated protection involved control of viral load and prevented organ damage. ?? T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3??/? mice from CMV-induced death confirming the protective antiviral role of ?? T cells. As observed in humans, different ?? T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27? ?? T cells. NK cells were the largely preponderant producers of IFN? and cytotoxic granules throughout the infection, suggesting that the protective role of ?? T cells did not principally rely on either of these two functions. Finally, ?? T cells were strikingly sufficient to fully protect Rag?/??c?/? mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of ?? T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new ?? T cell based therapies, especially useful in ?? T cell compromised patients. PMID:25747674

  18. Doxorubicin treatment induces tumor cell death followed by immunomodulation in a murine neuroblastoma model

    PubMed Central

    INOUE, SEIICHIRO; SETOYAMA, YUMIKO; ODAKA, AKIO

    2014-01-01

    Chemotherapy of malignant tumors induces tumor cell death. Numerous antitumor agents induce apoptosis of tumor cells, which are subsequently engulfed by phagocytes, initiating an immune reaction. The induction of immunogenic cell death by antitumor agents may be advantageous for antitumor immunity. The purpose of this study was to determine whether doxorubicin is capable of inducing an immunogenic reaction in murine neuroblastoma cells. The murine neuroblastoma cell line (neuro-2a cells) was cultured in a medium containing doxorubicin or cisplatin (CDDP), and induction of cell death was confirmed by cell viability assays. Cluster of differentiation (CD)8?+ lymphocytes were co-cultured with neuro-2a cells that had died following treatment with either doxorubicin or CDDP, and CD11b+ spleen cells or bone marrow-derived dendritic cells (BM-DCs) were added to the culture. Proliferation of CD8?+ lymphocytes and interferon (IFN)-? production were evaluated. When CD8?+ cells were co-cultured with doxorubicin-treated neuro-2a cells and BM-DCs, CD8?+ cells reacted to anti-CD3/CD28 antibody stimulation, proliferated and increased IFN-? production. IFN-? production was more effectively promoted by co-culture with doxorubicin-treated neuro-2a cells than by co-culture with CDDP-treated neuro-2a cells. These findings suggest that doxorubicin is capable of inducing immunogenic cell death in neuroblastoma cells, and thus has an immunological advantage for chemotherapy of neuroblastoma compared with CDDP. BM-DCs are considered to be the key antigen-presenting cells in the immune reaction following the induction of immunogenic neuroblastoma cell death and phagocytosis. PMID:24520271

  19. Conjugated linoleic acid induces apoptosis of murine mammary tumor cells via Bcl-2 loss

    PubMed Central

    Ou, Lihui; Ip, Clement; Lisafeld, Barbara; Ip, Margot M.

    2007-01-01

    Conjugated linoleic acid (CLA) is a powerful anticancer agent in a number of tumor model systems; however, its precise mechanism of action remains elusive. Here, we report that t10,c12 CLA, a component of synthetic CLA supplements, induced apoptosis and G1 arrest of p53 mutant TM4t murine mammary tumor cells. Furthermore, t10,c12-CLA induced a time- and concentration-dependent cleavage of caspases-3 and -9, and release of cytochrome c from mitochondria to cytosol. Levels of Bcl-2 protein were decreased both in total cellular lysates and in mitochondria after t10,c12-CLA treatment; however, there was no significant change in Bax or Bak. Overexpression of Bcl-2 attenuated apoptosis in response to t10,c12-CLA treatment. These results demonstrate that t10,c12-CLA triggers apoptosis of p53 mutant murine mammary tumor cells through the mitochondrial pathway by targeting Bcl-2. PMID:17400188

  20. The Human Lung Adenocarcinoma Cell Line EKVX Produces an Infectious Xenotropic Murine Leukemia Virus

    PubMed Central

    Cmarik, Joan L.; Troxler, Jami A.; Hanson, Charlotte A.; Zhang, Xiang; Ruscetti, Sandra K.

    2011-01-01

    The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells. PMID:22355448

  1. The human lung adenocarcinoma cell line EKVX produces an infectious xenotropic murine leukemia virus.

    PubMed

    Cmarik, Joan L; Troxler, Jami A; Hanson, Charlotte A; Zhang, Xiang; Ruscetti, Sandra K

    2011-12-01

    The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells. PMID:22355448

  2. Timing is everything: dendritic cell subsets in murine Leishmania infection.

    PubMed

    Ashok, Devika; Acha-Orbea, Hans

    2014-10-01

    Mouse models of Leishmania major infection have shown that a predominant CD4(+) T helper type 1 cell (Th1) response leads to protection, while T helper type 2 cell (Th2) predominance confers susceptibility. Dendritic cells (DCs) are antigen-presenting cells that orchestrate the T cell response. The immune response to L. major involves direct antigen presentation by migrating DCs or transfer of antigens to resident DCs to prime T cells. In this review, we discuss the timing and consequences of antigen presentation by DC subsets and how this affects Leishmania susceptibility. We propose a model where dermal DCs and Langerhans cells play a role early in infection, followed by inflammatory monocyte-derived DC and lymph node (LN)-resident DCs at later time points of infection to establish the resistant Th1 response. PMID:25190685

  3. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi

    PubMed Central

    Val, Stéphanie; Burgett, Katelyn; Brown, Kristy J.; Preciado, Diego

    2016-01-01

    Background Chronic Otitis Media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute Otitis Media (OM) pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line. Methods NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC) cultured at air-liquid interface over 48 hours– 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling. Results Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05). The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface. Conclusions NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level. PMID:26859300

  4. Anti-(human LFA-1) monoclonal antibodies bind P815 murine tumour cells.

    PubMed

    Palisson, M J; Altemeyer, A; Moosbrugger, I; Warter, S; Hauptmann, G; Bischoff, P

    1992-01-01

    Using anti-CD11a and anti-CD18 monoclonal antibodies (mAbs) directed respectively against the alpha and the beta chains of LFA-1, we obtained an important and specific staining of P815 murine tumour cells. Both ascitic and cultured cells displayed a positive staining. Other murine tumours of haematopoietic origin, as well as lymphocytes or lymphoblasts from DBA/2 mice, were not labelled by the same monoclonal antibodies. These results were surprising since, to our knowledge, no case of cross-reaction between species has been reported with LFA-1. Moreover, competition assays showed that epitopes recognized by the two anti-CD11a antibodies were different from those identified by H35.89.9, a mAb raised against the murine LFA-1 alpha chain. Using allogeneic cytotoxic T lymphocytes, we also showed that anti-(human LFA-1) mAbs were unable to block the lysis of P815 by these effector cells. Thus, the putative functional properties of these structures, as well as their importance from an antigeneic point of view, remain to be assessed. PMID:1373342

  5. Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

    SciTech Connect

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

    1986-05-01

    Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN/sup -/) for murine Cu-Zn-SOD was determined to be 6.8 x 10/sup -6/ M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied.

  6. Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease.

    PubMed

    Flynn, Ryan; Allen, Jessica L; Luznik, Leo; MacDonald, Kelli P; Paz, Katelyn; Alexander, Kylie A; Vulic, Ante; Du, Jing; Panoskaltsis-Mortari, Angela; Taylor, Patricia A; Poe, Jonathan C; Serody, Jonathan S; Murphy, William J; Hill, Geoffrey R; Maillard, Ivan; Koreth, John; Cutler, Corey S; Soiffer, Robert J; Antin, Joseph H; Ritz, Jerome; Chao, Nelson J; Clynes, Raphael A; Sarantopoulos, Stefanie; Blazar, Bruce R

    2015-06-25

    Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD. PMID:25852057

  7. Isolation and Characterization of Murine Mandibular Condylar Cartilage Cell Populations

    PubMed Central

    Chen, J.; Utreja, A.; Kalajzic, Z.; Sobue, T.; Rowe, D.; Wadhwa, S.

    2012-01-01

    Objectives The mandibular condylar cartilage is a heterogeneous tissue containing cells at various stages of chondrocyte maturation organized into 4 zones: superficial, polymorphic, flattened, and hypertrophic. The goal of this study was to use transgenic mice containing chondrocyte maturation markers fused to fluorescent protein transgenes to isolate and characterize homogenous cell populations of the mandibular condylar cartilage. Methods Fluorescent reporter expression in the mandibular condylar cartilage of transgenic mice containing the 3.6-kb fragment of the rat collagen type 1 promoter fused to a topaz-fluorescent protein (Col3.6-tpz), collagen type 2 promoter fused to a cyan-fluorescent protein (Col2-cyan), and/or collagen type 10 promoter fused to cherry-fluorescent protein (Col10-cherry) was examined. Mandibular condylar cartilage cells were analyzed by fluorescence-activated cell sorting (FACS) and either used for gene expression analysis or plated in cell cultures and exposed to adipogenic, osteogenic, or chondrogenic conditions. To determine cell fate, transgenic mice containing the Col3.6-cre recombinase were bred with cre reporter mice. Results Localization and analysis of gene expression revealed that Col3.6-tpz-positive cells corresponded to the polymorphic/flattened zones and Col2-cyan-positive cells corresponded to the flattened/hypertrophic zones of the mandibular condylar cartilage. Mandibular condylar cartilage FACS-sorted Col3.6-tpz-positive cells have the potential to differentiate into bone, cartilage, and fat. Cell fate mapping revealed that Col3.6 cells are precursors of some of the hypertrophic chondrocytes in the mandibular condylar cartilage. Conclusion Col3.6-tpz cells represent an earlier stage of the mandibular condylar cartilage maturation pathway. PMID:21646777

  8. Regulation of Glycan Structures in Murine Embryonic Stem Cells

    PubMed Central

    Nairn, Alison V.; Aoki, Kazuhiro; dela Rosa, Mitche; Porterfield, Mindy; Lim, Jae-Min; Kulik, Michael; Pierce, J. Michael; Wells, Lance; Dalton, Stephen; Tiemeyer, Michael; Moremen, Kelley W.

    2012-01-01

    The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased ?-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas ?-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development. PMID:22988249

  9. Electrochemotherapy with bleomycin induces hallmarks of immunogenic cell death in murine colon cancer cells.

    PubMed

    Calvet, Christophe Y; Famin, Delphine; Andr, Franck M; Mir, Lluis M

    2014-01-01

    Electrochemotherapy (ECT) is a local cancer treatment that has been used over the course of more than 2 decades for the removal of cutaneous and subcutaneous tumors. Several lines of evidence support the premise that the immune system is an important factor underlying anticancer treatment efficacy, potentially including patient responses to ECT. The concept of immunogenic cell death (ICD) arose a few years ago, stating that some cancer treatments generate danger-associated molecular patterns (DAMPs) that trigger an adaptive immune response against tumors. Hence, dying cancer cells behave as a therapeutic vaccine, eliciting a cytotoxic immune response against surviving malignant cells. In our study, we sought to evaluate the ability of ECT to generate cancer cell death encompassing the immunostimulatory characteristics of ICD. To this end, we assayed CT26 murine colon cancer cells in vitro in response to either electric pulses (EPs) application only or in combination with the anticancer drug bleomycin (that is ECT) by quantification of calreticulin (CRT) membrane externalization, as well as the liberation of adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) protein. We show here that cell permeabilizing yet non-lethal electric pulses induce CRT exposure on the cell surface of EP-only treated cancer cells, as well as ATP release. However, the association of electric pulses along with the chemotherapeutic agent bleomycin was mandatory for HMGB1 release coincident with regimen-induced cell death. These data obtained in vitro were then substantiated by vaccination protocols performed in immunocompetent mice, showing that the injection of dying ECT-treated cells elicits an antitumor immune response that prevents the growth of a subsequent administration of viable cancer cells. We also confirmed previous results showing ECT treatment is much more efficient in immunocompetent animals than in immunodeficient ones, causing complete regressions in the former but not in the latter. This supports a central role for immunity in this beneficial outcome. In conclusion, we show that ECT not only possesses an intrinsic cytotoxic property toward cancer cells but also generates a systemic anticancer immune response via the activation of ICD. Hence, ECT may represent an interesting approach to treat solid tumors while preventing recurrence and metastasis, possibly in combination with immunostimulating agents. PMID:25083316

  10. The effects of simulated hypogravity on murine bone marrow cells

    NASA Technical Reports Server (NTRS)

    Lawless, Desales

    1989-01-01

    Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

  11. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  12. AUGMENTATION OF MURINE NATURAL KILLER CELL ACTIVITY BY MANGANESE CHLORIDE

    EPA Science Inventory

    Natural Killer (NK) cell activity of spleen cells from male CBA/J mice was augmented by a single parenteral injection of MnCl2 administered 1 day prior to testing by in vitro and in vivo isotope release assays. Increased cytotoxic activity was observed in vitro against both NK-se...

  13. Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

    PubMed

    Lu, Ming-Yu; Li, Zhihong; Hwang, Shiaw-Min; Linju Yen, B; Lee, Gwo-Bin

    2015-09-01

    This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future. PMID:26396661

  14. Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

    PubMed Central

    Tsunoda, Kazuyuki; Nakagawa, Taneaki; Tsubota, Kazuo

    2016-01-01

    Purpose Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). Methods SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. Results SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. Conclusions Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium. PMID:26800086

  15. Study on the therapeutic effect of neural progenitor cells in mice of a glioma murine model

    PubMed Central

    XU, GUOZHENG; LIU, YING; ZHANG, YI; YANG, QIAN; DIAO, BO

    2016-01-01

    Glioma is a common malignacy of the brain that affects elderly patients in particular. Despite treatment, however, the survival rate is 12 months. The aim of the present study was to examine the therapeutic effect of neural progenitor cells (NPCs) on a glioma murine model, and to determine the possible mechanism of action. A glioma murine model was constructed and the tumor volume and tumor growth rate were measured. The therapeutic effect of cell injection on the glioma mouse model mice was confirmed. The quantitative polymerase chain reaction method was used to detect the expression of proto-oncogene and tumor suppressor gene. Intracranial injection of NPCs was performed to determine cell apoptosis. Preliminary results showed the mechanism of cell therapy effect at the transcription and cellular level. Compared with the model group, the tumor volume of the mice of the cell therapy group was significantly reduced from the 6th to 8th week, and the tumor growth rate was downregulated. The mechanism of action identified that NPCs regulate gene expression in tumor tissues, increase the expression of tumor suppressor gene, downregulate the gene expression of tumor cells, and reverse the proto-oncogene and imbalance of gene expression in gliomas. In conclusion, the new type of cell injection method can regulate the proto-oncogene of tumor tissue and tumor suppressor gene, improve the function phenotype of the cell, and effectively improve the clinical symptoms of mice with gliomas.

  16. Targeting the inhibitory receptor CTLA-4 on T cells increased abscopal effects in murine mesothelioma model

    PubMed Central

    Wu, Licun; Wu, Matthew Onn; De la Maza, Luis; Yun, Zhihong; Yu, Julie; Zhao, Yidan; Cho, John; de Perrot, Marc

    2015-01-01

    We previously demonstrated that blockade of immune suppressive CTLA-4 resulted in tumor growth delay when combined with chemotherapy in murine mesothelioma. Tumor-infiltrating T cells (TIT) after local radiotherapy (LRT) play critical roles in abscopal effect against cancer. We attempt to improve the local and abscopal effect by modulating T cell immunity with systemic blockade of CTLA-4 signal. The growth of primary tumors was significantly inhibited by LRT while CTLA-4 antibody enhanced the antitumor effect. Growth delay of the second tumors was achieved when the primary tumor was radiated. LRT resulted in more T cell infiltration into both tumors, including Treg and cytotoxic T cells. Interestingly, the proportion of Treg over effector T cells in both tumors was reversed after CTLA-4 blockade, while CD8 T cells were further activated. The expression of the immune-related genes was upregulated and cytokine production was significantly increased. LRT resulted in an increase of TIT, while CTLA-4 blockade led to significant reduction of Tregs and increase of cytotoxic T cells in both tumors. The abscopal effect is enhanced by targeting the immune checkpoints through modulation of T cell immune response in murine mesothelioma. PMID:25980578

  17. Identification and Analysis of Natural Killer Cells in Murine Nasal Passages

    PubMed Central

    Okada, Kazunari; Sato, Shintaro; Sato, Ayuko; Mandelboim, Ofer; Yamasoba, Tatsuya; Kiyono, Hiroshi

    2015-01-01

    Background Natural killer (NK) cells in the upper respiratory airways are not well characterized. In the current study, we sought to characterize and functionally assess murine nasal NK cells. Methods Using immunohistochemistry and flow cytometry, we compared the nasal NK cells of Ncr1GFP/+ knock-in mice, whose NK cells produced green fluorescent protein, with their splenic and pulmonary counterparts. In addition, we functionally analyzed the nasal NK cells of these mice in vitro. To assess the in vivo functions of nasal NK cells, C57BL/6 mice depleted of NK cells after treatment with PK136 antibody were nasally infected with influenza virus PR8. Results Immunohistochemical analysis confirmed the presence of NK cells in the lamina propria of nasal mucosa, and flow cytometry showed that these cells were of NK cell lineage. The expression patterns of Ly49 receptor, CD11b/CD27, CD62L and CD69 revealed that nasal NK cells had an immature and activated phenotype compared with that of their splenic and pulmonary counterparts. Effector functions including degranulation and IFN(interferon)-γ production after in vitro stimulation with phorbol 12-myristate-13-acetate plus ionomycin or IL(interleukin)-12 plus IL-18 were dampened in nasal NK cells, and the depletion of NK cells led to an increased influenza virus titer in nasal passages. Conclusions The NK cells of the murine nasal passage belong to the conventional NK cell linage and characteristically demonstrate an immature and activated phenotype. Despite their hyporesponsiveness in vitro, nasal NK cells play important roles in the host defense against nasal influenza virus infection. PMID:26575399

  18. Murine mechanical ventilation stimulates alveolar epithelial cell proliferation.

    PubMed

    Chess, Patricia Rose; Benson, Randi Potter; Maniscalco, William M; Wright, Terry W; O'Reilly, Michael A; Johnston, Carl J

    2010-08-01

    High tidal volume mechanical ventilation can cause inflammation and lung damage. Mechanical strain is also necessary for normal lung growth. The current work was performed to determine if mechanical ventilation with clinically utilized tidal volumes stimulates a proliferative response in the lung. Six- to 8-week-old C57/Bl6 mice, anesthetized with ketamine/xylozine, were ventilated for 6 hours with 10 mL/kg tidal volume, positive end-expiratory pressure (PEEP) 3cm H(2)O. Pulmonary function testing demonstrated decreased compliance within 3 hours of ventilation. Assessment of bronchoalveolar lavage (BAL) demonstrated no significant increase in lactate dehydrogenase, total lavagable cell number, or total protein after ventilation. There was evidence of inflammation in the lungs of ventilated mice, with an increased percentage of lymphocytes and neutrophils in BAL, and an increase in macrophage inflammatory protein (MIP)-2 and interleukin (IL)-1beta message in lung tissue. Immunohistochemistry of inflation-fixed lungs demonstrated increased alveolar cell proliferation, as measured by both proliferating cell nuclear antigen and Ki67 staining. Dual staining confirmed that proliferating cells labeled with proSP-B, demonstrating that ventilation induces proliferation of alveolar type II cells. Ventilation did not increase apoptosis in alveolar type II cells, as measured by TUNEL staining. Ventilation at low tidal volumes leads to a mild inflammatory response and alveolar epithelial cell proliferation. PMID:20653468

  19. Cultured human bone marrow-derived mast cells, their similarities to cultured murine E-mast cells.

    PubMed Central

    Gilead, L; Rahamim, E; Ziv, I; Or, R; Razin, E

    1988-01-01

    Homogeneous populations of human mast cells were differentiated and grown by culturing bone marrow cells in the presence of conditioned medium derived from lectin-stimulated human peripheral blood mononuclear cells. The cells obtained were similar in ultrastructure, proteoglycan type and lipid products generated upon calcium ionophore A23187, and immunological activation to the murine E-mast cells (E-MC) differentiated in culture containing IL-3. Fluorescence analysis revealed that the human E-MC expressed IgE-Fc receptors which retained bound IgE through several washes. These cells did not express cell-surface lymphoid determinants (T11, T4, T8 and B4) and myeloid determinants 'My'. However, 40% of these cells expressed monocytic surface determinants, such as M-1. The amount of histamine that was found per 10(6) cells was 525 +/- 106 ng (mean +/- SE, n = 4). These cultured mast cells possessed granular chondroitin sulphate E proteoglycan of about 180,000 MW. Following activation with either calcium ionophore A23187 or anti-hIgE challenge, these mast cells released their preformed mediators and generated mainly leukotriene C4 leukotriene B4, and platelet-activating factor. In conclusion, according to all of these criteria, these human cultured mast cells show many similarities to the murine cultured E-mast cells, and therefore could be considered as the culture analogue of the human intestinal E-mast cells identified recently. Images Figure 1 PMID:3130309

  20. Neuropilin-1 Identifies Endothelial Precursors in Human and Murine Embryonic Stem Cells Prior to CD34 Expression

    PubMed Central

    Cimato, Thomas; Beers, Jeanette; Ding, Shunli; Ma, Mingchao; McCoy, J. Phillip; Boehm, Manfred; Nabel, Elizabeth G.

    2009-01-01

    Background In murine embryonic stem cells (ESCs), the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human ESCs express VEGFR2, and VEGFR2 expression persists upon differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine ESCs. Methods and Results We report expression of the VEGF co-receptor NRP-1 coincides with expression of Brachyury and VEGFR-2, and identifies endothelial precursors in murine and human ESCs prior to CD31 or CD34 expression. When sorted and differentiated, VEGFR-2+NRP-1+ cells form endothelial-like colonies that express CD31, CD34 seven fold more efficiently than NRP-1? cells. Finally, antagonism of both the VEGF and Semaphorin-binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells. Conclusions The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP1 have important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP1 in a model of vasculogenesis. PMID:19364973

  1. Enhanced gene expression of the murine ecotropic retroviral receptor and its human homolog in proliferating cells.

    PubMed Central

    Yoshimoto, T; Yoshimoto, E; Meruelo, D

    1992-01-01

    The receptor for gp70 envelope glycoprotein of murine ecotropic leukemia virus is essential for virus entry into the host cell and has been recently demonstrated to function as a cationic amino acid transporter. In the experiments reported herein, we compared the gene expression of the murine ecotropic retroviral receptor (ERR) and its human homolog (H13) in rapidly proliferating cells versus resting cells using four different systems. (i) The expression of ERR gene is enhanced during activation of T and B lymphocytes by concanavalin A and lipopolysaccharide, respectively. Similar enhancement is observed by adding phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). These phenomena appear to involve protein kinase C; two PMA analogs, 4 alpha-phorbol and 4 alpha-PMA, lacking the ability to activate protein kinase C fail to induce elevated levels of gene expression, and the protein kinase C inhibitor, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride[, inhibits the enhancement induced by PMA. (ii) Friend murine leukemia virus induces rapid splenomegaly, and acute erythroleukemia in sensitive mice. Concomitantly with splenomegaly, ERR gene expression in spleen cells increases dramatically. (iii) The level of expression of the ERR or H13 gene in a variety of tumor cells is highly elevated compared with the level in noncancerous cells. (iv) H13 gene expression decreases upon terminal differentiation of the human promyelocytic leukemia cell line HL-60 into granulocytes or macrophages by dimethyl sulfoxide or PMA, respectively. These results suggest that ERR and H13 genes play an important role in cellular proliferation. Images PMID:1318407

  2. Development and function of murine B cells lacking RANK.

    PubMed

    Perlot, Thomas; Penninger, Josef M

    2012-02-01

    RANKL-RANK signaling regulates numerous physiologic processes such as bone remodeling, lymph node organogenesis, central thermoregulation, and formation of a lactating mammary gland in pregnancy. Recently, a receptor activator of NF-?B ligand (RANKL)-blocking Ab has been approved for human use in potentially millions of osteoporosis and cancer patients. However, germline deficiencies in RANKL or receptor activator of NF-?B (RANK) also lead to strong B cell defects in mice and human patients, suggesting that RANKL-RANK inhibition could interfere with B cell physiology and thereby trigger immunologic side-effects. To address this key question--that is, whether RANKL-RANK signaling affects B cell physiology directly or the observed defects are secondary because of the severe osteopetrosis--we generated B cell-specific RANK knockout mice. We show that B cells deficient for RANK undergo normal development and do not show any obvious defects in Ab secretion, class switch recombination, or somatic hypermutation. Our data indicate that ablation of the RANKL-RANK pathway has no direct adverse effect on B cell physiology. PMID:22219325

  3. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed Central

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-01-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  4. A kinesin is present at unique sertoli/spermatid adherens junctions in rat and mouse testes.

    PubMed

    Vaid, Kuljeet S; Guttman, Julian A; Singaraja, Roshni R; Vogl, A Wayne

    2007-12-01

    During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20. PMID:17855729

  5. Murine CMV infection induces the continuous production of mucosal resident T cells

    PubMed Central

    Smith, Corinne J.; Caldeira-Dantas, Sofia; Turula, Holly; Snyder, Christopher M.

    2015-01-01

    Summary Cytomegalovirus (CMV) is a herpesvirus that persists for life and maintains extremely large numbers of T cells with select specificities in circulation. However, it is unknown how viral persistence impacts T cell populations in mucosal sites. We found that many murine (M)CMV-specific CD8s in mucosal tissues became resident memory T cells (TRM). These cells adopted an intraepithelial localization in the salivary gland that correlated with, but did not depend on, expression of the integrin CD103. MCMV-specific TRM cells formed early after infection and spleen-localized cells had reduced capacities to become TRM at late times. Surprisingly however, small numbers of new TRM cells were formed from the circulating pool throughout infection, favoring populations maintained at high levels in the blood and shifting the immunodominance within the TRM populations over time. These data show that mucosal TRM populations can be dynamically maintained by a persistent infection. PMID:26526996

  6. Sublethal oxidative stress inhibits tumor cell adhesion and enhances experimental metastasis of murine mammary carcinoma.

    PubMed

    Kundu, N; Zhang, S; Fulton, A M

    1995-01-01

    We have postulated that murine mammary tumor progression is fueled, in part, by tumor-associated macrophages that deliver sub-lethal oxidative stress to tumor cells. In the present study, we determined whether oxidative stress would affect murine mammary tumor cell attachment to laminin and fibronectin, critical functions in the metastatic process. Sublethal oxidative stress generated by exposure of cells to hydrogen peroxide (H2O2, 1-1000 microM/L) inhibited tumor cell attachment to immobilized laminin or fibronectin. This oxidant effect was blocked in the presence of catalase which removes H2O2. The inhibitory effect on attachment was rapid, with significant inhibition occurring at 5 min; total inhibition was achieved at 60 min with 1 mM H2O2. The oxidative stress effect was partially reversible at 20 h post-treatment and occurred at concentrations of H2O2 that do not adversely affect cell viability or growth. Pretreatment of tumor cells with H2O2 or hypoxanthanine and xanthine oxidase (to generate superoxide radical and H2O2) prior to intravenous injection, enhanced experimental lung tumor colony formation. The enhancement of experimental metastatic potential with enzyme-generated oxidative stress was completely reversed by catalase; the H2O2-mediated enhancement was only partially reversed with catalase. Thus, treatments that inhibit tumor cell attachment to extracellular matrix proteins in vitro enhance experimental metastasis in vivo. PMID:7820952

  7. 1.8 Astroms Structure of Murine GITR Ligand Dimer Expressed in Drosophila Melanogaster S2 Cells

    SciTech Connect

    Chattopadhyay, K.; Ramagopal, U; Nathenson, S; Almo, S

    2009-01-01

    Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique 'strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 {angstrom} resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical 'strand-exchanged' dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  8. Hydrolysates of egg white proteins modulate T- and B-cell responses in mitogen-stimulated murine cells.

    PubMed

    Lozano-Ojalvo, Daniel; Molina, Elena; Lpez-Fandio, Rosina

    2016-02-17

    This work assessed the effects of hydrolysates of ovalbumin (OVA), lysozyme (LYS), ovomucoid (OM) and whole egg white (EW) on cytokine secretion, antibody production, oxidative stress and proliferation of murine spleen and mesenteric lymph node cells stimulated with T- (concanavalin A - ConA) or B-cell mitogens (lipopolysaccharide - LPS). The hydrolysates of OVA, LYS and EW with alcalase reduced ConA-stimulated lymphocyte proliferation and production of Th2-biased cytokines, such as IL-13 and IL-10, and decreased the secretion of the Th1 cytokine TNF-?. In addition, these hydrolysates considerably inhibited IgG1-class switching induced by LPS and counteracted the release of reactive oxygen species. EW peptides modulated the immune responses of murine cells to mitogen stimuli, revealing potential activities that could be used for different purposes as Th1- or Th2-skewing mediators. PMID:26778535

  9. Hinokitiol induces autophagy in murine breast and colorectal cancer cells.

    PubMed

    Wang, Wei-Kuang; Lin, Song-Tao; Chang, Wen-Wei; Liu, Li-Wen; Li, Tom Yu-Tung; Kuo, Chun-Yu; Hsieh, Jeng-Long; Lee, Che-Hsin

    2016-01-01

    Hinokitiol is found in the heartwood of cupressaceous plants and possesses several biological activities. Hinokitiol may play an important role in anti-inflammation and antioxidant processes, making it potentially useful in therapies for inflammatory-mediated disease. Previously, the suppression of tumor growth by hinokitiol has been shown to occur through apoptosis. Programmed cell death can also occur through autophagy, but the mechanism of hinokitiol-induced autophagy in tumor cells is poorly defined. We used an autophagy inhibitor (3-methyladenine) to demonstrate that hinokitiol can induce cell death via an autophagic pathway. Further, we suggest that hinokitiol induces autophagy in a dose-dependent manner. Markers of autophagy were increased after tumor cells were treated with hinokitiol. In addition, immunoblotting revealed that the levels of phosphoprotein kinase B (P-AKT), phosphomammalian target of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70S6K) in tumor cells were decreased after hinokitiol treatment. In conclusion, our results indicate that hinokitiol induces the autophagic signaling pathway via downregulation of the AKT/mTOR pathway. Therefore, our findings show that hinokitiol may control tumor growth by inducing autophagic signaling. 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 77-84, 2016. PMID:25044443

  10. Engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation.

    PubMed

    van der Schaft, Daisy W J; van Spreeuwel, Ariane C C; Boonen, Kristel J M; Langelaan, Marloes L P; Bouten, Carlijn V C; Baaijens, Frank P T

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative (1). The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues (2,3). Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts (4), neonatal muscle derived progenitor cells (5), cells derived from adult muscle tissues from other species such as human (6) or even induced pluripotent stem cells (iPS cells) (7). Cell contractility causes alignment of the cells along the long axis of the construct (8,9) and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent (8). Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary. PMID:23542531

  11. Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

    PubMed Central

    van der Schaft, Daisy W. J.; van Spreeuwel, Ariane C. C.; Boonen, Kristel J. M.; Langelaan, Marloes L. P.; Bouten, Carlijn V. C.; Baaijens, Frank P. T.

    2013-01-01

    Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary. PMID:23542531

  12. Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

    SciTech Connect

    Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath; Lane, Thomas A.; Carrier, Ewa . E-mail: assrivastava@ucsd.edu

    2006-07-28

    Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.

  13. Granzyme D is a novel murine mast cell protease that is highly induced by multiple pathways of mast cell activation.

    PubMed

    Rönnberg, Elin; Calounova, Gabriela; Guss, Bengt; Lundequist, Anders; Pejler, Gunnar

    2013-06-01

    Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy. PMID:23529614

  14. Modeling the pathways of energy balance using the N1E-115 murine neuroblastoma cell line.

    PubMed

    Roth, Jonathan D; Yee, Daniel K; Kisley, Lori R; Fluharty, Steven J

    2002-06-30

    A good in vitro model within which to investigate molecular interactions between feeding relevant neuropeptide systems has been lacking. Consequently, we began using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen various neuronal cell lines for the presence of feeding relevant neuropeptides and receptors. N1E-115 murine neuroblastoma cells have emerged as an attractive candidate for further analysis because they contain mRNA for a variety of key systems implicated in the regulation of energy homeostasis. PMID:12106700

  15. Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine

    PubMed Central

    Koh, Sang Don; Sanders, Kenton M; Ward, Sean M

    1998-01-01

    Interstitial cells of Cajal (ICC) are pacemaker cells in the small bowel, and therefore this cell type must express the mechanism responsible for slow wave activity. Isolated ICC were cultured for 13 days from the murine small intestine and identified with c-Kit-like immunoreactivity (c-Kit-LI).Electrical recordings were obtained from cultured ICC with the whole-cell patch clamp technique. ICC were rhythmically active, producing regular slow wave depolarizations with waveforms and properties similar to slow waves in intact tissues.Spontaneous activity of c-Kit-LI cells was inhibited by reduced extracellular Na+, gadolinium, and reduced extracellular Ca2+. The activity was not affected by nisoldipine. Voltage clamp studies showed rhythmic inward currents that were probably responsible for the slow wave activity. The current-voltage relationship showed that the spontaneous currents reversed at about +17 mV. These observations are consistent with the involvement of a non-selective cation current in the generation of slow waves, but do not rule out contributions from other conductances or transporters.A Ba2+-sensitive inwardly rectifying K+ current in c-Kit-LI cells that may be involved in slow wave repolarization and maintenance of a negative potential between slow waves was also found. Similar pharmacology was observed in studies of intact murine intestinal muscles.Cultured ICC may be a useful model for studying the properties and pharmacology of some of the ionic conductances involved in spontaneous rhythmicity in the gastrointestinal tract. PMID:9782170

  16. IFN-?-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure.

    PubMed

    Chen, Jichun; Feng, Xingmin; Desierto, Marie J; Keyvanfar, Keyvan; Young, Neal S

    2015-12-10

    Interferon gamma (IFN-?) has been reported to have both negative and positive activity on hematopoietic cells, adding complexity to the interpretation of its pleiotropic functions. We examined the effects of IFN-? on murine hematopoietic stem cells (HSCs) and progenitors in vitro and in vivo by using mouse models. IFN-? treatment expanded bone marrow (BM) c-Kit(+)Sca1(+)Lin(-) (KSL) cell number but reduced BM KLCD150(+) and KLCD150(+)CD48(-) cells. IFN-?-expanded KSL cells engrafted poorly when tested by competitive repopulation in vivo. KSL, KLCD150(+), and KLCD150(+)CD48(-) cells from IFN-?-treated animals all showed significant upregulation in Fas expression. When cocultured with activated T cells in vitro, KSL and KLCD150(+) cells from IFN-?-treated donors showed increased apoptosis relative to those from untreated animals, and infusion of activated CD8 T cells into IFN-?-injected animals in vivo led to partial elimination of KSL cells. Exposure of BM cells or KSL cells to IFN-? increased expression of Fas, caspases, and related proapoptotic genes and decreased expression of Ets-1 and other hematopoietic genes. In mouse models of BM failure, mice genetically deficient in IFN-? receptor expression showed attenuation of immune-mediated marrow destruction, whereas effector lymphocytes from IFN-?-deficient donors were much less potent in initiating BM damage. We conclude that the activity of IFN-? on murine hematopoiesis is context dependent. IFN-?-augmented apoptotic gene expression facilitates destruction of HSCs and progenitors in the presence of activated cytotoxic T cells, as occurs in human BM failure. PMID:26491068

  17. Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

    SciTech Connect

    Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti; Barnett, Joey V.; Camenisch, Todd D.

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme. • Arsenic does not block TGFβ2 induced smooth muscle cell differentiation.

  18. Estrogen and progesterone together expand murine endometrial epithelial progenitor cells

    PubMed Central

    Janzen, DM; Cheng, D; Schafenacker, AM; Paik, DY; Goldstein, AS; Witte, ON; Jaroszewicz, A; Pellegrini, M; Memarzadeh, S

    2013-01-01

    Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multi-lineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Co-administration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. PMID:23341289

  19. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines.

    PubMed

    Bzivin, C; Tomasi, S; Lohzic-Le Dvhat, F; Boustie, J

    2003-01-01

    Eight lichens were extracted successively with n-hexane, diethyl ether and methanol using a Soxhlet process. The cytotoxic activity of the 24 lichen extracts was evaluated in vitro using two murine (the L1210: lymphocytic leukaemia, and the 3LL: Lewis lung carcinoma) and four human (the K-562: chronic myelogenous leukaemia, the U251: glioblastoma, the DU145: prostate carcinoma, and the MCF7: breast adenocarcinoma) cancer cell lines and non-cancerous cells, the Vero cell line (African green monkey kidney cell line). The MTT assay revealed significant cytotoxicity (IC50 < or = 20 microg/ml) on one of the tested cancer cell lines for at least one extract of each lichen species. Some extracts of Cladonia convoluta, Cladonia rangiformis, Parmelia caperata, Platismatia glauca and Ramalina cuspidata demonstrated interesting activities particularly on human cancer cell lines as good selectivity indices were recorded (SI > 3). PMID:13678234

  20. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

    SciTech Connect

    Feurstein, D.; Holst, K.; Fischer, A.; Dietrich, D.R.

    2009-01-15

    Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 {mu}M) and BSP (50 {mu}M). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

  1. Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand

    SciTech Connect

    Humtsoe, Joseph O.; Bowling, Rodney A.; Feng, Shu; Wary, Kishore K. . E-mail: kwary@ibt.tamhsc.edu

    2005-09-30

    Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE). Whether the RGE motif of murine Lpp3 mediates cell-cell interaction has not been studied. In this report, we test the hypothesis that the cell adhesion function of the LPP3 protein is conserved across mouse and human. A glutathione S-transferase (GST) fusion protein of the proposed second extracellular loop of the murine Lpp3 sequence (GST-mLpp3-RGE) promoted attachment of cells in a long-term cell adhesion assay. GST-mLpp3-RGE interacted with {alpha}{sub 5}{beta}{sub 1} and {alpha}{sub v}{beta}{sub 3} integrins in a solid-phase ELISA, while a mutant control, GST-hLPP3-RAD, did not. Long-term adhesion of endothelial cells to GST-mLpp3-RGE induced phosphorylation of FAK, SHC, and CAS, whereas adhesion to GST-hLPP3-RAD failed to do so. Upon long-term adhesion both the GST-hLPP3-RGD and GST-mLpp3-RGE substrates bound to the {alpha}{sub 5}{beta}{sub 1} integrin of FRT-{alpha}{sub 5}(+) cells, an interaction that was inhibited by an anti-{alpha}{sub 5} integrin antibody. In addition, a cell aggregation assay showed that the intact mLpp3-RGE protein interacts with {alpha}{sub 5}{beta}{sub 1} and {alpha}{sub v}{beta}{sub 3} integrins expressed by adjacent cells, an interaction that can be blocked by GRGDSP peptides and anti-LPP3-RGD antibodies. These data, together with the known importance of integrins in angiogenesis, provide a mechanism for the function of LPP3 in cell-cell interactions in both human and mouse.

  2. Differential effects of protoporphyrin and uroporphyrin on murine mast cells

    SciTech Connect

    Lim, H.W.; Gigli, I.; Wasserman, S.I.

    1987-03-01

    To investigate the mechanisms responsible for the distinct cutaneous manifestations of erythropoietic protoporphyria and porphyria cutanea tarda, the effects of protoporphyrin (PP) and uroporphyrin (URO), the predominant porphyrins in the respective disease, on mast cells were examined. Release of preformed and generated mediators was assessed by the release of radioactivity from cells labeled with (/sup 3/H)serotonin and (/sup 14/C)arachidonic acid, respectively. Clinically relevant doses of PP (25-500 ng/ml) and 396-407 nm irradiation (3-16 X 10(2)J/m2) induced maximal net release of preformed mediators ,f 44.52 +/- 6.6 to 58.01 +/- 4.0% (mean +/- SE). In contrast, irradiation in the presence of URO (50-5000 ng/ml) resulted in less than 5% net release. (3H)Serotonin release induced by PP and irradiation was calcium-independent, and was not enhanced by phorbol 12-myristate 13-acetate, a known activator of protein kinase C. This release was suppressed by catalase, a scavenger of hydrogen peroxide. Furthermore, irradiation in the presence of PP, but not in the presence of URO, resulted in perturbation of cell membrane. Irradiation in the presence of PP also resulted in a maximal net release of generated mediators of 9.98 +/- 3.5% (mean +/- SE), whereas similar treatment in the presence of URO induced less than 0.5% net release. These results suggested that the burning, stinging, erythema, and edema experienced by patients with erythropoietic protoporphyria following sun exposure, and the lack of such findings in patients with porphyria cutanea tarda, may be explained, at least in part, by the differential effects of PP and URO on mast cells.

  3. Alveolar epithelial cells orchestrate DC function in murine viral pneumonia

    PubMed Central

    Unkel, Barbara; Hoegner, Katrin; Clausen, Bjrn E.; Lewe-Schlosser, Peter; Bodner, Johannes; Gattenloehner, Stefan; Janen, Hermann; Seeger, Werner; Lohmeyer, Juergen; Herold, Susanne

    2012-01-01

    Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal that GM-CSFdependent cross-talk between IV-infected AECs and CD103+ DCs is crucial for effective viral clearance and recovery from injury, which has potential implications for GM-CSF treatment in severe IV pneumonia. PMID:22996662

  4. Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.

    PubMed

    Kubaczka, Caroline; Senner, Claire E; Cierlitza, Monika; Arazo-Bravo, Marcos J; Kuckenberg, Peter; Peitz, Michael; Hemberger, Myriam; Schorle, Hubert

    2015-11-01

    Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation. PMID:26412560

  5. Effects of glial cell line-derived neurotrophic factor on cultured murine retinal progenitor cells

    PubMed Central

    Wang, Jinmei; Yang, Jing; Gu, Ping

    2010-01-01

    Purpose Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective of retinal neurons, and transduced retinal progenitor cells (RPCs) can deliver this cytokine for the treatment of retinal diseases, yet the potential effects of GDNF on RPCs have received little attention. Methods Murine RPCs were assessed under multiple conditions in the presence or absence of epidermal growth factor (EGF, 20 ng/ml) and/or GDNF (10 ng/ml) using a variety of techniques, including live-cell imaging, caspase-3 activity assay, whole genome microarray, quantitative polymerase chain reaction (qPCR), and western blotting. Results Live monitoring revealed that formation of initial aggregates resulted largely from the collision and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres enlarged in size and number, with more reaching the threshold criteria for cross-sectional areas in the EGF+GDNF condition. Proliferation was measurably augmented in association with EGF+GDNF, and Ki-67 expression was modestly increased (1.07 fold), as were hairy and enhancer of split 5 (Hes5), mammalian achaete-scute homolog 1 (Mash1), and Vimentin. However, global gene expression did not reveal a notable treatment-related response, and the expression of the majority of progenitor and lineage markers examined remained stable. GDNF reduced RPC apoptosis, compared to complete growth-factor withdrawal, although it could not by itself sustain mitotic activity. Conclusions These data support the feasibility of developing GDNF-transduced RPCs as potential therapeutic agents for use in retinal diseases. PMID:21203407

  6. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  7. An ES-Like Pluripotent State in FGF-Dependent Murine iPS cells

    PubMed Central

    Ungaro, Federica; Prigione, Alessandro; Chen, Hsu-Hsin; Welling, Maaike; Eijpe, Maureen; Mostoslavsky, Gustavo; Tesar, Paul; Adjaye, James; Geijsen, Niels; Broccoli, Vania

    2010-01-01

    Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a nave LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display nave ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. PMID:21209851

  8. Index sorting resolves heterogeneous murine hematopoietic stem cell populations

    PubMed Central

    Schulte, Reiner; Wilson, Nicola K.; Prick, Janine C.M.; Cossetti, Chiara; Maj, Michal K.; Gottgens, Berthold; Kent, David G.

    2015-01-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information. PMID:26051918

  9. Ex vivo 3D osteocyte network construction with primary murine bone cells

    PubMed Central

    Sun, Qiaoling; Gu, Yexin; Zhang, Wenting; Dziopa, Leah; Zilberberg, Jenny; Lee, Woo

    2015-01-01

    Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 m. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models. PMID:26421212

  10. A novel cholinergic epithelial cell with chemosensory traits in the murine conjunctiva.

    PubMed

    Wiederhold, Stephanie; Papadakis, Tamara; Chubanov, Vladimir; Gudermann, Thomas; Krasteva-Christ, Gabriela; Kummer, Wolfgang

    2015-11-01

    We recently identified a specialized cholinergic cell type in tracheal and urethral epithelium that utilizes molecules of the canonical taste transduction signaling cascade to sense potentially harmful substances in the luminal content. Upon stimulation, this cell initiates protective reflexes. Assuming a sentinel role of such cells at mucosal surfaces exposed to bacteria, we hypothesized their occurrence also in ocular mucosal surfaces. Utilizing a mouse strain expressing eGFP under the promoter of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT-eGFP), we observed a cholinergic cell in the murine conjunctiva. Singular cholinergic cells reaching the epithelial surface with slender processes were detected in fornical, but neither in bulbar nor palpebral epithelia. These cells were found neither in the lacrimal canaliculi, nor in the lacrimal sac and the nasolacrimal duct. Cholinergic conjunctival epithelial cells were immunoreactive for components of the canonical taste transduction signaling cascade, i.e. ?-gustducin, phospholipase C?2 and the monovalent cation channel TRPM5. Calcitonin gene-related peptide- and substance P-immunoreactive sensory nerve fibers were observed extending into the conjunctival epithelium approaching slender ChAT-eGFP-positive cells. In addition, we noted both ChAT-eGFP expression and ?-gustducin-immunoreactivity, albeit in different cell populations, in occasionally occurring lymphoid follicles of the nictitating membrane. The data show a previously unidentified cholinergic cell in murine conjunctiva with chemosensory traits that presumably utilizes acetylcholine for signaling. In analogy to similar cells described in the respiratory and urethral epithelium, it might serve to detect bacterial products and to initiate protective reflexes. PMID:26119492

  11. Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

    PubMed Central

    Moss, Catherine E.; Glass, Leslie L.; Diakogiannaki, Eleftheria; Pais, Ramona; Lenaghan, Carol; Smith, David M.; Wedin, Marianne; Bohlooly-Y, Mohammad; Gribble, Fiona M.; Reimann, Frank

    2016-01-01

    Aims/hypothesis Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. Methods GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. Results L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. Conclusions/interpretation GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion. PMID:26144594

  12. Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

    PubMed

    Schfer, Katja; Bain, Judith M; Di Pietro, Antonio; Gow, Neil A R; Erwig, Lars P

    2014-01-01

    Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host. PMID:25025395

  13. An In Vitro Murine Model of Vascular Smooth Muscle Cell Mineralization.

    PubMed

    Kelynack, Kristen J; Holt, Stephen G

    2016-01-01

    Vascular calcification (VC) is seen ubiquitously in aging blood vessels and prematurely in disease states like renal failure. It is thought to be driven by a number of systemic and local factors that lead to extra-osseous deposition of mineral in the vascular wall and valves as a common endpoint. The response of resident vascular smooth muscle cell to these dystrophic signals appears to be important in this process. Whilst in vivo models allow the observation of global changes in a pro-calcific environment, identifying the specific cells and mechanisms involved has been largely garnered from in vitro experiments, which provide added benefits in terms of reproducibility, cost, and convenience. Here we describe a 7-21 day cell culture model of calcification developed using immortalized murine vascular smooth muscle cells (MOVAS-1). This model provides a method by which vascular smooth muscle cell involvement and manipulation within a mineralizing domain can be studied. PMID:26676135

  14. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification

    PubMed Central

    Lai, D.; Ding, J.; Smith, G.W.; Smith, G.D.; Takayama, S.

    2015-01-01

    STUDY QUESTION Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? SUMMARY ANSWER The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. WHAT IS KNOWN ALREADY The use of more steps of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. STUDY DESIGN, SIZE, DURATION Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using KedemKatchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. MAIN RESULTS AND THE ROLE OF CHANCE The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P < 0.001), more spherical cellular morphology (P < 0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (P < 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P < 0.05) and improved developmental competence of vitrified and warmed murine zygotes (P < 0.05) than CPA exposure using the current clinically used manual pipetting method. LIMITATIONS, REASONS FOR CAUTION It is necessary to design the microfluidic device to be more user-friendly for widespread use. WIDER IMPLICATIONS OF THE FINDINGS The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology. STUDY FUNDING/COMPETING INTEREST(S) This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest. PMID:25355589

  15. Murine Hematopoietic Stem Cells and Multipotent Progenitors Express Truncated Intracellular Form of c-kit Receptor

    PubMed Central

    ZAYAS, JENNIFER; SPASSOV, DANISLAV S.; NACHTMAN, RONALD G.; JURECIC, ROLAND

    2011-01-01

    The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors. PMID:18447649

  16. Murine hematopoietic stem cells and multipotent progenitors express truncated intracellular form of c-kit receptor.

    PubMed

    Zayas, Jennifer; Spassov, Danislav S; Nachtman, Ronald G; Jurecic, Roland

    2008-04-01

    The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors. PMID:18447649

  17. Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model.

    PubMed

    Rothweiler, Sonja; Dill, Michael T; Terracciano, Luigi; Makowska, Zuzanna; Quagliata, Luca; Hlushchuk, Ruslan; Djonov, Valentin; Heim, Markus H; Semela, David

    2015-03-01

    Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing. PMID:25418579

  18. Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell

    PubMed Central

    Kawakita, Tetsuya; Shimmura, Shigeto; Hornia, Armand; Higa, Kazunari; Tseng, Scheffer C G

    2008-01-01

    The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily-isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum-free low [Ca2+] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm2) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage-independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air-medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up-regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine lim-bal/corneal epithelium-derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision. PMID:18318692

  19. Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells

    SciTech Connect

    Shalinsky, D.R.

    1988-01-01

    In efforts to exploit the differential oxygen levels within the subcompartments of solid neoplasms, this project has focused on modulating prostaglandin (PG) biosynthesis under aerobic and hypoxic conditions. Mammary adenocarcinoma tumor cells (Line 4526), either intact or sonicated, were incubated with either 2.0 uM {sup 14}C-arachidonic acid (AA) or 20.0 uM {sup 14}C-PGH{sub 2}, respectively. Following metabolism, products were extracted, separated by thin layer chromatography and analyzed by radiochromatographic scan. PGE{sub 2} was predominantly formed with minimal amounts of PGF{sub 2a} or PGD{sub 2}. Indomethacin and ibuprofen inhibited the PGE{sub 2} formation from AA with an IC{sub 50} value of 6.3 {times} 10{sup {minus}8} and 9.6 {times} 10{sup {minus}5}M, respectively. Suspended cells in glass vials were made hypoxic by flushing with N{sub 2} for varying time intervals to study AA metabolism. A time-dependent inhibition of PG biosynthesis was observed under hypoxia, and by 30 min, the PGE{sub 2} synthesis was reduced by 50% which was further inhibited by indomethacin. Misonidazole, a 2-nitroimidazole analogue, partially reversed the inhibition of PGE{sub 2} synthesis under hypoxia by 49% at 100 uM. However, misonidazole did not affect PG biosynthesis under aerobic conditions. The stimulation of PGE{sub 2} biosynthesis by misonidazole under hypoxia was blocked by indomethacin, suggesting that misonidazole can not act independently of the cyclooxygenase.

  20. IL-33 promotes MHC class II expression in murine mast cells

    PubMed Central

    Ito, Tomonobu; Egusa, Chizu; Maeda, Tatsuo; Numata, Takafumi; Nakano, Nobuhiro; Nishiyama, Chiharu; Tsuboi, Ryoji

    2015-01-01

    Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of FcεRI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis. PMID:26417437

  1. IL-33 promotes MHC class II expression in murine mast cells.

    PubMed

    Ito, Tomonobu; Egusa, Chizu; Maeda, Tatsuo; Numata, Takafumi; Nakano, Nobuhiro; Nishiyama, Chiharu; Tsuboi, Ryoji

    2015-09-01

    Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of Fc?RI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis. PMID:26417437

  2. Crucial roles of mesodermal cell lineages in a murine embryonic stem cell-derived in vitro liver organogenesis system.

    PubMed

    Ogawa, Shinichiro; Tagawa, Yoh-ichi; Kamiyoshi, Akiko; Suzuki, Akihiro; Nakayama, Jun; Hashikura, Yasuhiko; Miyagawa, Shinichi

    2005-08-01

    Recent studies in the field of regenerative medicine have exploited the pluripotency of embryonic stem (ES) cells to generate a variety of cell lineages. However, the target has always been only a single lineage, which was isolated from other differentiated cell populations. In the present study, we selected sublines with a high capability for differentiation to contracting cardiomyocytes and also produced germ-line chimeric mice from a parent ES line. We also succeed in establishing embryoid bodies prepared from the ES cells that differentiated into not only hepatocytes but also at least two mesodermal lineages: cardiomyocytes that supported liver development and endothelial cells corresponding to sinusoids. This allowed the development of an in vitro system using murine ES cells that approximated the events of liver development in vivo. The expression of albumin was significantly higher in cardiomyocytes that had arisen in differentiated ES cells than in those that had not. Our in vitro system for liver organogenesis consists of a blood/sinusoid vascular-like network and hepatocyte layers and shows higher levels of hepatic function, such as albumin production and ammonia degradation, than hepatic cell lines and primary cultures of murine adult hepatocytes. This innovative system will lead to the development of second-generation regenerative medicine techniques using ES cells and is expected to be useful for the development of bioartificial liver systems and drug-metabolism assays. PMID:16043458

  3. Detection of increased tyrosine phosphorylation in murine Langerhans cells after stimulation with contact sensitizers.

    PubMed

    Neisius, U; Brand, P; Plochmann, S; Saloga, J; Knop, J; Becker, D

    1999-01-01

    The signalling pathways in epidermal Langerhans cells (LC) during activation by contact sensitizers are poorly understood. Recently, we have described an increased phosphorylation of tyrosine residues in human MHC class II-positive cells in vitro following stimulation with contact sensitizers. In the study reported here the formation of phosphotyrosine (p-tyr) in murine epidermal LC upon stimulation with contact sensitizers was examined. By the use of a flow cytometric technique a significant increase in p-tyr was demonstrated in LC stimulated in vitro with the strong contact sensitizers TNCB (2,4,6-trinitro-chlorobenzene) and MCI/MI (5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone) but not after treatment with the irritants sodium lauryl sulphate or benzoic acid. The protein tyrosine kinase inhibitors genistein and tyrphostin A9, but not tyrphostin AG 1288, were able to block this process significantly. Similar results were obtained using the LC-like dendritic cell line XS52. In addition, Western blot analysis on XS52 cells revealed a selective phosphorylation of two protein bands with a molecular weight between 50 and 60 kDa following stimulation with TNCB. These results demonstrate that contact sensitizers induce an increased phosphorylation of tyrosine residues in murine LC and can be used as the basis for in vivo studies using inhibitors for signal transduction pathways for prevention of primary sensitization to contact sensitizers. PMID:10025724

  4. Cinnamon extract reduces symptoms, inflammatory mediators and mast cell markers in murine IL-10(-/-) colitis.

    PubMed

    Hagenlocher, Yvonne; Hösel, Angela; Bischoff, Stephan C; Lorentz, Axel

    2016-04-01

    Inflammatory bowel disease (IBD) shows an increasing prevalence and harm in western countries. Conventional therapies are associated with bad compliance and adverse side effects. Natural substances like cinnamon extract (CE) could be an additional therapy. We found recently that CE acts anti-inflammatory on mast cells - discussed of being relevant in IBD. Here, we analysed the effects of CE on murine IL-10(-/-) colitis as model for IBD. Mice were treated 12 weeks with or without CE in drinking water. Clinical scores and disease activity index were assessed. Colonic tissue samples were analysed for infiltration, tissue damage, bowel wall thickness, expression of pro-inflammatory mediators, mast cell proteases, tight junction proteins, and NF-κB signaling. Following treatment with CE, symptoms of murine colitis as well as increased infiltration of immune cells, tissue damage and bowel wall thickness in colon tissue of IL-10(-/-) mice were diminished significantly. MIP-2, TNF, IFNγ, CCL2, CCL3, CCL4 and IL-1β as well as MC-CPA, MCP-1 and MCP-4 were strongly upregulated in IL-10(-/-) mice compared to WT, but noteworthy not in CE group. Expression of tight junction proteins was not influenced by CE. Phosphorylation of IκB was slightly down-regulated in CE treated IL-10(-/-) mice compared to IL-10(-/-) controls. In summary, CE decreases inflammatory symptoms and expression of inflammatory markers in murine IL-10(-/-) colitis. CE has no influence on tight junction proteins, but seems acting via reducing pro-inflammatory mediators and recruitment of neutrophil granulocytes probably by inhibiting NF-κB signaling. PMID:27012624

  5. Suppression of properties associated with malignancy in murine melanoma-melanocyte hybrid cells.

    PubMed Central

    Wakeling, W. F.; Greetham, J.; Devlin, L. M.; Bennett, D. C.

    1992-01-01

    Murine and human melanoma cells differ relatively reliably from non-tumorigenic melanocytes in certain biological properties. When cultured at low pH, melanocytes tend to be pigmented and melanoma cells unpigmented. The growth of virtually all metastatic melanoma cells is inhibited by phorbol esters such as TPA (12-O-tetradecanoyl phorbol-13-acetate), which stimulate melanocyte growth. Melanocytes fail to grow in suspension culture or produce tumours when implanted in animals, while many melanoma lines can do both. Here we studied which of these properties were dominant in hybrid cells formed by fusion of drug-resistant murine B16-F10RR melanoma cells to melanocytes of the albino and brown lines, melan-c and melan-b. The albino melanocytes are unpigmented but well-differentiated, the brown melanocytes produce pale brown pigment and the melanoma cells are unpigmented under the conditions used. All hybrid colonies observed produced black pigment, except some melan-b/melanoma hybrids when growing sparsely with TPA. Thus pigmentation was generally dominant. 14/15 hybrid lines showed stimulation of proliferation by TPA, as do melanocytes. Most hybrid lines showed no or reduced capacity for growth in suspension, though some grew better in suspension when TPA was present. There was marked suppression of the tumorigenicity of the parental melanoma cells in 4/8 hybrids examined, and tumorigenicity was reduced in the others, despite considerable chromosome loss by the passage level tested. Thus most properties of the non-tumorigenic pigment cells were dominant, as often observed for other cell lineages, and providing further evidence for gene loss in the genesis of malignant melanoma. Images Figure 1 Figure 3 PMID:1562463

  6. Accessing the Genomic Effects of Naked Nanoceria in Murine Neuronal Cells

    PubMed Central

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

    2011-01-01

    Cerium oxide nanoparticles (nanoceria) are versatile engineered nanoparticles (ENPs) due to their unique redox properties. We and others have previously demonstrated naked nanoceria could act as antioxidants to protect cells against oxidative damage. While the redox properties may be beneficial, the genome-wide effects of nanoceria on gene transcription and associated biological processes remain elusive. Here we applied functional genomic approach to examine the genome-wide effects of nanoceria on global gene transcription and cellular functions in mouse neuronal cells. Importantly, we demonstrated nanoceria induced chemical- and size-specific changes in the murine neuronal cell transcriptome. The nanoceria specially contributed more than 83% of uniquely altered gene population and associate with a unique spectrum of genes related to neurological disease, cell cycle control and growth. These observations suggest an in-depth assessment of potential health effects of naked nanoceria and other naked nanoparticles is both necessary and imminent. PMID:21889474

  7. Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

    PubMed Central

    Forni, Maria Fernanda; Ramos Maia Lobba, Aline; Pereira Ferreira, Alexandre Hamilton; Sogayar, Mari Cleide

    2015-01-01

    The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft. PMID:26462205

  8. Sca-1 Identifies a Distinct Androgen-Independent Murine Prostatic Luminal Cell Lineage with Bipotent Potential

    PubMed Central

    Kwon, Oh-Joon; Zhang, Li; Xin, Li

    2016-01-01

    Recent lineage tracing studies support the existence of prostate luminal progenitors that possess extensive regenerative capacity, but their identity remains unknown. We show that Sca-1 (Stem Cell Antigen-1) identifies a small population of murine prostate luminal cells that reside in the proximal prostatic ducts adjacent to the urethra. Sca-1+ luminal cells do not express Nkx3.1. They do not carry the secretory function, although they express the androgen receptor. These cells are enriched in the prostates of castrated mice. In the in vitro prostate organoid assay, a small fraction of the Sca-1+ luminal cells are capable of generating budding organoids that are morphologically distinct from those derived from other cell lineages. Histologically, this type of organoid is composed of multiple inner layers of luminal cells surrounded by multiple outer layers of basal cells. When passaged, these organoids retain their morphological and histological features. Finally, the Sca-1+ luminal cells are capable of forming small prostate glands containing both basal and luminal cells in an in vivo prostate regeneration assay. Collectively, our study establishes the androgen-independent and bipotent organoid-forming Sca-1+ luminal cells as a functionally distinct cellular entity. These cells may represent a putative luminal progenitor population and serve as a cellular origin for castration resistant prostate cancer. PMID:26418304

  9. Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

    PubMed

    Kusewitt, D F; Budge, C L; Nolla, H A; Edwards, B S; Ley, R D

    1992-09-01

    Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes. PMID:1380650

  10. Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

    PubMed

    Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

    2015-01-01

    Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 m/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. PMID:24966117

  11. Caspase-2 Mediated Apoptotic and Necrotic Murine Macrophage Cell Death Induced by Rough Brucella abortus

    PubMed Central

    Chen, Fang; He, Yongqun

    2009-01-01

    Brucella species are Gram-negative, facultative intracellular bacteria that cause zoonotic brucellosis. Survival and replication inside macrophages is critical for establishment of chronic Brucella infection. Virulent smooth B. abortus strain 2308 inhibits programmed macrophage cell death and replicates inside macrophages. Cattle B. abortus vaccine strain RB51 is an attenuated rough, lipopolysaccharide O antigen-deficient mutant derived from smooth strain 2308. B. abortus rough mutant RA1 contains a single wboA gene mutation in strain 2308. Our studies demonstrated that live RB51 and RA1, but not strain 2308 or heat-killed Brucella, induced both apoptotic and necrotic cell death in murine RAW264.7 macrophages and bone marrow derived macrophages. The same phenomenon was also observed in primary mouse peritoneal macrophages from mice immunized intraperitoneally with vaccine strain RB51 using the same dose as regularly performed in protection studies. Programmed macrophage cell death induced by RB51 and RA1 was inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK). Caspase-2 enzyme activation and cleavage were observed at the early infection stage in macrophages infected with RB51 and RA1 but not strain 2308. The inhibition of macrophage cell death promoted the survival of rough Brucella cells inside macrophages. The critical role of caspase-2 in mediating rough B. abortus induced macrophage cell death was confirmed using caspase-2 specific shRNA. The mitochondrial apoptosis pathway was activated in macrophages infected with rough B. abortus as demonstrated by increase in mitochondrial membrane permeability and the release of cytochrome c to cytoplasm in macrophages infected with rough Brucella. These results demonstrate that rough B. abortus strains RB51 and RA1 induce apoptotic and necrotic murine macrophage cell death that is mediated by caspase-2. The biological relevance of Brucella O antigen and caspase-2-mediated macrophage cell death in Brucella pathogenesis and protective Brucella immunity is discussed. PMID:19714247

  12. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells

    PubMed Central

    Padilla-Nash, Hesed M.; McNeil, Nicole E.

    2013-01-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  13. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

    PubMed

    Padilla-Nash, Hesed M; McNeil, Nicole E; Yi, Ming; Nguyen, Quang-Tri; Hu, Yue; Wangsa, Danny; Mack, David L; Hummon, Amanda B; Case, Chanelle; Cardin, Eric; Stephens, Robert; Difilippantonio, Michael J; Ried, Thomas

    2013-08-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  14. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q. , E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  15. Histopathology and host response associated with reduced tumorigenicity of 5-bromodeoxyuridine--treated murine melanoma cells.

    PubMed Central

    Gyi, K. K.; Wrathall, J. R.

    1983-01-01

    Growth of B16 murine melanoma cells (clone B559) with low concentrations of 5-bromodeoxyuridine (BrdUrd) has previously been shown to have little effect upon cell proliferation in culture but to significantly reduce the tumorigenicity of the cells when they are injected into adult syngeneic mice. To determine the fate of BrdUrd-grown cells in vivo, we compared the sites of injection of control, fully tumorigenic B559 cells, and BrdUrd-grown cells (3 micrograms/ml for 3 days, termed 3BRM cells) with greatly reduced tumorigenicity (only 8%). We found no evidence of even a transitory period of progressive growth in vivo of BrdUrd-grown cells. At sites of injection of 3BRM cells, as early as Day 1 after injection, there was a significantly lower proliferation rate of the melanoma cells, significantly greater numbers of infiltrating host mononuclear cells, and a significantly higher ratio of host mononuclear to melanoma cells. There was no evidence of endothelial cell proliferation or neovascularization at the sites of 3BRM cells. When sites of injection at 1 day after injection were examined electron-microscopically, evidence of host melanoma cell interactions was more frequently observed at sites of 3BRM cells. The results suggest that the reduced tumorigenicity of BrdUrd-grown melanoma cells may be due to cytostatic and/or cytotoxic influences generated by an enhanced mononuclear cell response. Because the host response occurs so quickly, the involvement of natural killer cells is postulated. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:6824061

  16. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  17. Murinization of Internalin Extends Its Receptor Repertoire, Altering Listeria monocytogenes Cell Tropism and Host Responses

    PubMed Central

    Tsai, Yu-Huan; Disson, Olivier; Bierne, Hlne; Lecuit, Marc

    2013-01-01

    Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been murinized to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlAm) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlAm-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlAm-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen. PMID:23737746

  18. Cyclic-radiation response of murine fibrosarcoma cells grown as pulmonary nodules

    SciTech Connect

    Grdina, D.J.; Hunter, N.

    1982-10-01

    The radiation age response of murine fibrosarcoma (FSa) cells grown as pulmonary nodules in C/sub 3/Hf/Kam mice was determined. FSa cells were irradiated in vivo either with 10 Gy as 14 day-old lung tumors (i.e., artificial macrometastases) prior to cell separation or with 5 Gy as single cells trapped in the lungs of recipient mice (i.e., artificial micrometastases) following cell separation and synchronization by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine cell-cycle parameters and the relative synchrony of the separated populations, as well as the percent contamination of normal diploid cells in each of the tumor cell populations. Tumor populations containing up to 90% G/sub 1/, 60% S-, and 75% G/sub 2/+M-phase tumor cells were obtained. Cell clonogenicity, determined using a lung colony assay, ranged from 0.7 to 6% for control FSa cells from the various elutriator fractions. The radiation sensitivity of these separated cell populations varied by a factor of 6, regardless of whether the cells were irradiated as artificial micro or macro-metastases. In each experiment, tumor populations most enriched in s-phase cells exhibited the greatest radiation sensitivity. To confirm that these populations were highly enriched in S-phase cells and to demonstrate that they were more radiosensitive than FSa cells in other parts of the cell cycle, the elutriated tumor populations were exposed to either suicide labeling by high specific activity tritiated thymidine or hydroxyurea. The resultant age response curves were qualitatively similar to those obtained following irradiation and reflected the S-phase sensitivity of FSa cells to these agents.

  19. Identification of molecular markers of bipolar cells in the murine retina

    PubMed Central

    Kim, Douglas S; Ross, Sarah E; Trimarchi, Jeffrey M; Aach, John; Greenberg, Michael E; Cepko, Constance L

    2008-01-01

    Retinal bipolar neurons serve as relay interneurons that connect rod and cone photoreceptor cells to amacrine and ganglion cells. They exhibit diverse morphologies essential for correct routing of photoreceptor cell signals to specific postsynaptic amacrine and ganglion cells. The development and physiology of these interneurons have not been completely defined molecularly. Despite previous identification of genes expressed in several bipolar cell subtypes, molecules that mark each bipolar cell type still await discovery. In this report, novel genetic markers of murine bipolar cells were found. Candidates were initially generated by using microarray analysis of single bipolar cells and mining of retinal serial analysis of gene expression (SAGE) data. These candidates were subsequently tested for expression in bipolar cells by RNA in situ hybridization. Ten new molecular markers were identified, five of which are highly enriched in their expression in bipolar cells within the adult retina. Double-labeling experiments using probes for previously characterized subsets of bipolar cells were performed to identify the subtypes of bipolar cells that express the novel markers. Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found. From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells. Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons. PMID:18260140

  20. Transferrin, carbonic anhydrase C and ferritin in dissociated murine brain cell cultures.

    PubMed

    Griot, C; Vandevelde, M

    1988-07-01

    It is shown here that transferrin (Tf), the iron transport protein and carbonic anhydrase C (CA C) are specifically located within oligodendrocytes in murine brain cell cultures. Ferritin (F), the major iron storage protein, was demonstrated in oligodendrocytes, as well as in astrocytes and microglial cells and was more prominent in the former. CA C and Tf were seen first after 6-7 days in culture. CA C and F positivity increased rapidly and at day 20, 80-85% of galactocerebroside + oligodendrocytes were positive for both proteins. Only a small number of oligodendrocytes was Tf+ up to day 14, after which their numbers increased rapidly until day 20, when 67% of the oligodendrocytes were Tf+. Because of the presence of Tf and F in oligodendrocytes it is suggested that these cells may play an important role in the metabolism of iron within the central nervous system. PMID:3133394

  1. Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells

    SciTech Connect

    Weston, W.M.; Greene, R.M. )

    1991-01-01

    Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolin-stimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that,in contrast to what has been observed in other systems, ethanol does not affect the production or activity of G{alpha}s in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome.

  2. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

    PubMed Central

    Nunes-Alves, Cludio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Klverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

    2015-01-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCR? bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

  3. Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones.

    PubMed Central

    Deepe, G S; Smith, J G; Sonnenfeld, G; Denman, D; Bullock, W E

    1986-01-01

    Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously in response to histoplasmin; the T-cell lines and 6 clones also were reactive with heterologous fungal antigens prepared from either Blastomyces dermatitidis or Coccidioides immitis. Recognition of antigen by T cells was H-2 restricted; in the absence of antigen, four clones demonstrated alloreactivity. All T-cell clones conferred local delayed-type hypersensitivity responses when injected with antigen into footpads of mice. Ten of 12 T-cell clones released interleukin-2 after stimulation with antigen, and all clones tested secreted interferon. Moreover, culture supernatants from antigen-stimulated clones armed peritoneal macrophages to inhibit intracellular growth of H. capsulatum yeast cells. All clones assayed exerted nonspecific help. Thus, development of T-cell clones should facilitate analysis of the regulatory properties of Histoplasma-specific T cells. PMID:2430887

  4. Immunoglobulin heavy chain gene rearrangement and transcription in murine T cell hybrids and T lymphomas.

    PubMed Central

    Ziga, M C; D'Eustachio, P; Ruddle, N H

    1982-01-01

    We have examined the arrangement of immunoglobulin heavy chain constant (CH) and joining (JH) region genes in murine T cell hybrid lines and in T lymphomas. CH genes derived from both parental cell types were present in all hybrids for which polymorphism in sequences flanking CH genes permitted us to distinguish parental CH genes. All T lymphomas and T cell hybrids retained the C alpha gene in germ-line configuration and all but one cell line had germ-line C mu genes. Novel DNA fragments reactive with JH probes were observed in six of nine T cell hybrids, as well as in two T lymphomas, WEHI7.1 and YAC-1, but not in the fusion parent, BW5147. No RNA homologous to C gamma 2b, C alpha, or lambda genes was detected in any of the T cell lines. T cell lines contained poly(A)+ RNA homologous to a C mu cDNA probe. More importantly, in several cell lines the C mu RNAs were associated with membrane-bound polyribosomes. These results suggest that both JH rearrangements and C mu RNA production occur in at least some mature, antigen-specific T cells. They may therefore reflect events in normal T cell development and function related to those involved in the generation of the T receptor for antigen. Images PMID:6806823

  5. Perforin Gene Transfer Into Hematopoietic Stem Cells Improves Immune Dysregulation in Murine Models of Perforin Deficiency

    PubMed Central

    Carmo, Marlene; Risma, Kimberly A; Arumugam, Paritha; Tiwari, Swati; Hontz, Adrianne E; Montiel-Equihua, Claudia A; Alonso-Ferrero, Maria E; Blundell, Michael P; Schambach, Axel; Baum, Christopher; Malik, Punam; Thrasher, Adrian J; Jordan, Michael B; Gaspar, H Bobby

    2015-01-01

    Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-? secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL. PMID:25523759

  6. Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency.

    PubMed

    Carmo, Marlene; Risma, Kimberly A; Arumugam, Paritha; Tiwari, Swati; Hontz, Adrianne E; Montiel-Equihua, Claudia A; Alonso-Ferrero, Maria E; Blundell, Michael P; Schambach, Axel; Baum, Christopher; Malik, Punam; Thrasher, Adrian J; Jordan, Michael B; Gaspar, H Bobby

    2015-04-01

    Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8(+) T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8(+) lymphoblasts had reduced interferon-? secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL. PMID:25523759

  7. Interleukin 21 Signaling in B Cells Is Required for Efficient Establishment of Murine Gammaherpesvirus Latency

    PubMed Central

    Collins, Christopher M.; Speck, Samuel H.

    2015-01-01

    The human gammaherpesviruses take advantage of normal B cell differentiation pathways to establish life-long infection in memory B cells. Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice also leads to life-long infection in memory B cells. To gain access to the memory B cell population, MHV68 infected B cells pass through the germinal center reaction during the onset of latency and require signals from T follicular helper (TFH) cells for proliferation. Interleukin 21 (IL-21), one of the secreted factors produced by TFH cells, plays an important role in both the maintenance of the germinal center response as well as in the generation of long-lived plasma cells. Using IL-21R deficient mice, we show that IL-21 signaling is required for efficient establishment of MHV68 infection. In the absence of IL-21 signaling, fewer infected splenocytes are able to gain access to either the germinal center B cell population or the plasma cell population the latter being a major site of MHV68 reactivation. Furthermore, the germinal center B cell population in IL-21R-/- mice is skewed towards the non-proliferating centrocyte phenotype, resulting in reduced expansion of infected B cells. Additionally, the reduced frequency of infected plasma cells results in a significant reduction in the frequency of splenocytes capable of reactivating virus. This defect in establishment of MHV68 infection is intrinsic to B cells, as MHV68 preferentially establishes infection in IL-21R sufficient B cells in mixed bone marrow chimeric mice. Taken together, these data indicate that IL-21 signaling plays multiple roles during establishment of MHV68 infection, and identify IL-21 as a critical TFH cell-derived factor for efficient establishment of gammaherpesvirus B cell latency. PMID:25875847

  8. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jrg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grnwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  9. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    PubMed

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers. PMID:26206856

  10. Dendritic Cell-Based Vaccination in Cancer: Therapeutic Implications Emerging from Murine Models

    PubMed Central

    Mac Keon, Soledad; Ruiz, María Sol; Gazzaniga, Silvina; Wainstok, Rosa

    2015-01-01

    Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment. PMID:26042126

  11. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models.

    PubMed

    Mac Keon, Soledad; Ruiz, Mara Sol; Gazzaniga, Silvina; Wainstok, Rosa

    2015-01-01

    Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment. PMID:26042126

  12. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

    PubMed

    Bunin, Anna; Sisirak, Vanja; Ghosh, Hiyaa S; Grajkowska, Lucja T; Hou, Z Esther; Miron, Michelle; Yang, Cliff; Ceribelli, Michele; Uetani, Noriko; Chaperot, Laurence; Plumas, Joel; Hendriks, Wiljan; Tremblay, Michel L; Hcker, Hans; Staudt, Louis M; Green, Peter H; Bhagat, Govind; Reizis, Boris

    2015-08-18

    Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-?. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation. PMID:26231120

  13. Effects of Murine and Human Bone Marrow-Derived Mesenchymal Stem Cells on Cuprizone Induced Demyelination

    PubMed Central

    Gudi, Viktoria; Hoffmann, Andrea; Salinas Tejedor, Laura; Janen, Stefanie; Prajeeth, Chittappen Kandiyil; Baumgrtner, Wolfgang; Kavelaars, Annemieke; Heijnen, Cobi J.; van Velthoven, Cindy; Hansmann, Florian; Skripuletz, Thomas; Stangel, Martin

    2013-01-01

    For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes. PMID:23922802

  14. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

    PubMed

    Nunes-Alves, Cludio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Klverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

    2015-05-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCR? bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

  15. Embryotoxic effects of the marine biotoxin okadaic acid on murine embryonic stem cells.

    PubMed

    Ehlers, Anke; Stempin, Sandra; Al-Hamwi, Regina; Lampen, Alfonso

    2010-04-01

    Okadaic acid (OA), a marine toxin produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, consumption of OA induces acute toxic effects like diarrhoea, nausea, vomiting and abdominal pain. OA is a potent inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A), enzymes that are known to be critical regulators of embryonic development. To determine the embryotoxic potential of OA, we performed two independent cellular in-vitro assays, both of which are applicable for the detection of teratogenic compounds: (i) the validated embryonic stem cell test (EST) based on the morphological analysis of beating cardiomyocytes in embryoid bodies and (ii) the F9 cell assay quantifying the induction of cell differentiation by measuring the emitted luminescence of a reporter gene. In the presence of OA, beating cardiomyocytes in the EST were inhibited and the reporter gene in transiently transfected F9 cells was activated. Furthermore, OA treatment led to rapid morphological changes including cell rounding, the loss of cell-cell contacts and changed electrical impedance as monitored in real time by the xCELLigence system. The two independent bioassays (EST and F9 cell test) detected OA as a potential embryotoxic compound, since OA influences the differentiation process of cultured murine embryonic cells. PMID:20026154

  16. Erythropoietin acts as an anti-inflammatory signal on murine mast cells.

    PubMed

    Wiedenmann, Tanja; Ehrhardt, Stefanie; Cerny, Daniela; Hildebrand, Dagmar; Klein, Sabrina; Heeg, Klaus; Kubatzky, Katharina F

    2015-05-01

    Recently it was found that the erythropoietin receptor (EpoR) is expressed on innate immune cells, such as dendritic cells and macrophages. We found that murine bone marrow-derived mast cells express the EpoR and that its expression is increased under hypoxic conditions. Interestingly, Epo stimulation of the cells did not activate signal transducer and activator of transcription molecules, nor did we find differences in the expression of typical STAT-dependent genes, the proliferation rate, and the ability to differentiate or to protect the cells from apoptosis. Instead, we demonstrate that stimulation of mast cells with Epo leads to phosphorylation of the receptor tyrosine kinase c-kit. We hypothesize that this is due to the formation of a receptor complex between the EpoR and c-kit. The common beta chain of the IL-3 receptor family, which was described as part of the tissue protective receptor (TPR) on other non-erythroid cells, however is not activated. To investigate whether the EpoR/c-kit complex has tissue protective properties, cells were treated with the Toll-like receptor ligand LPS. Combined Epo and LPS treatment downregulated the inflammatory response of the cells as detected by a decrease in IL-6 and TNF-? secretion. PMID:25645506

  17. CD154-CD40 interactions in the control of murine B cell hematopoiesis

    PubMed Central

    Carlring, Jennifer; Altaher, Hala M.; Clark, Susan; Chen, Xi; Latimer, Sarah L.; Jenner, Tracey; Buckle, Anne-Marie; Heath, Andrew W.

    2011-01-01

    Interactions between CD40 and CD154 play a very important role in control of immune responses, including the delivery of T cell help to B cells and other APCs. Thus far, there has been no role postulated for CD40-CD154 interactions in hematopoiesis. We show here that CD40 is expressed on murine pro-B cells and that its ligation enhances pro-B cell proliferation in vitro and in vivo. In addition, CD154 mRNA is present in the BM. Moreover, we show that a deficiency in CD154 expression has effects on B cell hematopoiesis. Aged, CD154-deficient mice have significantly lower levels of B hematopoietic subsets downstream of pro-B cells in the BM. In addition, B lineage cells reconstitute more slowly following BMT into CD154-deficient recipients. We hypothesize that CD154 is expressed by radio-resistant cells in the BM and plays a role in fine-tuning B cell hematopoiesis. PMID:21330346

  18. Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality.

    PubMed

    Schneidawind, Dominik; Baker, Jeanette; Pierini, Antonio; Buechele, Corina; Luong, Richard H; Meyer, Everett H; Negrin, Robert S

    2015-05-28

    Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation. PMID:25795920

  19. Cyclic-radiation response of murine fibrosarcoma cells grown as pulmonary nodules

    SciTech Connect

    Grdina, D.J.; Hunter, N.

    1982-10-01

    The radiation age response of murine fibrosarcoma (FSa) cells grown as pulmonary nudules in C/sub 3/Hf/Kam mice was determined. FSa cells were irradiated in vivo either with 10 Gy as 14 day-old lung tumors (i.e., artifical micrometastases) following cell separation and synchronization by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine cell-cycle parameters and the relative synchrony of the separated populations, as well as the percent contamination of normal diploid cells in each of the tumor cells populations. Tumor populations containing up to 90% G/sub 1/-, 60% S-, and 75% G/sub 2/+M-phase tumor cells were obtained. Cell clonogenicity, determined using a lung colony assay, ranged from 0.7 to 6% for control FSa cells from the various elutriator fractions. The radiation sensitivity of these separated cell populations varied by a factor of 6, regardless of whether the cells were irradiated as artifical micro or macro-metastases. In each experiment, tumor population most enriched in S-phase cells exhibited the greatest radiation sensitivity. To confirm that these populations were highly enriched in S-phase cells and to demonstrate that they were more radiosensitive than FSa cells in other parts of the cell cycle, the elutriated tumor population were exposed to either suicide labeling by high specific activity tritated thymidine or hydroxyurea. The resultant age response curves were qualitatively similar to those obtained following irradiation and reflected the S-phase sensitivity of FSa cells to these agents.

  20. Murine mesenchymal progenitor cells from different tissues differentiated via mesenchymal microspheres into the mesodermal direction

    PubMed Central

    2009-01-01

    Background Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues. Results We introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR. Conclusions We were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells. PMID:20021685

  1. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    SciTech Connect

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  2. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cells from the osteoblastic precursor pool in the body, a gradual decrease of bone mass in weightlessness may be attributed to synergistic effects of radiation and weightlessness.

  3. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.

    PubMed

    Kochenderfer, James N; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A

    2010-11-11

    Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)-expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19-CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19-CAR-transduced T cells completely eliminated normal B cells from mice for at least 143 days. Anti-CD19-CAR-transduced T cells eradicated intraperitoneally injected lymphoma cells and large subcutaneous lymphoma masses. The antilymphoma efficacy of anti-CD19-CAR-transduced T cells was critically dependent on irradiation of mice before anti-CD19-CAR-transduced T-cell infusion. Anti-CD19-CAR-transduced T cells had superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived. Our results demonstrated impressive antilymphoma activity and profound destruction of normal B cells caused by anti-CD19-CAR-transduced T cells in a clinically relevant murine model. PMID:20631379

  4. Anti-inflammatory effects of natural product formulations on murine dendritic cells

    PubMed Central

    Miller, Andrea K.; Benson, Jenna M.; Muanza, Dave N.; Smith, Jerry R.; Shepherd, David M.

    2011-01-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being intensively studied, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cats claw) and Phytolens on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. To address this hypothesis, we evaluated the effects of BRC-301, 304, and 306 on DC2.4 cells and further assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). LPS stimulation of DC2.4 cells and bmDCs induced production of NO, TNF-?, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NO or IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of FITC-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  5. Anti-inflammatory effects of natural product formulations on murine dendritic cells.

    PubMed

    Miller, Andrea K; Benson, Jenna M; Muanza, Dave N; Smith, Jerry R; Shepherd, David M

    2011-03-01

    The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being studied intensively, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations, including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts, and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat's claw) and Phytolens() on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. In order to address this hypothesis, we evaluated the effects of BRC-301, BRC-304, and BRC-306 on DC2.4 cells and assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). Lipopolysaccharide (LPS) stimulation of DC2.4 cells and bmDCs induced production of nitric oxide (NO), TNF-?, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NOor IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of fluorescein isothiocyanate-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity. PMID:21399725

  6. Exploring the translational disconnect between the murine and human inflammatory response: analysis of LPS doseresponse relationship in murine versus human cell lines and implications for translation into murine models of sepsis

    PubMed Central

    McCarron, Eamon P; Williams, Dominic P; Antoine, Daniel J; Kipar, Anja; Lemm, Jana; Stehr, Sebastian; Welters, Ingeborg D

    2015-01-01

    Background Inflammation forms an important part of the human innate immune system and is largely dependent on the activation of the classical NF-?B pathway through Toll-like receptors (TLRs). Understanding this has allowed researchers to explore roles of therapeutic targets in managing conditions such as sepsis. Recapitulating an inflammatory response using lipopolysaccharide (LPS), a sterile technique, can provide information that is dissimilar to the clinical condition. By examining NF-?B activation (through immunoblotting of the p65 subunit) in two separate cell lines (murine and human) and analyzing two murine models of sepsis (intraperitoneal [IP] LPS and IP stool inoculation), an evaluation of the translational disconnect between experimental and clinical sepsis can be made. Methods THP-1 (human) cells and RAW 264.7 (murine) cells were dosed with concentrations of LPS (human, 1 pg/mL to 100 ng/mL; murine, 30 pg/mL to 1,000 ng/mL) and nuclear actin and p65 were immunoblotted to measure changes in nuclear density. In vivo, C57BL/6 mice received either IP injection of stool suspension (5 L/g) or LPS (25 mg/kg) or saline (1 mL/kg). Animals