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Sample records for mouse oocyte killing

  1. Microinjection of Follicle-Enclosed Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

  2. Quantitative Microinjection of Mouse Oocytes and Eggs

    NASA Astrophysics Data System (ADS)

    Kline, Douglas

    Quantitative microinjection is used to introduce known quantities of molecules or probes into single cells to examine cellular function. The relatively large mammalian oocyte or egg is easily manipulated and can be injected with impermeant reagents including a variety of signaling molecules and fluorescent probes. Techniques have been developed to inject picoliter quantities of solution into oocytes and eggs with precision and reliability. The methods described here outline the quantitative injection procedures as they are used to inject mouse oocytes and eggs in a culture dish on the stage on an inverted microscope. The techniques are applicable to the oocytes, eggs, and early embryos of most mammalian species. Included are some general instructions on fabrication of transfer pipettes, holding pipettes, beveled injection pipettes, and equipment for quantitative injection.

  3. Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells.

    PubMed

    Colonna, R; Cecconi, S; Tatone, C; Mangia, F; Buccione, R

    1989-05-01

    The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte. PMID:2707483

  4. SIRT1, 2, 3 protect mouse oocytes from postovulatory aging

    PubMed Central

    Zhang, Teng; Zhou, Yang; Li, Li; Wang, Hong-Hui; Ma, Xue-Shan; Qian, Wei-Ping; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

    2016-01-01

    The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation. PMID:26974211

  5. Mitofusin-2 is required for mouse oocyte meiotic maturation.

    PubMed

    Zhang, Jing-Hua; Zhang, Teng; Gao, Si-Hua; Wang, Ke; Yang, Xiu-Yan; Mo, Fang-Fang; Na Yu; An, Tian; Li, Yu-Feng; Hu, Ji-Wei; Jiang, Guang-Jian

    2016-01-01

    Mitofusin-2 (Mfn2) is essential for embryonic development, anti-apoptotic events, protection against free radical-induced lesions, and mitochondrial fusion in many cells. However, little is known about its mechanism and function during oocyte maturation. In this study, we found that Mfn2 was expressed in the cytoplasm during different stages of mouse oocyte maturation. Mfn2 was mainly associated with α-tubulin during oocyte maturation. Knockdown of Mfn2 by specific siRNA injection into oocytes caused the mitochondrial morphology and quantity to change, resulting in severely defective spindles and misaligned chromosomes. This led to metaphase I arrest and the failure of first polar body extrusion. Furthermore, Mfn2 depletion from GV stage oocytes caused the redistribution of p38 MAPK in oocyte cytoplasm. These findings provide insights into potential mechanisms of Mfn2-mediated cellular alterations, which may have significant implications for oocyte maturation. PMID:27485634

  6. Mitofusin-2 is required for mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Jing-Hua; Zhang, Teng; Gao, Si-Hua; Wang, Ke; Yang, Xiu-Yan; Mo, Fang-Fang; Na Yu; An, Tian; Li, Yu-Feng; Hu, Ji-Wei; Jiang, Guang-Jian

    2016-01-01

    Mitofusin-2 (Mfn2) is essential for embryonic development, anti-apoptotic events, protection against free radical-induced lesions, and mitochondrial fusion in many cells. However, little is known about its mechanism and function during oocyte maturation. In this study, we found that Mfn2 was expressed in the cytoplasm during different stages of mouse oocyte maturation. Mfn2 was mainly associated with α-tubulin during oocyte maturation. Knockdown of Mfn2 by specific siRNA injection into oocytes caused the mitochondrial morphology and quantity to change, resulting in severely defective spindles and misaligned chromosomes. This led to metaphase I arrest and the failure of first polar body extrusion. Furthermore, Mfn2 depletion from GV stage oocytes caused the redistribution of p38 MAPK in oocyte cytoplasm. These findings provide insights into potential mechanisms of Mfn2-mediated cellular alterations, which may have significant implications for oocyte maturation. PMID:27485634

  7. Differences in oocyte development and estradiol sensitivity among mouse strains.

    PubMed

    Pepling, Melissa E; Sundman, Emily A; Patterson, Nicole L; Gephardt, Grant W; Medico, Leonard; Wilson, Krystal I

    2010-02-01

    Mouse oocytes develop in clusters of interconnected cells called germline cysts. Shortly after birth, the majority of cysts break apart and primordial follicles form, consisting of one oocyte surrounded by granulosa cells. Concurrently, oocyte number is reduced by two-thirds. Exposure of neonatal females to estrogenic compounds causes multiple oocyte follicles that are likely germline cysts that did not break down. Supporting this idea, estrogen disrupts cyst breakdown and may regulate normal oocyte development. Previously, the CD-1 strain was used to study cyst breakdown and oocyte survival, but it is unknown if there are differences in these processes in other mouse strains. It is also unknown if there are variations in estrogen sensitivity during oocyte development. Here, we examined neonatal oocyte development in FVB, C57BL/6, and F2 hybrid (Oct4-GFP) strains, and compared them with the CD-1 strain. We found variability in oocyte development among the four strains. We also investigated estrogen sensitivity differences, and found that C57BL/6 ovaries are more sensitive to estradiol than CD-1, FVB, or Oct4-GFP ovaries. Insight into differences in oocyte development will facilitate comparison of mice generated on different genetic backgrounds. Understanding variations in estrogen sensitivity will lead to better understanding of the risks of environmental estrogen exposure in humans. PMID:19846484

  8. Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

    PubMed Central

    Lee, Jae Won; Lee, Geun-Kyung; Suh, Chang Suk; Kim, Kwang Pyo; Lim, Hyunjung Jade

    2016-01-01

    The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes. PMID:26881843

  9. High survival of mouse oocytes using an optimized vitrification protocol

    PubMed Central

    Zhou, Cheng-Jie; Wang, Dong-Hui; Niu, Xin-Xin; Kong, Xiang-Wei; Li, Yan-Jiao; Ren, Jing; Zhou, Hong-Xia; Lu, Angeleem; Zhao, Yue-Fang; Liang, Cheng-Guang

    2016-01-01

    The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes. PMID:26781721

  10. The type and extent of injuries in vitrified mouse oocytes.

    PubMed

    Liang, Yang; Ning, Fang-Yong; Du, Wen-Jing; Wang, Chun-Sheng; Piao, Shan-Hua; An, Tie-Zhu

    2012-04-01

    To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws. PMID:22202671

  11. PTK2b function during fertilization of the mouse oocyte

    SciTech Connect

    Luo, Jinping; McGinnis, Lynda K.; Carlton, Carol; Beggs, Hilary E.; Kinsey, William H.

    2014-08-01

    Highlights: • PTK2b is expressed in oocytes and is activated following fertilization. • PTK2b suppression in oocytes prevents fertilization, but not parthenogenetic activation. • PTK2b suppression prevents the oocyte from fusing with or incorporating bound sperm. • PTK2b suppressed oocytes that fail to fertilize do not exhibit calcium oscillations. - Abstract: Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

  12. DNA damage response during mouse oocyte maturation.

    PubMed

    Mayer, Alexandra; Baran, Vladimir; Sakakibara, Yogo; Brzakova, Adela; Ferencova, Ivana; Motlik, Jan; Kitajima, Tomoya S; Schultz, Richard M; Solc, Petr

    2016-01-01

    Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation. PMID:26745237

  13. Active diffusion positions the nucleus in mouse oocytes.

    PubMed

    Almonacid, Maria; Ahmed, Wylie W; Bussonnier, Matthias; Mailly, Philippe; Betz, Timo; Voituriez, Raphaël; Gov, Nir S; Verlhac, Marie-Hélène

    2015-04-01

    In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function. PMID:25774831

  14. Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy

    SciTech Connect

    Mailhes, J.B.; Yuan, Z.P.

    1987-01-01

    Unpublished results from our laboratory showed that colchicine increased the incidence of hyperploid mouse metaphase II (MII) oocytes when injected at the same time as human chorionic gonadotrophin (HCG). The objective of the present study was to determine whether the time of administering colchicine influenced the incidence of aneuploidy in MII oocytes. CD-1 mice were given pregnant mare's serum (PMS) and, 48 hr later, HCG. An intraperitoneal injection of 0.2 mg/kg colchicine was given at +4, +2, 0, -2, or -4 hr relative to HCG. Oocytes were collected 17 hr post-HCG and processed, and chromosomes were subsequently C-banded. The percentage of hyperploid oocytes was 0.77, 2.56, 5.71, 7.79, 3.54, and 2.70 for control, +4, +2, 0, -2, or -4 hr pre/post-HCG, respectively. Chi-square analyses of these data demonstrated that colchicine significantly increases the proportion of aneuploid oocytes, and that the relative sensitivity of colchicine-induced aneuploidy depends upon the time that this drug is administered relative to HCG.

  15. KL/KIT co-expression in mouse fetal oocytes.

    PubMed

    Doneda, Luisa; Klinger, Francesca-Gioia; Larizza, Lidia; De Felici, Massimo

    2002-12-01

    The tyrosine kinase receptor, KIT, and its ligand, KL are important regulators of germ cell development. The aim of this study was to examine in detail the expression of the genes encoding these proteins (White and Steel, respectively) during the fetal period (14.5-18.5 days post coitum, dpc) and the two weeks after birth in mouse ovaries using the highly sensitive in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). KL and KIT mRNAs were not detected in 14.5-15.5 dpc ovaries but, between 16.5 and 17.5 dpc, most of the oocytes in the outer regions of the ovaries positively stained for both mRNAs. The majority of the co-expressing oocytes were identified at the zygotene/pachytene stage of meiotic prophase I. At 18.5 dpc, positive staining for KL mRNA was present only in the somatic cells in the outer regions of the ovaries. At birth, faint KL mRNA-labelled somatic cells were mainly found in the central region of the ovaries and, by P7-14, a higher level of expression was detected in the follicle cells of one- and two-layered growing follicles. Between 17.5 dpc and birth, most of the oocytes expressed KIT mRNA and, from P7 onward, there was a considerable accumulation of transcripts in the growing oocytes. The results of in situ RT-PCR were confirmed by RT-PCR on purified populations of oocytes, and at protein level by means of immunohistochemistry. The co-expression of KL and KIT in a fraction of fetal oocytes suggests that the KL/KIT system, besides the well known paracrine functions on germ cells, may exert a novel autocrine role during the mid-stage of the oocyte meiotic prophase. The possibility that this autocrine loop plays a role in sustaining the survival of fetal oocytes in this stage is supported by the finding that the addition to the culture medium of anti-KL or anti-KIT antibodies led to a significant increase in oocyte apoptosis in the absence of exogenous KL. PMID:12533025

  16. Impact of tightly focused femtosecond laser pulses on nucleolus-like bodies of mouse GV oocyte and the ability of mouse oocytes to mature.

    PubMed

    Astafev, A A; Zalesskiy, A D; Zatsepina, O V; Kostrov, A N; Krivoharchenko, A S; Osychenko, A A; Serobyan, G A; Nadtochenko, V A

    2016-03-01

    Using femtosecond laser radiation, nucleolus-like bodies (NLBs) of mouse oocytes were locally dissected without damage to zona pellucida, cytoplasmic membrane, nuclear membrane, and nucleoplasm surrounding NLB. It was found that, after dissection of 2.7 × 10(-11) cm(3) of NLB material, which is approximately 5.2% of 10 μm NLB volume, the probability of germinal vesicle oocyte development to metaphase II stage of meiosis decreased 3-7 times compared to the non-treated oocytes. This result indicates that NLB material organization is significant for mouse oocyte maturation. PMID:27193718

  17. Antioxidant supplementation overcomes the deleterious effects of maternal restraint stress-induced oxidative stress on mouse oocytes.

    PubMed

    Lian, Hua-Yu; Gao, Yan; Jiao, Guang-Zhong; Sun, Ming-Ju; Wu, Xiu-Fen; Wang, Tian-Yang; Li, Hong; Tan, Jing-He

    2013-12-01

    In this study, using a mouse model, we tested the hypothesis that restraint stress would impair the developmental potential of oocytes by causing oxidative stress and that antioxidant supplementation could overcome the adverse effect of stress-induced oxidative stress. Female mice were subjected to restraint stress for 24 h starting 24 h after equine chorionic gonadotropin injection. At the end of stress exposure, mice were either killed to recover oocytes for in vitro maturation (IVM) or injected with human chorionic gonadotropin and caged with male mice to observe in vivo development. The effect of antioxidants was tested in vitro by adding them to IVM medium or in vivo by maternal injection immediately before restraint stress exposure. Assays carried out to determine total oxidant and antioxidant status, oxidative stress index, and reactive oxygen species (ROS) and glutathione levels indicated that restraint stress increased oxidative stress in mouse serum, ovaries, and oocytes. Whereas the percentage of blastocysts and number of cells per blastocyst decreased significantly in oocytes from restraint-stressed mice, addition of antioxidants to IVM medium significantly improved their blastocyst development. Supplementation of cystine and cysteamine to IVM medium reduced ROS levels and aneuploidy while increasing glutathione synthesis and improving pre- and postimplantation development of oocytes from restraint-stressed mice. Furthermore, injection of the antioxidant epigallocatechin gallate into restraint-stressed mice significantly improved the blastocyst formation and postimplantation development of their oocytes. In conclusion, restraint stress at the oocyte prematuration stage impaired the developmental potential of oocytes by increasing oxidative stress and addition of antioxidants to IVM medium or maternal antioxidant injection overcame the detrimental effect of stress-induced oxidative stress. The data reported herein are helpful when making attempts to

  18. Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes

    PubMed Central

    Liu, Rui; Liu, Yu; Zhang, Fei; Zhang, Zhen; Shen, Yu-Ting; Xu, Lin; Chen, Ming-Huang; Wang, Ya-Long; Xu, Bai-Hui; Yang, Xiang-Jun; Wang, Hai-Long

    2016-01-01

    Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes. PMID:27284927

  19. Preparation of Cell Lysate from Mouse Oocytes for Western Blotting Analysis.

    PubMed

    Marangos, Petros

    2016-01-01

    Western Blotting has been used extensively for the identification of the protein factors that regulate mammalian oocyte meiosis. However, the limitations in collecting sufficient numbers of oocytes can hinder the efficiency of the technique. Here we provide a detailed protocol for the accurate preparation of mouse oocyte samples for Western Blotting analysis. PMID:27557583

  20. Aurora kinase A controls meiosis I progression in mouse oocytes

    PubMed Central

    Saskova, Adela; Solc, Petr; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M.; Motlik, Jan

    2011-01-01

    Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition. PMID:18677115

  1. Vitrification, in vitro fertilization, and development of Atg7 deficient mouse oocytes

    PubMed Central

    Bang, Soyoung; Lee, Geun-Kyung; Shin, Hyejin; Suh, Chang Suk

    2016-01-01

    Objective Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Methods Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. Results The survival rate of vitrified-warmed Atg7f/f;Zp3-Cre (Atg7d/d) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7f/f) oocytes. Fertilization and development in the Atg7d/d oocytes were significantly lower than the Atg7f/f oocytes, comparable to the Atg5d/d oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7d/d MII oocytes when compared to fresh Atg7d/d oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. Conclusion We confirmed that the LC3-positive signal is nearly absent in Atg7d/d oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses. PMID:27104152

  2. Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols.

    PubMed

    Men, H S; Chen, J C; Ji, W Z; Shang, E Y; Yang, S C; Zou, R J

    1997-05-01

    The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 degrees C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P < 0.01). The results of IVF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P > 0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants. PMID:16728088

  3. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  4. Generation of live offspring from vitrified mouse oocytes of C57BL/6J strain.

    PubMed

    Kohaya, Natsuki; Fujiwara, Katsuyoshi; Ito, Junya; Kashiwazaki, Naomi

    2013-01-01

    In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility. PMID:23516430

  5. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  6. Effective protein inhibition in intact mouse oocytes through peptide nanoparticle-mediated antibody transfection

    PubMed Central

    Li, Ruichao; Jin, Zhen; Gao, Leilei; Liu, Peng

    2016-01-01

    Female meiosis is a fundamental area of study in reproductive medicine, and the mouse oocyte model of in vitro maturation (IVM) is most widely used to study female meiosis. To investigate the probable role(s) of an unknown protein in female meiosis, the method traditionally used involves microinjecting a specific antibody into mouse oocytes. Recently, in studies on somatic cells, peptide nanoparticle-mediated antibody transfection has become a popular tool because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, untill now no researchers have tried using this technique on mouse oocytes because the zona pellucida surrounding the oocyte membrane (vitelline membrane) is usually thought or proved to be a tough barrier to macromolecules such as antibodies and proteins. Therefore, we attempted to introduce an antibody into mouse oocytes using a peptide nanoparticle. Here we show for the first time that with our optimized method, an antibody can be effectively delivered into mouse oocytes and inhibit its target protein with high specificity. We obtained significant results using small GTPase Arl2 as a test subject protein. We propose peptide nanoparticle-mediated antibody transfection to be a superior alternative to antibody microinjection for preliminary functional studies of unknown proteins in mouse oocytes. PMID:27114861

  7. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  8. NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse

    PubMed Central

    PENG, Hui; LIN, Xiujiao; LIU, Fang; WANG, Cheng; ZHANG, Wenchang

    2015-01-01

    Nlrp9a, Nlrp9b and Nlrp9c are preferentially expressed in oocytes and early embryos in the mouse. Simultaneous genetic ablation of Nlrp9a and Nlrp9c does not affect early embryonic development, but the function of Nlrp9b in the process of oocyte maturation and embryonic development has not been elucidated. Here we show that both Nlrp9b mRNA and its protein are expressed in ovaries and the small intestine. Moreover, the NLRP9B protein was restricted to oocytes in the ovary and declined with oocyte aging. After ovulation and fertilization, NLRP9B protein was found in preimplantation embryos. Confocal microscopy demonstrated that it was mainly localized in the cytoplasm in the oocytes and blastomeres. Thus, this protein might play a role in oocyte maturation and early embryonic development. However, knockdown of Nlrp9b expression in GV-stage oocytes using RNA interference did not affect oocyte maturation or subsequent parthenogenetic development after Nlrp9b-deficient oocytes were activated. Furthermore, Nlrp9b knockdown zygotes could reach the blastocyst stage after being cultured for 3.5 days in vitro. These results provide the first evidence that the NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse. PMID:26411641

  9. L-proline: a highly effective cryoprotectant for mouse oocyte vitrification.

    PubMed

    Zhang, Lu; Xue, Xu; Yan, Jie; Yan, Li-Ying; Jin, Xiao-Hu; Zhu, Xiao-Hui; He, Zhi-Zhu; Liu, Jing; Li, Rong; Qiao, Jie

    2016-01-01

    Recent studies have shown that L-proline is a natural osmoprotectant and an antioxidant to protect cells from injuries such as that caused by freezing and thawing in many species including plant, ram sperm and human endothelial cells. Nevertheless, this nontoxic cryoprotectant has not yet been applied to mammalian oocyte vitrification. In this study we evaluated the efficiency and safety of the new cryoprotectant in oocyte vitrification. The results indicated that L-proline improves the survival rate of vitrified oocytes, protects mitochondrial functions and could be applied as a new cryoprotectant in mouse oocyte vitrification. PMID:27412080

  10. L-proline: a highly effective cryoprotectant for mouse oocyte vitrification

    PubMed Central

    Zhang, Lu; Xue, Xu; Yan, Jie; Yan, Li-Ying; Jin, Xiao-Hu; Zhu, Xiao-Hui; He, Zhi-Zhu; Liu, Jing; Li, Rong; Qiao, Jie

    2016-01-01

    Recent studies have shown that L-proline is a natural osmoprotectant and an antioxidant to protect cells from injuries such as that caused by freezing and thawing in many species including plant, ram sperm and human endothelial cells. Nevertheless, this nontoxic cryoprotectant has not yet been applied to mammalian oocyte vitrification. In this study we evaluated the efficiency and safety of the new cryoprotectant in oocyte vitrification. The results indicated that L-proline improves the survival rate of vitrified oocytes, protects mitochondrial functions and could be applied as a new cryoprotectant in mouse oocyte vitrification. PMID:27412080

  11. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro

    PubMed Central

    Liu, Shuzhen; Jiang, Ligang; Zhong, Tao; Kong, Shuhui; Zheng, Rongbin; Kong, Fengyun; Zhang, Cong; Zhang, Lei; An, Liguo

    2015-01-01

    Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro. PMID:26275143

  12. Effect of mycotoxin-containing diets on epigenetic modifications of mouse oocytes by fluorescence microscopy analysis.

    PubMed

    Zhu, Cheng-Cheng; Hou, Yan-Jun; Han, Jun; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-08-01

    Mycotoxins, such as aflatoxin (AF), fumonisin B1, zearalenone (ZEA), and deoxynivalenol (DON), are commonly found in many food commodities. Mycotoxins have been shown to increase DNA methylation levels in a human intestinal cell line. We previously showed that the developmental competence of oocytes was affected in mice that had been fed a mycotoxin-containing diet. In this study, we explored possible mechanisms of low mouse oocyte developmental competence after mycotoxin treatment in an epigenetic modification perspective. Mycotoxin-contaminated maize (DON at 3,875 μg/kg, ZEA at 1,897 μg/kg, and AF at 806 μg/kg) was included in diets at three different doses (mass percentage: 0, 15, and 30%) and fed to mice for 4 weeks. The fluorescence intensity analysis showed that the general DNA methylation levels increased in oocytes from high dose mycotoxin-fed mice. Mouse oocyte histone methylation was also altered. H3K9me3 and H4K20me3 level increased in oocytes from mycotoxin-fed mice, whereas H3K27me3 and H4K20me2 level decreased in oocytes from mycotoxin-fed mice. Thus, our results indicate that naturally occurring mycotoxins have effects on epigenetic modifications in mouse oocytes, which may be one of the reasons for reduced oocyte developmental competence. PMID:24810297

  13. Effects of ovarian endometriotic fluid exposure on fertilization rate of mouse oocytes and subsequent embryo development

    PubMed Central

    2013-01-01

    Background Accidental exposure of oocyte/cumulus complex to endometriotic fluid is not uncommon during oocyte retrieval. Only two studies were available on this subject and they gave conflicting results. In this study, we used a mouse model to evaluate the effect of controlled exposure of oocytes to ovarian endometriotic fluid. Methods Mouse oocytes/cumulus complexes (n = 862) were divided into 4 groups, and were exposed to endometriotic fluid (group 1), pooled sera from subjects without endometrioma (group 2), phosphate-buffered saline (group 3), and fertilization medium (controls). After five minutes, oocytes were washed and inseminated. Embryo development was observed daily. The quality of hatching blastocysts was assessed by counting the number of inner cell mass (ICM) and trophectoderm (TE) cells. Results The fertilization, cleavage and blastocyst formation rates in the four groups were not statistically different. The proportions of hatching/hatched blastocysts from fertilized oocytes in groups 1 and 2 were significantly lower than those in group 3 and controls (P = 0.015). Hatching blastocysts from all groups showed no significant difference in the number of ICM and TE cells. Conclusions Exposure of mouse oocytes/cumulus complexes to endometriotic fluid had subtle detrimental effects on subsequent blastocyst development. However, one should be cautious in projecting the results of this study to contaminated human oocytes in a clinical setting. PMID:23332096

  14. p90Rsk is not involved in cytostatic factor arrest in mouse oocytes

    PubMed Central

    Dumont, Julien; Umbhauer, Muriel; Rassinier, Pascale; Hanauer, André; Verlhac, Marie-Hélène

    2005-01-01

    Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos–MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos−/− strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos−/− oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes. PMID:15837801

  15. Mouse oocytes differentiate through organelle enrichment from sister cyst germ cells.

    PubMed

    Lei, Lei; Spradling, Allan C

    2016-04-01

    Oocytes differentiate in diverse species by receiving organelles and cytoplasm from sister germ cells while joined in germline cysts or syncytia. Mouse primordial germ cells form germline cysts, but the role of cysts in oogenesis is unknown. We find that mouse germ cells receive organelles from neighboring cyst cells and build a Balbiani body to become oocytes, whereas nurselike germ cells die. Organelle movement, Balbiani body formation, and oocyte fate determination are selectively blocked by low levels of microtubule-dependent transport inhibitors. Membrane breakdown within the cyst and an apoptosis-like process are associated with organelle transfer into the oocyte, events reminiscent of nurse cell dumping in Drosophila We propose that cytoplasmic and organelle transport plays an evolutionarily conserved and functionally important role in mammalian oocyte differentiation. PMID:26917595

  16. Rab6a is a novel regulator of meiotic apparatus and maturational progression in mouse oocytes

    PubMed Central

    Hou, Xiaojing; Zhang, Jiaqi; Li, Ling; Ma, Rujun; Ge, Juan; Han, Longsen; Wang, Qiang

    2016-01-01

    Rab family GTPases have been well known to regulate intracellular vesicle transport, however their function in mammalian oocytes has not been addressed. In this study, we report that when Rab6a is specifically knockdown, mouse oocytes are unable to progress normally through meiosis, arresting at metaphase I. Moreover, in these oocytes, the defects of chromosome alignment and spindle organization are readily observed during maturation, and resultantly increasing the aneuploidy incidence. We further reveal that kinetochore-microtubule attachments are severely compromised in Rab6a-depleted oocytes, which may in part mediate the meiotic phenotypes described above. In addition, when Rab6a function is altered, BubR1 levels on the kinetochores are markedly increased in metaphase oocytes, indicating the activation of spindle assembly checkpoint. In sum, we identify Rab6a as an important player in modulating oocyte meiosis, specifically the chromosome/spindle organization and metaphase-anaphase transition. PMID:26915694

  17. Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation.

    PubMed

    Hou, Yan-Jun; Zhu, Cheng-Cheng; Xu, Yin-Xue; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2015-09-01

    Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. PMID:24733567

  18. Essential Role for Endogenous siRNAs during Meiosis in Mouse Oocytes

    PubMed Central

    Stein, Paula; Rozhkov, Nikolay V.; Li, Fan; Cárdenas, Fabián L.; Davydenk, Olga; Vandivier, Lee E.; Gregory, Brian D.; Hannon, Gregory J.; Schultz, Richard M.

    2015-01-01

    The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals. PMID:25695507

  19. Suppression of Chemically Induced and Spontaneous Mouse Oocyte Activation by AMP-Activated Protein Kinase1

    PubMed Central

    Ya, Ru; Downs, Stephen M.

    2013-01-01

    ABSTRACT Oocyte activation is an important process triggered by fertilization that initiates embryonic development. However, parthenogenetic activation can occur either spontaneously or with chemical treatments. The LT/Sv mouse strain is genetically predisposed to spontaneous activation. LT oocytes have a cell cycle defect and are ovulated at the metaphase I stage instead of metaphase II. A thorough understanding of the female meiosis defects in this strain remains elusive. We have reported that AMP-activated protein kinase (PRKA) has an important role in stimulating meiotic resumption and promoting completion of meiosis I while suppressing premature parthenogenetic activation. Here we show that early activation of PRKA during the oocyte maturation period blocked chemically induced activation in B6SJL oocytes and spontaneous activation in LT/SvEiJ oocytes. This inhibitory effect was associated with high levels of MAPK1/3 activity. Furthermore, stimulation of PRKA partially rescued the meiotic defects of LT/Sv mouse oocytes in concert with correction of abnormal spindle pole localization of PRKA and loss of prolonged spindle assembly checkpoint activity. Altogether, these results confirm a role for PRKA in helping sustain the MII arrest in mature oocytes and suggest that dysfunctional PRKA contributes to meiotic defects in LT/SvEiJ oocytes. PMID:23390161

  20. Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Ma, Rujun; Zhang, Jiaqi; Liu, Xiaohui; Li, Ling; Liu, Honglin; Rui, Rong; Gu, Ling; Wang, Qiang

    2016-01-01

    Rab GTPases have been reported to define the identity and transport routes of vesicles. Rab6 is one of the most extensively studied Rab proteins involved in regulating organelle trafficking and integrity maintenance. However, to date, the function of Rab6 in mammalian oocytes has not been addressed. Here we report severe disorganization of endoplasmic reticulum upon specific knockdown of Rab6a in mouse oocytes. In line with this finding, intracellular Ca2+ stores are accordingly reduced in Rab6a-depleted oocytes. Furthermore, in these oocytes, we observe the absence of cortical granule free domain, which is a kind of special organelle in matured oocytes and its exocytosis is calcium dependent. On the other hand, following Rab6a knockdown, the prominent defects of cytoskeletal structures are detected during oocyte meiosis. In particular, the majority of Rab6a-depleted oocytes fail to form the actin cap, and the frequency of spindle defects and chromosome misalignment is significantly elevated. In summary, our data reveal that Rab6a not only participates in modulating the organization of oocyte organelles, but also is a novel regulator of meiotic apparatus in mammalian oocytes. PMID:27030207

  1. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice.

    PubMed

    Seki, Shinsuke; Mazur, Peter

    2008-10-01

    Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One took place in oocytes that were partly dehydrated by an initial hold for 12 min at -25 degrees C. They were then cooled rapidly to -70 degrees C and warmed slowly, or they were warmed rapidly to intermediate temperatures and held. These oocytes underwent no IIF during cooling but blackened from IIF during warming. The blackening rate increased about 5-fold for each five-degree rise in temperature. Upon thawing, they were dead. The second aspect involved oocytes that had been vitrified by cooling to -196 degrees C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140 degrees C/min to 3300 degrees C/min. Survivals after warming at 140 degrees C/min and 250 degrees C/min were low (<30%). Survivals after warming at > or =2200 degrees C/min were high (80%). When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed. PMID:18562703

  2. Maturation, Fertilization, and the Structure and Function of the Endoplasmic Reticulum in Cryopreserved Mouse Oocytes1

    PubMed Central

    Lowther, Katie M.; Weitzman, Vanessa N.; Maier, Donald; Mehlmann, Lisa M.

    2009-01-01

    Oocyte cryopreservation is a promising technology that could benefit women undergoing assisted reproduction. Most studies examining the effects of cryopreservation on fertilization and developmental competence have been done using metaphase II-stage oocytes, while fewer studies have focused on freezing oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation. Herein, we examined the effects of vitrifying GV-stage mouse oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes necessary for proper fertilization and early embryonic development. We examined the endoplasmic reticulum (ER) as one indicator of cytoplasmic structure, as well as the ability of oocytes to develop Ca2+ release mechanisms following vitrification and in vitro maturation. Vitrified GV-stage oocytes matured in culture to metaphase II at a rate comparable to that of controls. These oocytes had the capacity to release Ca2+ following injection of inositol 1,4,5-trisphosphate, demonstrating that Ca2+ release mechanisms developed during meiotic maturation. The ER remained intact during the vitrification procedure as assessed using the lipophilic fluorescent dye DiI. However, the reorganization of the ER that occurs during in vivo maturation was impaired in oocytes that were vitrified before oocyte maturation. These results show that vitrification of GV-stage oocytes does not affect nuclear maturation or the continuity of the ER, but normal cytoplasmic maturation as assessed by the reorganization of the ER is disrupted. Deficiencies in factors that are responsible for proper ER reorganization during oocyte maturation could contribute to the low developmental potential previously reported in vitrified in vitro-matured oocytes. PMID:19299317

  3. Disruption of Bidirectional Oocyte-Cumulus Paracrine Signaling During In Vitro Maturation Reduces Subsequent Mouse Oocyte Developmental Competence1

    PubMed Central

    Yeo, Christine X.; Gilchrist, Robert B.; Lane, Michelle

    2009-01-01

    Oocyte-cumulus cell bidirectional communication is essential for normal development of the oocyte and cumulus cells (CCs) within the follicle. We showed recently that addition of recombinant growth differentiation factor 9 (GDF9), which signals through the SMAD2/3 pathway, during mouse oocyte in vitro maturation (IVM) increased fetal viability. This study thus aimed to observe the effects of disrupting oocyte-CC bidirectional communication during IVM on oocyte developmental competence and fetal outcomes. Cumulus-oocyte complexes (COCs) from equine chorionic gonadotropin-primed prepubertal (CBA/C57BL6) mice were cultured with or without 50 mIU/ml follicle-stimulating hormone (FSH) and 10 ng/ml epidermal growth factor (EGF) or 4 μM SMAD2/3 inhibitor SB-431542. Cumulus expansion and first polar body extrusion were then assessed, or COCs were fertilized and stained to evaluate sperm entry or cultured to the blastocyst stage. Embryo development and blastocyst quality were assessed, and Day 4.5 blastocysts were transferred to pseudopregnant recipients to analyze fetal outcomes. SMAD2/3 inhibition or FSH/EGF absence during IVM resulted in decreased cumulus expansion. First polar body extrusion and sperm entry were decreased in the absence of FSH/EGF, whereas only sperm entry was affected in SB-431542-matured COCs. Embryo development and blastocyst rates were unaffected; however, blastocyst quality was significantly altered, with reduced inner cell mass cell numbers in embryos derived from COCs matured in both treatments. When COCs were matured with SB-431542 in the absence of FSH/EGF, cumulus expansion was reduced, but fertilization, embryo development, and embryo quality were not. Inhibition of SMAD2/3 signaling in the presence of FSH/EGF significantly reduced fetal survival but had no effect on implantation or fetal and placental dimensions and morphology. PMID:19144958

  4. MOUSE VERSUS RAT: PROFOUND DIFFERENCES IN MEIOTIC REGULATION AT THE LEVEL OF THE ISOLATED OOCYTE

    PubMed Central

    Downs, Stephen M.

    2011-01-01

    Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO) or dissected follicles were obtained 44–48 h after priming immature mice (20–23-days-old) with 5 IU or immature rats (25–27-days-old) with 12.5 IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. The guanylate cyclase activator, atrial natriuretic peptide, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to undergo germinal vesicle breakdown (GVB) by follicle stimulating hormone (FSH) and amphiregulin, while human chorionic gonadotropin (hCG) was ineffective in both species. Nevertheless, FSH and amphiregulin stimulated cumulus expansion in both species. FSH and hCG were both effective inducers of GVB in cultured mouse and rat follicles while amphiregulin was stimulatory only in mouse follicles. Changing the culture medium or altering macromolecular supplementation had no effect on FSH-induced maturation in rat CEO. The AMP-activated protein kinase (AMPK) activator, AICAR, was a potent stimulator of maturation in mouse CEO and DO, but only marginally stimulatory in rat CEO and ineffective in rat DO. The AMPK inhibitor, compound C, blocked meiotic induction more effectively in hCG-treated mouse follicles and heat-treated mouse CEO. Both agents produced contrasting results on polar body formation in cultured CEO in the two species. Active AMPK was detected in germinal vesicles of immature mouse, but not rat, oocytes prior to hCG-induced maturation in vivo; it colocalized with chromatin after GVB in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. Finally, cultured mouse and rat CEO displayed disparate maturation responses to energy substrate

  5. Role of Na+/Ca2+ exchanger (NCX) in modulating postovulatory aging of mouse and rat oocytes.

    PubMed

    Zhang, Chuan-Xin; Cui, Wei; Zhang, Min; Zhang, Jie; Wang, Tian-Yang; Zhu, Jiang; Jiao, Guang-Zhong; Tan, Jing-He

    2014-01-01

    We studied the role of the Na+/Ca2+ exchanger (NCX) in modulating oocyte postovulatory aging by observing changes in NCX contents and activities in aging mouse and rat oocytes. Whereas the NCX activity was measured by observing oocyte activation following culture with NCX inhibitor or activator, the NCX contents were determined by immunohistochemical quantification. Although NCX was active in freshly-ovulated rat oocytes recovered 13 h post hCG injection and in aged oocytes recovered 19 h post hCG in both species, it was not active in freshly-ovulated mouse oocytes. However, NCX became active when the freshly-ovulated mouse oocytes were activated with ethanol before culture. Measurement of cytoplasmic Ca2+ revealed Ca2+ increases always before NCX activation. Whereas levels of the reactive oxygen species (ROS) and the activation susceptibility increased, the density of NCX member 1 (NCX1) decreased significantly with oocyte aging in both species. While culture with H2O2 decreased the density of NCX1 significantly, culture with NaCl supplementation sustained the NCX1 density in mouse oocytes. It was concluded that (a) the NCX activity was involved in the modulation of oocyte aging and spontaneous activation; (b) ROS and Na+ regulated the NCX activity in aging oocytes by altering its density as well as functioning; and (c) cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte. PMID:24695407

  6. In vitro maturation and in vitro fertilization of mouse oocytes and preimplantation embryo culture.

    PubMed

    Kidder, Benjamin L

    2014-01-01

    Epigenetic regulation of gene expression in the germline is important for reproductive success of mammals. Misregulation of genes whose expression is correlated with reproductive success may result in subfertility or infertility. To study epigenetic events that occur during oocyte maturation and preimplantation embryo development, it is important to generate large numbers of ovarian follicles and embryos. Oocyte maturation can be modeled using in vitro maturation (IVM), which is a system of maturing ovarian follicles in a culture dish. In addition, fertilization and early embryogenesis can be modeled using in vitro fertilization (IVF), which involves the fertilization of mature oocytes with capacitated sperm in a culture dish. Here, we describe protocols for in vitro maturation (IVM) and in vitro fertilization (IVF) of mouse oocytes and preimplantation embryo culture. These protocols are suitable for the study of oocyte and embryo biology and the production of embryonic mice. PMID:24743999

  7. Development of mouse and rat oocytes in chimeric reaggregated ovaries after interspecific exchange of somatic and germ cell components.

    PubMed

    Eppig, J J; Wigglesworth, K

    2000-10-01

    The germ cell and somatic cell compartments of newborn rat and mouse ovaries, which contain only primordial stage follicles, were completely exchanged and reaggregated to produce xenogeneic chimeric ovaries. The reaggregated ovaries were grafted beneath the renal capsules of ovariectomized SCID mice to develop for periods up to 21 days. Xenogeneic follicles developed with essentially normal morphological characteristics. Both rat and mouse oocytes with species-specific characteristics grew within follicles that were composed of somatic cells exclusively of the alternative species. Rat oocytes grown in mouse follicles became competent to resume meiosis, and progressed to metaphase II when they were removed from follicles and cultured. In addition, mouse oocytes grown in rat follicles underwent fertilization and preimplantation development in vitro, and developed to term after embryos were transferred to pseudopregnant mouse foster mothers. Therefore, despite an estimated 11 million years of divergent evolution, oocytes and somatic cells of rat and mouse ovaries can be exchanged and can produce functional oocytes. It is concluded that factors involved in oocyte-somatic cell interactions necessary to support oocyte development and appropriate differentiation of the oocyte-associated granulosa cells are conserved between rats and mice. Moreover, although granulosa cells play important roles in oocyte development, the development of species-specific characteristics of oocytes occurs without apparent modification by a xenogeneic follicular environment. PMID:10993822

  8. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

    PubMed

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

    2016-06-15

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  9. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    PubMed Central

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  10. Growth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro

    PubMed Central

    Zhang, Zhi-Peng; Liang, Gui-Jin; Zhang, Xi-Feng; Zhang, Guo-Liang; Chao, Hu-He; Li, Lan; Sun, Xiao-Feng; Min, Ling-Jiang; Pan, Qing-Jie; Shi, Qing-Hua; Sun, Qing-Yuan; De Felici, Massimo; Shen, Wei

    2012-01-01

    In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage. PMID:22848595

  11. Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells

    SciTech Connect

    Cecconi, S.; Tatone, C.; Buccione, R.; Mangia, F.; Colonna, R. )

    1991-05-01

    The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: (a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and (b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

  12. Apoptotic effects on maturation of mouse oocytes, fertilization and fetal development by puerarin.

    PubMed

    Huang, Fu-Jen; Chan, Wen-Hsiung

    2016-10-01

    Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo. PMID:26712108

  13. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality.

    PubMed

    Shaeib, Faten; Khan, Sana N; Thakur, Mili; Kohan-Ghadr, Hamid-Reza; Drewlo, Sascha; Saed, Ghassan M; Pennathur, Subramaniam; Abu-Soud, Husam M

    2016-01-01

    Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes. PMID:26982351

  14. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality

    PubMed Central

    Shaeib, Faten; Khan, Sana N.; Thakur, Mili; Kohan-Ghadr, Hamid-Reza; Drewlo, Sascha; Saed, Ghassan M.; Pennathur, Subramaniam; Abu-Soud, Husam M.

    2016-01-01

    Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes. PMID:26982351

  15. Investigations of oocyte in vitro maturation within a mouse model.

    PubMed

    Chin, Alexis Heng Boon; Chye, Ng Soon

    2004-02-01

    This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage. PMID:15214575

  16. Exceptional material requirement for reproduction in mouse oocytes.

    PubMed

    Yu, L; Wang, S F; Zhai, Q Z; Yao, Y Q; Jiang, F; Lu, Y X

    2015-01-01

    Limited information on oocytes and fertilization prevents the efficient therapy of patients with infertility. The most important reason for this lack of understanding is a deficiency in research dedicated to oocytes and fertilization. Currently, we are concerned with the role of nutrition in the process of oocyte development to better understand the relationship between nutrition and infertility. The aim of this study was to explore the relationship between some exceptional materials and infertility to elucidate the role of these materials in oocyte development. We used proteomic analysis to identify numerous nutrition-related proteins in three developmental stages: the germinal vesicle stage, the metaphase II-arrested stage, and the fertilized oocyte-zygote stage. Specific proteins were abundantly expressed during the three stages. These proteins included astacin-like metalloendopeptidase, selenium-binding proteins, and other proteins involved in metabolic and signaling pathways. Other proteins were involved in the citrate cycle, the electron transport chain, the urea cycle, fatty acid metabolism, and the insulin signaling pathway. Almost all these proteins exhibited different expression levels in the three stages. The results of the present study provide a better understanding of the molecular mechanisms of early embryonic development and suggest new treatment methods for infertility. PMID:26600495

  17. Effects of Aroclor 1254 on In Vivo Oocyte Maturation in the Mouse

    PubMed Central

    Liu, ShuZhen; Jiang, LiGang; Meng, XiaoQian; Han, XiaoYing; Cheng, Dong; Zhang, TianLiang; Miao, YiLiang

    2014-01-01

    Polychlorinated biphenyls (PCBs) are stable, lipophilic compounds that accumulate in the environment and in the food chain. Though some studies provided evidence that PCBs had adverse effects on reproductive function, most of these results were from in vitro models. Therefore we investigated the effect of Aroclor 1254 (a commercial PCBs mixture) treatments on in vivo maturation and developmental potential of mouse oocytes. In the present study, female ICR mice were treated with different doses (12.5, 25 and 50 mg/kg) of Aroclor 1254 (a commercial PCB mixture) once every 72 hours by intraperitoneal injection for 9 days. After three treatments of Aroclor 1254, the mice were superovulated to collect oocytes one day after the last exposure. The effects of Aroclor 1254 on oocyte maturation, fertilization, and preimplantation embryonic development were investigated. Immunofluorescence-stained oocytes were observed under a confocal microscope to assess the effects of Aroclor 1254 on spindle morphology. Parthenogenic activation and the incidence of cumulus apoptosis in cumulus-oocyte complexes were observed as well. Oocytes exposed to different doses of Aroclor 1254 in vivo were associated with a significant decrease in outgrowth potential, abnormal spindle configurations, and the inhibition of parthenogenetic activation of ovulated oocytes. Furthermore, the incidence of apoptosis in cumulus cells was increased after exposed to Aroclor 1254. These results may provide reference for the treatment of reproductive diseases such as infertility or miscarriage caused by environmental contaminants. PMID:25013911

  18. TRAIP is involved in chromosome alignment and SAC regulation in mouse oocyte meiosis

    PubMed Central

    Yuan, Yi-Feng; Ren, Yi-Xin; Yuan, Peng; Yan, Li-Ying; Qiao, Jie

    2016-01-01

    Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore–microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation. PMID:27405720

  19. TRAIP is involved in chromosome alignment and SAC regulation in mouse oocyte meiosis.

    PubMed

    Yuan, Yi-Feng; Ren, Yi-Xin; Yuan, Peng; Yan, Li-Ying; Qiao, Jie

    2016-01-01

    Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore-microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation. PMID:27405720

  20. The effect of glucocorticoids on mouse oocyte in vitro maturation and subsequent fertilization and embryo development.

    PubMed

    González, Raquel; Ruiz-León, Yolanda; Gomendio, Montserrat; Roldan, Eduardo R S

    2010-02-01

    Increased glucocorticoid levels, due to medical therapy or stress-related, may affect reproduction via the hypothalamus-pituitary-axis or directly at the oocyte level. We examined the effects of natural (corticosterone) or synthetic (dexamethasone) glucocorticoids on mouse oocyte maturation and underlying changes in extracellular signal-regulated kinase (ERK) phosphorylation patterns. Fertilization and progression up to the blastocyst stage were also evaluated. Oocytes were exposed to corticosterone or dexamethasone (0, 0.25, 2.5, 25 or 250microM) for 17h during in vitro maturation. After maturation, ERK-1/2 activation in oocytes was assessed by SDS-PAGE and immunoblotting, and fertilization and developmental capacity were examined in vitro. Corticosterone exposure during oocyte maturation significantly decreased progression to metaphase II, fertilization and embryo development at the highest concentration. Corticosterone caused a concentration-dependent inhibition of ERK-1/2 activation, with the highest concentration resulting in considerable inhibition of oocyte ERK-1/2 phosphorylation and no blastocyst development. In contrast, dexamethasone had no effect on maturation, fertilization and cleavage, and no effect was seen on ERK-1/2 phosphorylation. Based on these in vitro findings, high glucocorticoid levels may have consequences for subsequent development, although a short exposure to physiologic or stress-related glucocorticoid levels may not represent a hazard to meiosis progression of the oocyte. PMID:19733225

  1. Intact fetal ovarian cord formation promotes mouse oocyte survival and development

    PubMed Central

    2010-01-01

    Background Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined. Results Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles. Conclusions Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the human fetal ovary, the

  2. Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Teng; Zhou, Yang; Qi, Shu-Tao; Wang, Zhen-Bo; Qian, Wei-Ping; Ouyang, Ying-Chun; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

    2015-01-01

    Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc6-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes. PMID:26054848

  3. Toxic effects of HT-2 toxin on mouse oocytes and its possible mechanisms.

    PubMed

    Zhu, Cheng-Cheng; Zhang, Yue; Duan, Xing; Han, Jun; Sun, Shao-Chen

    2016-06-01

    T-2 toxin is one of the type A trichothecene mycotoxins that is considered to be the most toxic of the trichothecenes. T-2 toxin has been shown to exert various toxic effects in farm animals and humans, as it induces lesions in the brain and in lymphoid, hematopoietic, and gastrointestinal tissues. HT-2 toxin is the major metabolite of T-2 toxin. There is little information regarding the effects of HT-2 toxin on the female reproductive system, particularly oocyte maturation. Thus, in this study, we investigated the toxic effects of HT-2 on mouse oocyte maturation and its possible mechanisms of action. HT-2 toxin exposure disrupted oocyte maturation, reduced actin expression in both the oocyte cortex and cytoplasm, and disrupted meiotic spindle morphology by reducing p-MAPK protein level. HT-2 toxin exposure also induced oxidative stress and resulted in oocyte apoptosis, as shown by ROS accumulation, increased SOD mRNA level, and the expression of the early apoptosis marker Annexin V and increased caspase-3 and bax mRNA levels. Additionally, HT-2 toxin exposure increased LC3 and ATG12 protein levels and lc3 and atg14 mRNA levels, which indicated that HT-2 toxin induced autophagy in mouse oocytes. We also examined for possible epigenetic modifications. Fluorescence intensity analysis showed that 5mC level increased after HT-2 toxin exposure, whereas H3K9me2 and H3K27me3 levels decreased after HT-2 toxin exposure, which indicated that DNA and histone methylations were altered. Thus, our results indicated that HT-2 toxin exposure reduced mouse oocyte maturation capability by affecting cytoskeletal dynamics, apoptosis/autophagy, oxidative stress, and epigenetic modifications. PMID:26138683

  4. Nek11 regulates asymmetric cell division during mouse oocyte meiotic maturation.

    PubMed

    Guo, Lei; Wang, Zhen-Bo; Wang, Hong-Hui; Zhang, Teng; Qi, Shu-Tao; Ouyang, Ying-Chun; Hou, Yi; Sun, Qing-Yuan

    2016-06-10

    Nek11, a member of the never in mitosis gene A (NIMA) family, is activated in somatic cells associated with G1/S or G2/M arrest. However, its function in meiosis is unknown. In this research, the expression, localization and functions of NEK11 in the mouse oocyte meiotic maturation were examined. Western blotting indicated that NEK11S was the major NEK11 protein in mouse oocyte. MYC-tagged Nek11 mRNA microinjection and immunofluorescent staining showed that NEK11 was localized to the meiotic spindles at MI and MII stage. Knockdown of Nek11 by microinjection of siRNA did not affect germinal vesicle breakdown (GVBD) and the first polar body extrusion, but caused formation of 2-cell-like eggs. These results demonstrate that Nek11 regulates asymmetric cell division during oocyte meiotic maturation. PMID:27150633

  5. Effect of organochlorine pesticides on maturation of starfish and mouse oocytes.

    PubMed

    Picard, André; Pahlavan, Golbahar; Palavan, Golbahar; Robert, Stéphanie; Pesando, Danielle; Ciapa, Brigitte

    2003-05-01

    Methoxychlor, lindane, and dieldrin are organochlorine pesticides that have been described as altering different reproductive functions in mammals and in invertebrates. However, few data have been published concerning the effects these pesticides have on oocyte maturation and fertilization. The aim of this study was to determine whether these compounds could affect maturation of mouse and starfish oocytes. We observed that germinal vesicle breakdown (GVBD) in starfish oocytes was significantly inhibited by the pesticides. Furthermore, formation of the first meiotic spindle and extrusion of the first polar body were also altered in mouse as well as in starfish. Our results suggest that the three pesticides act on common intracellular targets in invertebrates as well as in vertebrates. PMID:12700411

  6. Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing

    PubMed Central

    Zhang, Linlin; Zhou, Jiankui; Han, Jinxiong; Hu, Bian; Hou, Ningning; Shi, Yun; Huang, Xingxu

    2016-01-01

    The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing. PMID:27119535

  7. Effects of ochratoxin a on mouse oocyte maturation and fertilization, and apoptosis during fetal development.

    PubMed

    Huang, Fu-Jen; Chan, Wen-Hsiung

    2016-06-01

    We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1-10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked these OTA-triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase-dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA-pretreated oocytes, indicating that such cells undergo apoptosis via p53-, p21-, and caspase-3-dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724-735, 2016. PMID:25504763

  8. Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

    PubMed

    Hegele-Hartung, C; Kuhnke, J; Lessl, M; Grøndahl, C; Ottesen, J; Beier, H M; Eisner, S; Eichenlaub-Ritter, U

    1999-11-01

    Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization. PMID:10529286

  9. Factors affecting the survival, fertilization, and embryonic development of mouse oocytes after vitrification using glass capillaries.

    PubMed

    Tan, Xiuwen; Song, Enliang; Liu, Xiaomu; You, Wei; Wan, Fachun

    2009-09-01

    Cryopreservation of mammalian oocytes is an important way to provide a steady source of materials for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. However, oocytes cryopreservation has not been common used, as there still are some problems waiting to be solved on the repeatability, safety, and validity. Then, it is necessary to investigate the damage occurred from vitrification and find a way to avoid or repair it. In this study, mouse mature oocytes were firstly pretreated in different equilibrium media, such as 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO), 10% EG + 10% DMSO, and 15% EG + 15% DMSO in TCM199 supplemented with 20% fetal calf serum (FCS), for 1, 3, and 5 min, respectively, and then oocytes were transferred into vitrification solution (20% EG, 20% DMSO, 0.3 M sucrose, and 20% FCS in TCM199, M2, Dulbecco's phosphate buffered saline, and 0.9% saline medium, respectively) and immediately loaded into glass capillaries to be plunged into liquid nitrogen. After storage from 1 h to 1 wk, they were diluted in stepwise sucrose solutions. The surviving oocytes were stained for cortical granule, meiotic spindles, and chromosomes. Oocytes without treatments were used as controls. The results showed that oocytes pretreated in 5% EG +5% DMSO group for 3-5 min or in 10% EG + 10% DMSO group for 1-3 min were better than other treatments. Oocytes vitrified in TCM199 as basic medium showed higher survival and better subsequent embryonic development than other groups. When the concentration of FCS in vitrification solution reduced below 15%, the rates of survival, fertilization, and developing to blastocyst declined dramatically. The inner diameter (0.6 mm) of glass capillaries and amount of vitrification solution (1-3 microl) achieved more rapid cooling and warming and so reduce the injury to oocytes. Cropreservation led to the exocytosis of cortical granule of oocytes (about 10%) and serious disturbance of

  10. Depletion of the LINC complex disrupts cytoskeleton dynamics and meiotic resumption in mouse oocytes

    PubMed Central

    Luo, Yibo; Lee, In-Won; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2016-01-01

    The SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) proteins constitute the linker of nucleoskeleton and cytoskeleton (LINC) complex on the nuclear envelope. To date, the SUN1/KASH5 complex is known to function as meiotic-specific factors. In this study, gene-silencing methods were used to explore the roles of SUN1 and KASH5 in mouse oocytes after prophase. SUN1 was detected throughout the nucleus; however, KASH5 was dispersed through the cell. After germinal vesicle breakdown (GVBD), SUN1 and KASH5 migrated during spindle formation and localized to the spindle poles at the MII stage. Most oocytes were arrested at the germinal vesicle (GV) stage after depletion of either SUN1 or KASH5. The DNA damage response was triggered in SUN1-depleted oocytes and thus gave rise to the G2/M checkpoint protein, p-CHK1. Oocytes that underwent GVBD had relatively small and abnormal spindles and lower levels of cytoplasm F-actin mesh. Immunofluorescence results also indicated the dislocation of pericentrin and P150Glued after SUN1 or KASH5 depletion. Furthermore, KASH5 localized exclusively near the oocyte cortex after SUN1 depletion, but SUN1 localization was unaffected in KASH5-depleted oocytes. Taken together, the results suggest that SUN1 and KASH5 are essential factors in the regulation of meiotic resumption and spindle formation. PMID:26842404

  11. Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte

    PubMed Central

    Han, Longsen; Ge, Juan; Zhang, Liang; Ma, Rujun; Hou, Xiaojing; Li, Bin; Moley, Kelle; Wang, Qiang

    2015-01-01

    Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus. PMID:26481302

  12. Mouse Oocytes Transcribe Injected Xenopus 5S RNA Gene

    PubMed Central

    Brinster, Ralph L.; Chen, Howard Y.; Trumbauer, Myrna E.

    2016-01-01

    Transcripts produced after injection of the Xenopus 5S RNA gene into oocyte germinal vesicles of mice migrate electrophoretically with the 5S RNA marker, an indication that the gene is transcribed and processed with considerable accuracy, Approximately two 5S RNA molecules are transcribed per gene per hour. This system may be useful in studying DNA processing and gene regulation by the mammalian ovum and might be modified to allow permanent incorporation of specific genes into mice. PMID:7194505

  13. Vitrification by Cryotop and the Maturation, Fertilization, and Developmental Rates of Mouse Oocytes

    PubMed Central

    Abedpour, Neda; Rajaei, Farzad

    2015-01-01

    Background: Oocyte cryopreservation is an important part of modern fertility treatment. The effect of vitrification on the fertilization and developmental rates of embryo is still a matter of debate. Objectives: This study aimed to investigate the effect of vitrification on the success of mouse oocyte maturation, fertilization, and preimplantation development in vitro. Materials and Methods: In this experimental study, a total of 200 germinal vesicle (GV) and 200 metaphase II (MII) oocytes were obtained from ovaries and fallopian tubes of NMRI mice, respectively and divided into two control and experimental (vitrified) groups. Oocytes in the experimental group were vitrified by Cryotop using vitrification medium (Origio, Denmark) and kept in liquid nitrogen for one month. Then, they were cultured in maturation medium for 24 hours. In vitro maturated metaphase 2 (IVM-MII) and ovulated metaphase 2 (OV-MII) oocytes were inseminated and the fertilized embryos assessed until the hatching blastocyst stage. Outcomes were assessed for statistical significance by Chi-square test using SPSS software. Results: Vitrification caused a significant reduction in the maturation rate of oocytes. Of those that matured, the fertilization rate of vitrified IVM-MII (44.1%) and OV-MII oocytes (50%) was not significantly different from each other but both were significantly lower than the control group (P < 0.05). There was no significant difference in developmental rates of both vitrified groups and the control group. Conclusions: The present study showed that vitrification using Cryotop and freezing medium can damage oocytes by reducing the maturation and fertilization rates in both developmental stages. PMID:26568845

  14. The Defensive Role of Cumulus Cells Against Reactive Oxygen Species Insult in Metaphase II Mouse Oocytes.

    PubMed

    Shaeib, Faten; Khan, Sana N; Ali, Iyad; Thakur, Mili; Saed, Mohammed G; Dai, Jing; Awonuga, Awoniyi O; Banerjee, Jashoman; Abu-Soud, Husam M

    2016-04-01

    We investigated the ability of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical ((·)OH), and hypochlorous acid (HOCl), to overcome the defensive capacity of cumulus cells and elucidate the mechanism through which ROS differentially deteriorate oocyte quality. Metaphase II mouse oocytes with (n = 1634) and without cumulus cells (n = 1633) were treated with increasing concentration of ROS, and the deterioration in oocyte quality was assessed by the changes in the microtubule morphology and chromosomal alignment. Oocyte and cumulus cell viability and cumulus cell number were assessed by indirect immunofluorescence, staining of gap junction protein, and trypan blue staining. The treated oocytes showed decreased quality as a function of increasing concentrations of ROS when compared to controls. Cumulus cells show protection against H2O2 and (·)OH insult at lower concentrations, but this protection was lost at higher concentrations (>50 μmol/L). At higher H2O2 concentrations, treatment dramatically influenced the cumulus cell number and viability with resulting reduction in the antioxidant capacity making the oocyte more susceptible to oxidative damage. However, cumulus cells offered no significant protection against HOCl at any concentration used. In all circumstances in which cumulus cells did not offer protection to the oocyte, both cumulus cell number and viability were decreased. Therefore, the deterioration in oocyte quality may be caused by one or more of the following: a decrease in the antioxidant machinery by the loss of cumulus cells, the lack of scavengers for specific ROS, and/or the ability of the ROS to overcome these defenses. PMID:26468254

  15. Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing.

    PubMed

    Baumann, Claudia; Viveiros, Maria M

    2015-01-01

    Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down's syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization. PMID:26485537

  16. A three-step MTOC fragmentation mechanism facilitates bipolar spindle assembly in mouse oocytes.

    PubMed

    Clift, Dean; Schuh, Melina

    2015-01-01

    Assembly of a bipolar microtubule spindle is essential for accurate chromosome segregation. In somatic cells, spindle bipolarity is determined by the presence of exactly two centrosomes. Remarkably, mammalian oocytes do not contain canonical centrosomes. This study reveals that mouse oocytes assemble a bipolar spindle by fragmenting multiple acentriolar microtubule-organizing centres (MTOCs) into a high number of small MTOCs to be able to then regroup and merge them into two equal spindle poles. We show that MTOCs are fragmented in a three-step process. First, PLK1 triggers a decondensation of the MTOC structure. Second, BicD2-anchored dynein stretches the MTOCs into fragmented ribbons along the nuclear envelope. Third, KIF11 further fragments the MTOCs following nuclear envelope breakdown so that they can be evenly distributed towards the two spindle poles. Failure to fragment MTOCs leads to defects in spindle assembly, which delay chromosome individualization and congression, putting the oocyte at risk of aneuploidy. PMID:26147444

  17. Size of lethality target in mouse immature oocytes determined with accelerated heavy ions.

    PubMed

    Straume, T; Dobson, R L; Kwan, T C

    1989-01-01

    Mouse immature oocytes were irradiated in vivo with highly charged, heavy ions from the Bevalac accelerator at the Lawrence Berkeley Laboratory. The particles used were 670-MeV/nucleon Si14+, 570-MeV/nucleon Ar18+, and 450-MeV/nucleon Fe26+. The cross-sectional area of the lethality target in these extremely radiosensitive cells was determined from fluence-response curves and information on energy deposition by delta rays. Results indicate a target cross-section larger than that of the nucleus, one which closely approximates the cross-sectional area of the entire oocyte. For 450-MeV/nucleon Fe26+ particles, the predicted target cross-sectional area is 120 +/- 16 microns2, comparing well with the microscopically determined cross-sectional area of 111 +/- 12 microns2 for these cells. The present results are in agreement with our previous target studies which implicate the oocyte plasma membrane. PMID:2657842

  18. Improved Low-CPA Vitrification of Mouse Oocytes Using Quartz Microcapillary

    PubMed Central

    Choi, Jung Kyu; Huang, Haishui; He, Xiaoming

    2015-01-01

    Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management. PMID:25869750

  19. Improved low-CPA vitrification of mouse oocytes using quartz microcapillary.

    PubMed

    Choi, Jung Kyu; Huang, Haishui; He, Xiaoming

    2015-06-01

    Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management. PMID:25869750

  20. The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Calarco, Patricia G.

    2005-04-01

    Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes ([gamma]-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

  1. Next Generation Sequencing-Based Comprehensive Chromosome Screening in Mouse Polar Bodies, Oocytes, and Embryos.

    PubMed

    Treff, Nathan R; Krisher, Rebecca L; Tao, Xin; Garnsey, Heather; Bohrer, Chelsea; Silva, Elena; Landis, Jessica; Taylor, Deanne; Scott, Richard T; Woodruff, Teresa K; Duncan, Francesca E

    2016-04-01

    Advanced reproductive age is unequivocally associated with increased aneuploidy in human oocytes, which contributes to infertility, miscarriages, and birth defects. The frequency of meiotic chromosome segregation errors in oocytes derived from reproductively aged mice appears to be similar to that observed in humans, but a limitation of this important model system is our inability to accurately identify chromosome-specific aneuploidy. Here we report the validation and application of a new low-pass whole-genome sequencing approach to comprehensively screen chromosome aneuploidy in individual mouse oocytes and blastocysts. First, we validated this approach by using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in blastocysts derived from in vitro- and in vivo-matured oocytes in both young and reproductively aged mice. Using this next generation sequencing approach, we quantitatively assessed meiotic and mitotic segregation errors at the single chromosome level, distinguished between errors due to premature separation of sister chromatids and classical nondisjunction of homologous chromosomes, and quantified mitochondrial DNA (mtDNA) segregation in individual cells. This whole-genome sequencing technique, therefore, greatly improves the utility of the mouse model system for the study of aneuploidy and is a powerful quantitative tool with which to examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation in the context of reproductive aging and beyond. PMID:26911429

  2. Factor in urinary extracts from pregnant women that inhibit mouse oocyte maturation in vitro.

    PubMed

    Sakakibara, R; Sakai, K; Sakurai, Y; Kohnoura, T; Ishiguro, M

    1993-01-01

    Mouse oocyte maturation inhibitory factors, on the basis of inhibitory activity of spontaneous germinal vesicle breakdown (GVBD) of denuded mouse oocytes in culture, were extracted and partially purified by reversed-phase resin adsorption and Sephadex G-100 and G-50 column chromatographies from the urine of pregnant women. Denuded oocytes obtained from ovaries of ICR mice underwent spontaneous GVBD by cultivation for 3 h in modified Krebs-Ringer's buffered solution, while this spontaneous GVBD was found to be inhibited by adding the final preparation (U-D-4) of urine. The inhibition was dose dependent, ranging from 0.6 to 10 micrograms protein/ml medium. Oocytes treated with U-D-4 and resuspended in control medium resumed GVBD. The molecular mass of U-D-4 was estimated to be less than 2,000 Da with gel filtration. Ether treatment failed to extract inhibitory factor(s) from U-D-4 and pepsin treatment inactivated U-D-4, indicating that inhibitory factor(s) in U-D-4 are peptide-like substances. The inhibitory effect of U-D-4 on spontaneous GVBD was partially reversed in the presence of naloxone, a potent opioid antagonist. U-D-4s obtained from urine samples of pregnant women, nonpregnant women, and men showed the inhibitory effect on spontaneous GVBD; however, the activity of U-D-4 obtained from pregnancy urine was significantly more potent than those of the other urine samples. PMID:8418810

  3. Clathrin heavy chain 1 is required for spindle assembly and chromosome congression in mouse oocytes.

    PubMed

    Zhao, Jie; Wang, Lu; Zhou, Hong-Xia; Liu, Li; Lu, Angeleem; Li, Guang-Peng; Schatten, Heide; Liang, Cheng-Guang

    2013-10-01

    Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation. PMID:23816345

  4. Oocyte heterogeneity with respect to the meiotic silencing of unsynapsed X chromosomes in the XY female mouse.

    PubMed

    Taketo, Teruko; Naumova, Anna K

    2013-10-01

    In the XY pachytene spermatocyte, the sex chromosomes do not synapse except for the pseudoautosomal region and become transcriptionally silenced. It has been suggested that the meiotic silencing of unsynapsed chromatin (MSUC) also occurs in oocytes. In the XY sex-reversed female mouse, the sex chromosomes fail to pair in the majority of oocytes and a greater number of oocytes are eliminated during the meiotic prophase compared to the XX female. Yet, many XY oocytes survive to reach the second meiotic metaphase. The goal of our current study was to determine whether the single X chromosome shows the characteristics of asynapsis and meiotic silencing in a proportion of XY oocytes, which can explain the survival of the remaining oocytes. We first examined the accumulation of markers associated with asynapsis or transcriptional silencing, i.e., BRCA1, γH2AX, H3K9me3, and H3K27me3, at the single X chromosome in the XY oocyte. We found that γH2AX and BRCA1 were enriched on the single X chromosome whereas H3K9me3 was not, and H3K27me3 was enriched at all chromosomes in the majority of XY oocytes. We next examined the meiotic silencing of the single X chromosome using enrichment of the X-encoded ATRX protein. On average, ATRX enrichment was lower in XY oocytes than in XX oocytes as expected from its half gene dosage. However, the intensity of ATRX staining in XY oocytes harboring γH2AX domains showed a remarkable heterogeneity. We conclude that MSUC occurs with varying consequences, resulting in a heterogeneous population of oocytes with respect to protein enrichment in the XY female mouse. PMID:23760560

  5. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

    NASA Technical Reports Server (NTRS)

    Schatten, Heide; Schatten, Gerald; Balczon, Ron; Simerly, Calvin; Mazia, Daniel

    1986-01-01

    The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

  6. Bacterial artificial chromosome transgenesis through pronuclear injection of fertilized mouse oocytes.

    PubMed

    Vintersten, Kristina; Testa, Giuseppe; Naumann, Ronald; Anastassiadis, Konstantinos; Stewart, A Francis

    2008-01-01

    In the mouse, conventional transgenes often produced unpredictable results mainly because they were too small to recapitulate a natural gene context. Bacterial artificial chromosomes (BACs) are large enough to encompass the natural context of most mammalian genes and consequently deliver more reliable recapitulations of their endogenous counterparts. Furthermore, recombineering methods now make it easy to engineer precise changes in a BAC transgene. Consequently, BACs have become the preferred vehicle for mouse transgenesis. Here, we detail methods for BAC transgenesis through pronuclear injection of fertilized oocytes. PMID:18370149

  7. Tropomodulin-3 is essential in asymmetric division during mouse oocyte maturation.

    PubMed

    Jo, Yu-Jin; Jang, Woo-In; Kim, Nam-Hyung; Namgoong, Suk

    2016-01-01

    The dynamic polymerization and depolymerization of actin filaments is essential for various cellular processes such as cell migration, rotation, cytokinesis, and mammalian oocyte maturation. Tropomodulin 3 (Tmod3) binds to the slow-growing (pointed) ends of the actin filament, thereby protecting the filament from depolymerization. However, the roles of Tmod3 in mammalian oocyte maturation remain elusive. Tmod3 mRNA and protein is present at all stages of mouse oocyte maturation. Tmod3 protein is mainly localized in the cytoplasm and appears enriched near the chromosome during maturation. By knocking down or ectopically overexpressing Tmod3, we confirmed that Tmod3 regulate the level of the intracytoplasmic actin mesh and asymmetric spindle migration. Expression of N-terminal Tmod3 (correspond to 1-155 amino acids), which contains the tropomyosin-binding site, results in decreased density of the actin mesh, thereby demonstrating the importance of the interaction between tropomyosin and tropomodulin for the maintenance of the actin mesh. Taken together, these findings indicate that Tmod3 plays crucial roles in oocyte maturation, presumably by protecting the actin filament from depolymerization and thereby controlling the density of the cytoplasmic actin mesh. PMID:27374327

  8. Tropomodulin-3 is essential in asymmetric division during mouse oocyte maturation

    PubMed Central

    Jo, Yu-Jin; Jang, Woo-In; Kim, Nam-Hyung; Namgoong, Suk

    2016-01-01

    The dynamic polymerization and depolymerization of actin filaments is essential for various cellular processes such as cell migration, rotation, cytokinesis, and mammalian oocyte maturation. Tropomodulin 3 (Tmod3) binds to the slow-growing (pointed) ends of the actin filament, thereby protecting the filament from depolymerization. However, the roles of Tmod3 in mammalian oocyte maturation remain elusive. Tmod3 mRNA and protein is present at all stages of mouse oocyte maturation. Tmod3 protein is mainly localized in the cytoplasm and appears enriched near the chromosome during maturation. By knocking down or ectopically overexpressing Tmod3, we confirmed that Tmod3 regulate the level of the intracytoplasmic actin mesh and asymmetric spindle migration. Expression of N-terminal Tmod3 (correspond to 1–155 amino acids), which contains the tropomyosin-binding site, results in decreased density of the actin mesh, thereby demonstrating the importance of the interaction between tropomyosin and tropomodulin for the maintenance of the actin mesh. Taken together, these findings indicate that Tmod3 plays crucial roles in oocyte maturation, presumably by protecting the actin filament from depolymerization and thereby controlling the density of the cytoplasmic actin mesh. PMID:27374327

  9. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    SciTech Connect

    Downs, S.M. )

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  10. Rab3A, Rab27A, and Rab35 regulate different events during mouse oocyte meiotic maturation and activation.

    PubMed

    Wang, H H; Cui, Q; Zhang, T; Wang, Z B; Ouyang, Y C; Shen, W; Ma, J Y; Schatten, H; Sun, Q Y

    2016-06-01

    Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions. PMID:26791531

  11. Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes

    PubMed Central

    de Paola, Matilde; Bello, Oscar Daniel; Michaut, Marcela Alejandra

    2015-01-01

    Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model. PMID:26267363

  12. Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes.

    PubMed

    de Paola, Matilde; Bello, Oscar Daniel; Michaut, Marcela Alejandra

    2015-01-01

    Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model. PMID:26267363

  13. Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries

    PubMed Central

    Zhang, Yuanwei; Jiang, Xiaohua; Zhang, Huan; Ma, Tieliang; Zheng, Wei; Sun, Rui; Shen, Wei; Sha, Jiahao; Cooke, Howard J.; Shi, Qinghua

    2011-01-01

    Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer guanulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries. PMID:21253613

  14. Live Imaging of Intracellular Dynamics During Meiotic Maturation in Mouse Oocytes.

    PubMed

    Yoshida, Shuhei; Sakakibara, Yogo; Kitajima, Tomoya S

    2016-01-01

    Fluorescence live imaging is a powerful approach to study intracellular dynamics during cellular events such as cell division. By applying automated confocal live imaging to mouse oocytes, in which meiotic maturation can be induced in vitro after the introduction of fluorescent proteins through microinjection, the meiotic dynamics of intracellular structures, such as chromosomes, can be monitored at high resolution. A combination of this method with approaches for the perturbation of specific proteins opens up opportunities for understanding the molecular and intracellular basis of mammalian meiosis. PMID:27557586

  15. [The effect of in-situ nerve growth factor from different biological sources on the reinitiation of mouse oocyte meiotic maturation in culture and on parthenogenetic activation].

    PubMed

    Fedorushchenko, A N; Koval', T Iu; Khamidov, D Kh

    1999-01-01

    We studied the capacity of mouse oocytes to complete meiotic maturation in vitro and form the female pronucleus upon parthenogenetic activation by cycloheximide, in response to a single injection into the mouse ovaries in situ of a purified fraction of 2.5 S NGF from mouse submaxillary glands and beta-NGF from bovine sperm. Injection of NGF from both sources at 10 ng/ml with subsequent incubation of the ovaries for 1 h increased the capacity of matured oocytes for parthenogenetic formation of the pronucleus. The frequency of pronucleus formation in both "naked oocyte" and oocytes surrounded by the cumulus cells was four times that in the control. PMID:10624718

  16. Mouse oocytes suppress miR-322-5p expression in ovarian granulosa cells.

    PubMed

    Sumitomo, Jun-Ichi; Emori, Chihiro; Matsuno, Yuta; Ueno, Mizuki; Kawasaki, Kurenai; Endo, Takaho A; Shiroguchi, Katsuyuki; Fujii, Wataru; Naito, Kunihiko; Sugiura, Koji

    2016-08-25

    This study tested the hypothesis that oocyte-derived paracrine factors (ODPFs) regulate miRNA expression in mouse granulosa cells. Expression of mmu-miR-322-5p (miR-322) was higher in mural granulosa cells (MGCs) than in cumulus cells of the Graafian follicles. The expression levels of miR-322 decreased when cumulus cells or MGCs were co-cultured with oocytes denuded of their cumulus cells. Inhibition of SMAD2/3 signaling by SB431542 increased miR-322 expression by cumulus-oocyte complexes (COCs). Moreover, the cumulus cells but not the MGCs in Bmp15(-/-)/Gdf9(+/-) (double-mutant) mice exhibited higher miR-322 expression than those of wild-type mice. Taken together, these results show that ODPFs suppress the expression of miR-322 in cumulus cells. Gene ontology analysis of putative miR-322 targets whose expression was detected in MGCs with RNA-sequencing suggested that multiple biological processes are affected by miR-322 in MGCs. These results demonstrate that ODPFs regulate miRNA expression in granulosa cells and that this regulation may participate in the differential control of cumulus cell versus MGC functions. Therefore, the ODPF-mediated regulation of cumulus cells takes place at both transcriptional and post-transcriptional levels. PMID:27180925

  17. Mouse oocytes suppress miR-322-5p expression in ovarian granulosa cells

    PubMed Central

    SUMITOMO, Jun-ichi; EMORI, Chihiro; MATSUNO, Yuta; UENO, Mizuki; KAWASAKI, Kurenai; ENDO, Takaho A.; SHIROGUCHI, Katsuyuki; FUJII, Wataru; NAITO, Kunihiko; SUGIURA, Koji

    2016-01-01

    This study tested the hypothesis that oocyte-derived paracrine factors (ODPFs) regulate miRNA expression in mouse granulosa cells. Expression of mmu-miR-322-5p (miR-322) was higher in mural granulosa cells (MGCs) than in cumulus cells of the Graafian follicles. The expression levels of miR-322 decreased when cumulus cells or MGCs were co-cultured with oocytes denuded of their cumulus cells. Inhibition of SMAD2/3 signaling by SB431542 increased miR-322 expression by cumulus-oocyte complexes (COCs). Moreover, the cumulus cells but not the MGCs in Bmp15–/–/Gdf9+/– (double-mutant) mice exhibited higher miR-322 expression than those of wild-type mice. Taken together, these results show that ODPFs suppress the expression of miR-322 in cumulus cells. Gene ontology analysis of putative miR-322 targets whose expression was detected in MGCs with RNA-sequencing suggested that multiple biological processes are affected by miR-322 in MGCs. These results demonstrate that ODPFs regulate miRNA expression in granulosa cells and that this regulation may participate in the differential control of cumulus cell versus MGC functions. Therefore, the ODPF-mediated regulation of cumulus cells takes place at both transcriptional and post-transcriptional levels. PMID:27180925

  18. Mouse fertility is enhanced by oocyte-specific loss of core 1-derived O-glycans

    PubMed Central

    Williams, Suzannah A; Stanley, Pamela

    2010-01-01

    Regulation of the number of eggs ovulated by different mammalian species remains poorly understood. Here we show that oocyte-specific deletion at the primary follicle stage of core 1 β1,3galactosyltransferase (T-synthase; generates core 1-derived O-glycans), leads to a sustained increase in fertility. T-syn mutant females ovulated 30–50% more eggs and had a sustained increase in litter size compared to controls. Ovarian weights and follicle numbers were greater in mutants but follicular apoptosis was not decreased. The number of follicles entering the growing pool was unaltered and 3 week mutants ovulated less eggs suggesting that increased fertility results from prolonged follicle development. T-syn mutant ovaries also contained numerous multiple-oocyte follicles (MOFs) that appeared to form by adjacent, predominantly preantral, follicles joining - a new mechanism for MOF generation. Ovulation of multiple eggs from MOFs was not the reason for increased fertility based on ovulated egg and corpora lutea numbers. Thus the absence of T-synthase caused modified follicular development leading to the maturation and ovulation of more follicles, to MOF formation at late stages of folliculogenesis, and to increased fertility. These results identify novel roles for glycoprotein(s) from the oocyte as suppressor(s) of fertility and regulator(s) of follicular integrity in the mouse. PMID:18276833

  19. Nicotinamide Impairs Entry into and Exit from Meiosis I in Mouse Oocytes

    PubMed Central

    Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden

    2015-01-01

    Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and

  20. Regulation of Inositol 1,4,5-Trisphosphate Receptor Function During Mouse Oocyte Maturation

    PubMed Central

    Wakai, Takuya; Vanderheyden, Veerle; Yoon, Sook-Young; Cheon, Banyoon; Zhang, Nan; Parys, Jan B.; Fissore, Rafael A.

    2011-01-01

    At the time of fertilization, an increase in the intracellular Ca2+ concentration ([Ca2+]i) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP3R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca2+ release. The sensitivity of IP3R1, i.e. its Ca2+ releasing capability, is increased during oocyte maturation so that the optimum [Ca2+]i response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP3R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization and ER Ca2+ concentration ([Ca2+]ER). Here we evaluated using mouse oocytes how each of these factors affected IP3R1 sensitivity. The capacity for IP3-induced Ca2+ release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP3R1 sensitivity was underpinned by an increase in [Ca2+]ER and receptor phosphorylation(s) but not by changes in IP3R1 cellular distribution, as inhibition of the former factors reduced Ca2+ release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca2+]ER and IP3R1 phosphorylation during maturation enhance IP3R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP3R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca2+ homeostasis also shape the pattern of oscillations in mammalian eggs. PMID:21465476

  1. C-type natriuretic peptide improved vitrified-warmed mouse cumulus oocyte complexes developmental competence.

    PubMed

    Yang, Lei; Wei, Qiang; Li, Wei; Ge, Junbang; Zhao, Xiaoe; Ma, Baohua

    2016-04-01

    Cryopreservation of cumulus oocyte complexes (COCs) is important for reproductive medicine. However, the vitrified-warmed COCs have lower maturation rate and subsequent developmental competence compared with fresh COCs. The present study was aimed to evaluate the effects of supplementation of the maturation medium with C-type natriuretic peptide (CNP) on the developmental competence of vitrified-warmed mouse COCs. Addition of CNP to the maturation medium improved the maturation rate and enhanced the developmental competence of vitrified-warmed mouse COCs. The reason may be that vitrified COCs led to a decline in cyclic guanosine monophosphate (cGMP) levels. Furthermore, addition of CNP to the maturation medium elevated cGMP levels of the vitrified-warmed COCs. In conclusion, cryopreservation-associated lower maturation rate and developmental competence of COCs may be ameliorated by CNP during maturation culture after warming. PMID:26921772

  2. Ejaculated Mouse Sperm Enter Cumulus-Oocyte Complexes More Efficiently In Vitro than Epididymal Sperm

    PubMed Central

    Suarez, Susan S.

    2015-01-01

    The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca2+ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca2+ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo. PMID:25996155

  3. APCFZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

    PubMed Central

    Holt, Janet E.; Lane, Simon I. R.; Jennings, Phoebe; García-Higuera, Irene; Moreno, Sergio; Jones, Keith T.

    2012-01-01

    FZR1 is an anaphase-promoting complex (APC) activator best known for its role in the mitotic cell cycle at M-phase exit, in G1, and in maintaining genome integrity. Previous studies also established that it prevents meiotic resumption, equivalent to the G2/M transition. Here we report that mouse oocytes lacking FZR1 undergo passage through meiosis I that is accelerated by ∼1 h, and this is due to an earlier onset of spindle assembly checkpoint (SAC) satisfaction and APCCDC20 activity. However, loss of FZR1 did not compromise SAC functionality; instead, earlier SAC satisfaction was achieved because the bipolar meiotic spindle was assembled more quickly in the absence of FZR1. This novel regulation of spindle assembly by FZR1 led to premature bivalent attachment to microtubules and loss of kinetochore-bound MAD2. Bivalents, however, were observed to congress poorly, leading to nondisjunction rates of 25%. We conclude that in mouse oocytes FZR1 controls the timing of assembly of the bipolar spindle and in so doing the timing of SAC satisfaction and APCCDC20 activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction. PMID:22918942

  4. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    PubMed

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming. PMID:27472658

  5. Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

    PubMed

    Park, Yong Seok; You, Seung Yeop; Cho, Sungrae; Jeon, Hyuk-Joon; Lee, Sukchan; Cho, Dong-Hyung; Kim, Jae-Sung; Oh, Jeong Su

    2016-09-01

    The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes. PMID:27160095

  6. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes

    PubMed Central

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S.; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming. PMID:27472658

  7. WASH complex regulates Arp2/3 complex for actin-based polar body extrusion in mouse oocytes

    PubMed Central

    Wang, Fei; Zhang, Liang; Zhang, Guang-Li; Wang, Zhen-Bo; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-01-01

    Prior to their fertilization, oocytes undergo asymmetric division, which is regulated by actin filaments. Recently, WASH complex were identified as actin nucleation promoting factors (NPF) that activated Arp2/3 complex. However, the roles of WASH complex remain uncertain, particularly for oocyte polarization and asymmetric division. Here, we examined the functions of two important subunits of a WASH complex, WASH1 and Strumpellin, during mouse oocyte meiosis. Depleting WASH1 or disrupting Strumpellin activity by WASH1 morpholino (MO) injection or Strumpellin antibody injection decreased polar body extrusion and caused oocyte symmetric division, and this may have been due to spindle formation and migration defects. Time lapse microscopy showed that actin filaments distribution and relative amount at the membrane and in the cytoplasm of oocytes was significantly decreased after disrupting WASH complex. In addition, Arp2/3 complex expression was reduced after WASH1 depletion. Thus, our data indicated that WASH complex regulated Arp2/3 complex and were required for cytokinesis and following polar body extrusion during mouse oocyte meiotic maturation. PMID:24998208

  8. Relocalization of STIM1 in mouse oocytes at fertilization: early involvement of store-operated calcium entry.

    PubMed

    Gómez-Fernández, Carolina; Pozo-Guisado, Eulalia; Gañán-Parra, Miguel; Perianes, Mario J; Alvarez, Ignacio S; Martín-Romero, Francisco Javier

    2009-08-01

    Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization. PMID:19470709

  9. Reversible Disassembly of the Actin Cytoskeleton Improves the Survival Rate and Developmental Competence of Cryopreserved Mouse Oocytes

    PubMed Central

    Hosu, Basarab G.; Mullen, Steven F.; Critser, John K.; Forgacs, Gabor

    2008-01-01

    Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method. PMID:18665248

  10. VEGF and FGF2 Improve Revascularization, Survival, and Oocyte Quality of Cryopreserved, Subcutaneously-Transplanted Mouse Ovarian Tissues.

    PubMed

    Li, Sheng-Hsiang; Hwu, Yuh-Ming; Lu, Chung-Hao; Chang, Hsiao-Ho; Hsieh, Cheng-En; Lee, Robert Kuo-Kuang

    2016-01-01

    This study was conducted to investigate the effect of the vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) on revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue. Autologous subcutaneous transplantation of vitrified-thawed mouse ovarian tissues treated with (experimental group) or without (control group) VEGF and FGF2 was performed. After transplantation to the inguinal region for two or three weeks, graft survival, angiogenesis, follicle development, and oocyte quality were examined after gonadotropin administration. VEGF coupled with FGF2 (VEGF/FGF2) promoted revascularization and significantly increased the survival rate of subcutaneously-transplanted cryopreserved ovarian tissues compared with untreated controls. The two growth factors did not show long-term effects on the ovarian grafts. In contrast to the untreated ovarian grafts, active folliculogenesis was revealed as the number of follicles at various stages and of mature oocytes in antral follicles after gonadotropin administration were remarkably higher in the VEGF/FGF2-treated groups. Although the fertilization rate was similar between the VEGF/FGF2 and control groups, the oocyte quality was much better in the VEGF/FGF2-treated grafts as demonstrated by the higher ratio of blastocyst development. Introducing angiogenic factors, such as VEGF and FGF2, may be a promising strategy to improve revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue. PMID:27483256

  11. VEGF and FGF2 Improve Revascularization, Survival, and Oocyte Quality of Cryopreserved, Subcutaneously-Transplanted Mouse Ovarian Tissues

    PubMed Central

    Li, Sheng-Hsiang; Hwu, Yuh-Ming; Lu, Chung-Hao; Chang, Hsiao-Ho; Hsieh, Cheng-En; Lee, Robert Kuo-Kuang

    2016-01-01

    This study was conducted to investigate the effect of the vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) on revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue. Autologous subcutaneous transplantation of vitrified-thawed mouse ovarian tissues treated with (experimental group) or without (control group) VEGF and FGF2 was performed. After transplantation to the inguinal region for two or three weeks, graft survival, angiogenesis, follicle development, and oocyte quality were examined after gonadotropin administration. VEGF coupled with FGF2 (VEGF/FGF2) promoted revascularization and significantly increased the survival rate of subcutaneously-transplanted cryopreserved ovarian tissues compared with untreated controls. The two growth factors did not show long-term effects on the ovarian grafts. In contrast to the untreated ovarian grafts, active folliculogenesis was revealed as the number of follicles at various stages and of mature oocytes in antral follicles after gonadotropin administration were remarkably higher in the VEGF/FGF2-treated groups. Although the fertilization rate was similar between the VEGF/FGF2 and control groups, the oocyte quality was much better in the VEGF/FGF2-treated grafts as demonstrated by the higher ratio of blastocyst development. Introducing angiogenic factors, such as VEGF and FGF2, may be a promising strategy to improve revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue. PMID:27483256

  12. Effect of Rat Medicated Serum Containing You Gui Wan on Mouse Oocyte In Vitro Maturation and Subsequent Fertilization Competence

    PubMed Central

    Jiang, Xiao-Hui; Deng, Yan-li; Lu, Hua; Duan, Heng; Zhen, Xia; Hu, Xiang; Liang, Xin

    2014-01-01

    You Gui Wan (YGW) is a classic herbal formula in traditional Chinese medicine (TCM) used for the clinical treatment of infertility. This study was to explore whether YGW has an impact on mouse oocyte maturation in vitro and subsequent fertilization competence. Rat medicated serum containing YGW was prepared by orally administrating YGW. Mouse immature oocytes were cultured with YGW medicated serum and compared to those cultured with or without normal rat serum or follicle-stimulating hormone (FSH). YGW medicated serum significantly increased the percentages of matured oocytes when compared to the groups with or without normal rat serum (P < 0.01). Furthermore, YGW medicated serum increased the rate of in vitro fertilization (IVF) when compared to the groups treated with FSH and with or without normal rat serum (P < 0.001). YGW medicated serum also had significant effects on the mRNA expressions of PKA, CREB, MAPK, PKC, PKG, and MPF and the concentrations of cAMP, cGMP, and NO in matured oocytes. These results indicate that YGW can promote mouse oocyte maturation and IVF in vitro. Signaling pathways, such as the cAMP/PKA/MAPK, the PKC-MAPK, and the NO-cGMP-PKG pathway, which are similar to those induced by FSH, may be responsible for this action. PMID:25530775

  13. Structural and functional measurements of fertilized mouse oocytes with combined high-resolution OCT and inverted microscope (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Karnowski, Karol; Ajduk, Anna; Wojtkowski, Maciej; Szkulmowski, Maciej

    2016-03-01

    We present a comprehensive imaging methodology for 3D structural and functional measurements of fertilized mouse oocytes. In contrary to methods used for mouse zygote imaging so far OCT provides 3D data without z axis movement of sample or objective lens. Furthermore, complex scanning protocols used in this study give access to different scales of repetition times and thus may become a tool for investigation of a different dynamic processes. Additionally, proposed scanning approach via variety of statistic operations can be used to enhance the quality of structural images. OCT system capabilities are presented and compared to standard microscopy. With a single 3D measurements one can extract 3D structure of the oocytes as well as en-face images that correspond to both bright and dark field microscopy. As an example of dynamic oocyte imaging pronuclei motion during development is presented. Limitations and possibilities of the new system are discussed.

  14. Immunogenicity of killed Bordetella bronchiseptica vaccines in the mouse.

    PubMed

    Smith, I M; Baskerville, A J; Brothwell, E; Oliphant, J

    1982-03-01

    Two intramuscular injections (two weeks apart of graded doses of killed strains of Bordetella bronchiseptica from the pig (OLN 14 or LBF 1) or the dog (D1) had produced in mice circulating agglutinins ranging in mean titre (log2) per group from about 3.3 to 10.2 two weeks later. These levels depended partly on vaccinal strains and dose, and partly on the strain used as agglutinogen. Other such mice were challenged intraperitoneally with about 50 LD50 (approximately or equal to 10(7.4) viable bacteria) of two pig strains, one (293) from a British case of atrophic rhinitis and the other (N) from an American herd. Against challenge vaccinal strain OLN 14 was about 10 and LBF 1 about 100 times more immunogenic than vaccinal strain D1. In a separate experiment mice given intramuscularly amounts of LBF 1 or D1 vaccine estimated as being immunogenically equivalent were challenged intraperitoneally with one or other of seven pig or seven dog strains. On aggregate each vaccine protected to about the same extent against challenge by the pig strains, although LBF 1 vaccine was less effective than D1 vaccine against a strain of Danish origin. Both vaccines also protected more mice against challenge by the dog than the pig strans but LBF 1 vaccine was somewhat less effective than D1 vaccine, especially when challenged by strain D1. PMID:7079606

  15. Kid depletion in mouse oocytes associated with multinucleated blastomere formation and inferior embryo development.

    PubMed

    Egashira, Akiyoshi; Yamauchi, Nobuhiko; Islam, Md Rashedul; Yamagami, Kazuki; Tanaka, Asami; Suyama, Hikaru; El-Sayed, El-Sharawy Mohamed; Tabata, Shoji; Kuramoto, Takashi

    2016-08-01

    This study investigated the knockdown (KD) of Kid on maturation developmental competence and multinucleation of mouse germinal vesicle (GV) oocytes after parthenogenetic activation. Data revealed that Kid messenger RNA (mRNA) was expressed in GV and MII stage oocyte and 1- and 2-cell embryos. Additionally, Kid mRNA expression in the Kid KD group decreased by nearly 46% compared to the control small interfering RNA (siRNA) groups. The rate of multinucleated embryos in the Kid KD group (52.4%) was significantly higher (P < 0.05) than the control siRNA group (4.7%). Finally, the developmental rates were significantly lower in the Kid siRNA group at > 4-cell stage (28.6% vs. 53.5%) and the blastocyst stage (2.4% vs. 23.3%) compared to the control siRNA groups. Suppression of Kid using siRNA caused multinucleation in early embryos with high frequency and it may increase 2- to 4-cell arrested embryos and reduce the developmental competence to blastocyst. PMID:26890962

  16. Estrogen and bisphenol A disrupt spontaneous [Ca(2+)](i) oscillations in mouse oocytes.

    PubMed

    Mohri, Tatsuma; Yoshida, Shigeru

    2005-01-01

    The present work aims to study the effects of estrogen or endocrine disrupters (EDs) on the dynamic changes in intracellular Ca(2+) concentration of mouse immature oocytes (IOs) loaded with Ca(2+)-sensitive dye Fura-2 using an image analyzer. The majority of IOs isolated from the ovary exhibited spontaneous Ca(2+) oscillations at regular intervals. Entry of external Ca(2+), probably through gap junctions, contributes to Ca(2+) oscillations since they were reversibly inhibited by removing Ca(2+) from the bathing medium or by the application of a gap-junction inhibitor carbenoxolone (CBX, 30 microM). Both 17beta-estradiol (E2) and E2-BSA, a membrane impermeable estrogen, shortened the duration of Ca(2+) oscillations in a dose-dependent manner (1-1000 nM), and produced an irregular pattern of the oscillations, strongly suggesting that E2 acts on the plasma membrane of the oocyte. For bisphenol A (BPA), one of the estrogen-mimicking EDs, a 10,000-fold higher concentration (100 microM) was necessary to exert similar inhibitory action to that of E2. PMID:15567167

  17. Maternal Diet-Induced Obesity Alters Mitochondrial Activity and Redox Status in Mouse Oocytes and Zygotes

    PubMed Central

    Igosheva, Natalia; Abramov, Andrey Y.; Poston, Lucilla; Eckert, Judith J.; Fleming, Tom P.; Duchen, Michael R.; McConnell, Josie

    2010-01-01

    The negative impact of obesity on reproductive success is well documented but the stages at which development of the conceptus is compromised and the mechanisms responsible for the developmental failure still remain unclear. Recent findings suggest that mitochondria may be a contributing factor. However to date no studies have directly addressed the consequences of maternal obesity on mitochondria in early embryogenesis. Using an established murine model of maternal diet induced obesity and a live cell dynamic fluorescence imaging techniques coupled with molecular biology we have investigated the underlying mechanisms of obesity-induced reduced fertility. Our study is the first to show that maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and zygotes. Specifically, maternal diet-induced obesity in mice led to an increase in mitochondrial potential, mitochondrial DNA content and biogenesis. Generation of reactive oxygen species (ROS) was raised while glutathione was depleted and the redox state became more oxidised, suggestive of oxidative stress. These altered mitochondrial properties were associated with significant developmental impairment as shown by the increased number of obese mothers who failed to support blastocyst formation compared to lean dams. We propose that compromised oocyte and early embryo mitochondrial metabolism, resulting from excessive nutrient exposure prior to and during conception, may underlie poor reproductive outcomes frequently reported in obese women. PMID:20404917

  18. Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development

    PubMed Central

    2013-01-01

    Background The adverse effects of maternal diabetes on oocyte maturation and embryo development have been reported. Methods In this study, we used time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of diabetes on the ER’s structural dynamics during oocyte maturation, fertilization and early embryo development. Results We report that the ER first became remodeled into a dense ring around the developing MI spindle, and then surrounded the spindle during migration to the cortex. ER reorganization during mouse early embryo development was characterized by striking localization around the pronuclei in the equatorial section, in addition to larger areas of fluorescence deeper within the cytoplasm. In contrast, in diabetic mice, the ER displayed a significantly higher percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. In addition, a higher frequency of large ER aggregations was detected in GV oocytes and two cell embryos from diabetic mice. Conclusions These results suggest that the diabetic condition adversely affects the ER distribution pattern during mouse oocyte maturation and early embryo development. PMID:23597066

  19. Acute fasting decreases the expression of GLUT1 and glucose utilisation involved in mouse oocyte maturation and cumulus cell expansion.

    PubMed

    Han, Yingying; Yan, Jun; Zhou, Jinlian; Teng, Zhen; Bian, Fenghua; Guo, Meng; Mao, Guankun; Li, Junxia; Wang, Jianwei; Zhang, Meijia; Xia, Guoliang

    2012-01-01

    Acute fasting impairs meiotic resumption and glucose consumption in mouse cumulus cell and oocyte complexes (COCs). This study examines the effects of acute fasting on the regulation of glucose transporter 1 (GLUT1) expression and glucose consumption in oocyte maturation. Our results indicate that the restriction of glucose utilisation by 2-deoxyglucose (2-DG) mimicked the inhibitory effects of acute fasting on oocyte meiotic resumption and cumulus cell expansion, effects that were rescued by high glucose concentrations in the culture medium. GLUT1 protein levels were higher in cumulus cells compared with oocytes, and GLUT1 expression in COCs increased with FSH treatment in vitro. However, under acute fasting conditions, GLUT1 expression in COCs decreased and the response to FSH disappeared. Exposure to high glucose conditions (27.5mM and 55mM), significantly increased both glucose consumption and GLUT1 levels in COCs. Inhibition of GLUT1 function using an anti-GLUT1 antibody significantly inhibited FSH-induced oocyte meiotic resumption. Taken together, these results suggest that acute fasting decreases GLUT1 expression and glucose utilisation, inhibiting the processes of oocyte maturation and cumulus cell expansion. PMID:22697123

  20. Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

    SciTech Connect

    Bornslaeger, E.A.; Poueymirou, W.T.; Mattei, P.; Schultz, R.M.

    1986-01-01

    Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, oocytes were treated with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4..beta..-phorbol, 12,13-didecanoate (4..beta..-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC/sub 8/). An inactive phorbol ester, 4a-phorbol 12,13-didecanoate (4..cap alpha..-PDD), did not inhibit GVBD. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of a cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC/sub 8/ partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis.

  1. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.

    PubMed

    Tam, Oliver H; Aravin, Alexei A; Stein, Paula; Girard, Angelique; Murchison, Elizabeth P; Cheloufi, Sihem; Hodges, Emily; Anger, Martin; Sachidanandam, Ravi; Schultz, Richard M; Hannon, Gregory J

    2008-05-22

    Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. PMID:18404147

  2. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes

    PubMed Central

    Tam, Oliver H.; Aravin, Alexei A.; Stein, Paula; Girard, Angelique; Murchison, Elizabeth P.; Cheloufi, Sihem; Hodges, Emily; Anger, Martin; Sachidanandam, Ravi; Schultz, Richard M.; Hannon, Gregory J.

    2010-01-01

    Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways1. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. PMID:18404147

  3. Method of Euthanasia Influences the Oocyte Fertilization Rate with Fresh Mouse Sperm

    PubMed Central

    Hazzard, Karen C; Watkins-Chow, Dawn E; Garrett, Lisa J

    2014-01-01

    In vitro fertilization (IVF) is used to produce mouse embryos for a variety of reasons. We evaluated the effect of the method of euthanasia on the fertilization rate in 2 different IVF protocols. Oocytes collected from C57BL/6J female mice euthanized by CO2 inhalation or cervical dislocation were used in IVF with fresh sperm from either wild-type or genetically engineered C57BL/6J. Compared with CO2 inhalation, cervical dislocation improved the resulting rate of fertilization by 18% in an IVF method using Cook media and by 13% in an IVF method using methyl-B cyclodextrin and reduced glutathione. The lower fertilization rate due to euthanasia by CO2 inhalation was accompanied by changes in blood pH and body temperature despite efforts to minimize temperature drops. In our hands, euthanasia by cervical dislocation improved fertilization rates and consequently reduced the number of egg-donor mice required. PMID:25650969

  4. Effects of Simulated Weightlessness on Mammalian Development. Part 2: Meiotic Maturation of Mouse Oocytes During Clinostat Rotation

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1985-01-01

    In order to understand the role of gravity in basic cellular processes that are important during development, the effects of a simulated microgravity environment on mammalian gametes and early embryos cultured in vitro are examined. A microgravity environment is simulated by use of a clinostat, which essentially reorients cells relative to the gravity vector. Initial studies have focused on assessing the effects of clinostat rotation on the meiotic progression of mouse oocytes. Modifications centered on providing the unique in vitro culture of the clinostat requirements of mammalian oocytes and embryos: 37 C temperature, constant humidity, and a 5% CO2 in air environment. The oocytes are observed under the dissecting microscope for polar body formation and gross morphological appearance. They are then processed for cytogenetic analysis.

  5. Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation

    PubMed Central

    Mokhber Maleki, Elham; Eimani, Hussein; Bigdeli, Mohammad Reza; Golkar Narenji, Afsane; Abedi, Reyhane

    2016-01-01

    Background Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 μg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. Results Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse

  6. Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1, 2-propanediol.

    PubMed

    Li, Dun-Gao; Zhu, Yan; Xing, Feng-Ying; Li, Shan-Gang; Chen, Xue-Jin; Jiang, Man-Xi

    2013-01-01

    The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes. PMID:23594385

  7. The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model

    PubMed Central

    Lin, Juan; Chen, Fei; Sun, Ming-Ju; Zhu, Jiang; Li, You-Wei; Pan, Liu-Zhu; Zhang, Jie; Tan, Jing-He

    2016-01-01

    The epigenetic factors causing competence differences between SN (surrounded nucleolus) and NSN (non-surrounded nucleolus) oocytes, the significance for the increased histone acetylation and methylation in SN oocytes, and whether chromatin configuration or histone modification determines oocyte competence, are unclear. This study has addressed these issues by using the ovary-holding (OH) stress models where oocyte SN configuration was uncoupled from histone modifications and developmental potential. Prepubertal mouse ovaries containing high percentages of NSN oocytes were preserved at 37 or 39 °C for 1 or 2 h before examination for oocyte chromatin configuration, developmental competence, histone modification and apoptosis. Whereas 1-h OH at 37 °C caused a moderate apoptosis with increased oocyte competence, improved histone modification and a normal NSN-to-SN transition, harsher OH conditions induced a severe apoptosis with decreased oocyte competence, impaired histone modification and a pseudo (premature) NSN-to-SN transition. Observations on Fas/FasL expression and using the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations indicated that OH triggered oocyte apoptosis with activation of the Fas signaling. It was concluded that OH stress caused oocyte apoptosis with activation of the Fas/FasL system and that oocyte competence was more closely correlated with histone modification than with chromatin configuration. PMID:27321442

  8. Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes

    PubMed Central

    2013-01-01

    Background The cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts. Results Global gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR. Conclusions The developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte. PMID:23758669

  9. Closed vitrification of mouse oocytes using the CryoLogic vitrification method: A modification that improves developmental competence

    PubMed Central

    Jo, Jun Woo; Jee, Byung Chul; Kim, Seok Hyun

    2013-01-01

    Objective To compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested. Methods Mature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed. Results The modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts. Conclusion Closed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated. PMID:24505560

  10. Effects of 5α-Dihydrotestosterone and 17β-Estradiol on the Mouse Ovarian Follicle Development and Oocyte Maturation

    PubMed Central

    Tarumi, Wataru; Itoh, Masanori T.; Suzuki, Nao

    2014-01-01

    We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17β-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, 5α-dihydrotestosterone (DHT), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10−6 M) of DHT prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10−6 M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not DHT, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation. PMID:24911314

  11. Selective Disruption of Aurora C Kinase Reveals Distinct Functions from Aurora B Kinase during Meiosis in Mouse Oocytes

    PubMed Central

    Balboula, Ahmed Z.; Schindler, Karen

    2014-01-01

    Aurora B kinase (AURKB) is the catalytic subunit of the chromosomal passenger complex (CPC), an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT) attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I). We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction. PMID:24586209

  12. Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.

    PubMed

    Woods, Stephanie E; Qi, Peimin; Rosalia, Elizabeth; Chavarria, Tony; Discua, Allan; Mkandawire, John; Fox, James G; García, Alexis

    2014-01-01

    The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

  13. Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups

    PubMed Central

    Woods, Stephanie E.; Qi, Peimin; Rosalia, Elizabeth; Chavarria, Tony; Discua, Allan; Mkandawire, John; Fox, James G.; García, Alexis

    2014-01-01

    The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen

  14. Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

    PubMed Central

    Yang, Shang-Wu; Gao, Chen; Chen, Lei; Song, Ya-Li; Zhu, Jin-Liang; Qi, Shu-Tao; Jiang, Zong-Zhe; Wang, Zhong-Wei; Lin, Fei; Huang, Hao; Xing, Fu-Qi; Sun, Qing-Yuan

    2012-01-01

    Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage. PMID:23159858

  15. Inhibition of the Binding between RGS2 and β-Tubulin Interferes with Spindle Formation and Chromosome Segregation during Mouse Oocyte Maturation In Vitro

    PubMed Central

    Sun, Zhao-Gui; Zhang, Zhi; Zhu, Yan

    2016-01-01

    RGS2 is a negative regulator of G protein signaling that contains a GTPase-activating domain and a β-tubulin binding region. This study aimed to determine the localization and function of RGS2 during mouse oocyte maturation in vitro. Immunofluorescent staining revealed that RGS2 was widely expressed in the cytoplasm with a greater abundance on both meiotic spindles and first/second polar bodies from the fully-grown germinal vesicle (GV) stage to the MII stages. Co-expression of RGS2 and β-tubulin could also be detected in the spindle and polar body of mouse oocytes at the MI, AI, and MII stages. Inhibition of the binding site between RGS2 and β-tubulin was accomplished by injecting anti-RGS2 antibody into GV-stage oocytes, which could result in oocytes arrest at the MI or AI stage during in vitro maturation, but it did not affect germinal vesicle breakdown. Moreover, injecting anti-RGS2 antibody into oocytes resulted in a significant reduction in the rate of first polar body extrusion and abnormal spindle formation. Additionally, levels of phosphorylated MEK1/2 were significantly reduced in anti-RGS2 antibody injected oocytes compared with control oocytes. These findings suggest that RGS2 might play a critical role in mouse oocyte meiotic maturation by affecting β-tubulin polymerization and chromosome segregation. PMID:27463806

  16. Isolation of apoptotic mouse fetal oocytes by AnnexinV assay.

    PubMed

    Lobascio, Anna-Maria; Klinger, Francesca-Gioia; De Felici, Massimo

    2007-01-01

    Expression of phosphotidylserine by fetal oocytes in culture renders significant numbers of such cells able to bind AnnexinV-coated microbeads and allows their separation from Annexin V-negative oocytes on a Magnetic Cell Separation (MACS) column in a magnetic field. The majority of oocytes (> or =75%) which bound Annexin V-coated microbeads were viable, as indicated by their propidium iodine (PI) negativity. However, they showed apoptotic morphologies and were found to be TUNEL-positive. On the other hand, AnnexinV-negative oocytes, besides being PI negative, appeared morphologically healthy and TUNEL negative. Moreover, AnnexinV-positive oocytes showed a marked lower ratio of Bcl-xL/Bax transcripts in comparison to AnnexinV-negative oocytes. We conclude that the present method is able to separate fetal oocytes in two distinct populations: AnnexinV-positive oocytes showing features typical of apoptotic cells and AnnexinV-negative oocytes comprising for the most part viable non-apoptotic cells. This procedure should greatly facilitate studies aimed to identify the currently poorly understood molecular pathways governing apoptosis in mammalian fetal oocytes. PMID:17294366

  17. TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis.

    PubMed

    Jeon, Hyuk-Joon; You, Seung Yeop; Park, Yong Seok; Chang, Jong Wook; Kim, Jae-Sung; Oh, Jeong Su

    2016-04-01

    Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes. PMID:26802898

  18. Mouse Oocyte Control of Granulosa Cell Development and Function: Paracrine Regulation of Cumulus Cell Metabolism

    PubMed Central

    Su, You-Qiang; Sugiura, Koji; Eppig, John J.

    2009-01-01

    Bi-directional communication between oocytes and the companion granulosa cells is essential for the development and functions of both compartments. Oocytes are deficient in their ability to transport certain amino acids and in carrying out glycolysis and cholesterol biosynthesis, and require that cumulus cells provide them with the specific amino acids and the products in these metabolic pathways. Oocytes control metabolic activities in cumulus cells by promoting the expression of genes in cumulus cells encoding specific amino acid transporters and enzymes essential for the oocyte-deficient metabolic processes. Hence, oocytes outsource metabolic functions to cumulus cells to compensate for oocyte metabolic deficiencies. Oocyte control of granulosa cell metabolism may also participate in regulating the rate of follicular development in coordination with endocrine, paracrine and autocrine signals. Oocytes influence granulosa cell development mainly by secretion of paracrine factors although juxtacrine signals probably also participate. Key oocyte-derived paracine factors include growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) 15, and fibroblast growth factor 8B (FGF8B). PMID:19197803

  19. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3 complex-driven cytoplasmic streaming in mouse oocytes

    PubMed Central

    Yi, Kexi; Unruh, Jay R.; Deng, Manqi; Slaughter, Brian D.; Rubinstein, Boris; Li, Rong

    2012-01-01

    Mature mammalian oocytes are poised for the completion of second polar body extrusion upon fertilization by positioning the metaphase spindle in close proximity to an actomyosin-rich cortical cap. Loss of this spindle position asymmetry is often associated with poor oocyte quality and infertility 1–3. Here, we report a novel role for the Arp2/3 actin nucleation complex in the maintenance of asymmetric spindle position in mature mouse oocytes. The Arp2/3 complex localizes to the cortical cap in a Ran GTPase-dependent manner and accounts for the nucleation of the majority of actin filaments in both the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 complex activity or localization leads to rapid dissociation of the spindle from the cortex. High resolution live imaging and spatiotemporal image correlation spectroscopy (STICS) analysis reveal that in normal oocytes actin filaments flow continuously away from the Arp2/3-rich cortex, generating a cytoplamic streaming that results in a net pushing force on the spindle toward the actomyosin cap. Arp2/3 inhibition not only diminishes this actin flow and cytoplamic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, leading to spindle movement away from the cortex. We conclude that the Arp2/3 complex maintains asymmetric meiotic spindle position by generating an actin polymerization-driven cytoplamic streaming and by suppressing a counteracting force from myosin-II-based contractility. PMID:21874009

  20. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos.

    PubMed

    Yang, Qiyuan; Lin, Jimin; Liu, Miao; Li, Ronghong; Tian, Bin; Zhang, Xue; Xu, Beiying; Liu, Mofang; Zhang, Xuan; Li, Yiping; Shi, Huijuan; Wu, Ligang

    2016-06-01

    Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3' mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos. PMID:27500274

  1. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos

    PubMed Central

    Yang, Qiyuan; Lin, Jimin; Liu, Miao; Li, Ronghong; Tian, Bin; Zhang, Xue; Xu, Beiying; Liu, Mofang; Zhang, Xuan; Li, Yiping; Shi, Huijuan; Wu, Ligang

    2016-01-01

    Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3′ mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos. PMID:27500274

  2. 1,2-propanediol-induced premature centromere separation in mouse oocytes and aneuploidy in one-cell zygotes.

    PubMed

    Mailhes, J B; Young, D; London, S N

    1997-07-01

    Aneuploidy in germ cells results in reproductive failure and mental and physical disorders in humans. Unfortunately, little is known about the causes and mechanisms of aneuploidy induction. The objective of this study was to test the hypothesis that propylene glycol (1,2-propanediol; PG) induces cytogenetic aberrations in mouse metaphase II (MII) oocytes that predispose zygotes to aneuploidy. Female ICR mice received 7.5 IU eCG and 5.0 IU hCG 48 h later. PG doses of 1300, 2600, and 5200 mg/kg body weight were given 3 h post-hCG; controls received the solvent deionized water. Ovulated oocytes were collected 16 h after administration of PG and processed for cytogenetic analysis. For the one-cell zygote cytogenetic study, females were given PG and paired (1:1) with ICR males for 16 h. Females that mated were given 2 x 10(-3) M colchicine 22 h post-PG, and zygotes were collected 18 h later. PG significantly (p < 0.05) increased both the proportion of MII oocytes with premature centromere separation (PCS) and the proportion of aneuploid one-cell zygotes. These results support the hypothesis that PG-induced PCS in MII oocytes predisposes zygotes to aneuploidy. PMID:9209085

  3. Meiosis-activating sterol promotes resumption of meiosis in mouse oocytes cultured in vitro in contrast to related oxysterols.

    PubMed

    Grøndahl, C; Ottesen, J L; Lessl, M; Faarup, P; Murray, A; Grønvald, F C; Hegele-Hartung, C; Ahnfelt-Rønne, I

    1998-05-01

    The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in

  4. FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence.

    PubMed

    Franciosi, Federica; Manandhar, Shila; Conti, Marco

    2016-02-01

    A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3' untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Pten(fl/fl):Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality. PMID:26653334

  5. Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1

    PubMed Central

    Kryzak, Cassie A.; Moraine, Maia M.; Kyle, Diane D.; Lee, Hyo J.; Cubeñas-Potts, Caelin; Robinson, Douglas N.; Evans, Janice P.

    2013-01-01

    ABSTRACT Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm. PMID:23863404

  6. Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

    PubMed Central

    Tatone, Carla; Carbone, Maria Cristina

    2006-01-01

    Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the

  7. Application of oocyte cryopreservation technology in TALEN-mediated mouse genome editing.

    PubMed

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Nakagata, Naomi; Yamasaki, Sho; Takeda, Naoki; Ohmuraya, Masaki; Yamamoto, Takashi

    2014-01-01

    Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8-12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95-100% when either 5-week-old or 8-12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies. PMID:25077765

  8. Leptin and ObRa/MEK signalling in mouse oocyte maturation and preimplantation embryo development.

    PubMed

    Ye, Yinghui; Kawamura, Kazuhiro; Sasaki, Mitsue; Kawamura, Nanami; Groenen, Peter; Sollewijn Gelpke, Maarten D; Kumagai, Jin; Fukuda, Jun; Tanaka, Toshinobu

    2009-08-01

    Recent studies indicate that LH stimulates production of ovarian paracrine factors that induce meiosis of the oocyte. DNA microarray analyses of ovarian transcripts were performed in mice and major increases of a short isoform of leptin receptor, ObRa, were identified by the preovulatory LH/human chorionic gonadotrophin (HCG) surge. In oocytes, the level of ObRa transcripts was increased shortly after HCG stimulation, whereas the level of ObRb transcripts was not changed. Leptin was produced by cumulus, granulosa, theca and interstitial cells of ovaries and its transcript level was not regulated during gonadotrophin treatment. Treatment with leptin promoted germinal vesicle breakdown (GVBD) in oocytes within preovulatory follicles, and enhance first polar body extrusion in both cumulus-oocyte complexes and denuded oocytes. The leptin-promoted GVBD and first polar body extrusion were blocked by a mitogen-activated protein kinase extracellular signal regulated kinase kinases (MEK)1/2 inhibitor, U0126, but not its inactive analogue U0124. Furthermore, leptin promoted fertilization of oocytes and the in-vitro development of zygotes to preimplantation embryos. These findings suggest paracrine roles of leptin in the enhancement of nuclear maturation of oocytes through MEK1/2 signalling, and in the promotion of cytoplasmic maturation essential for successful oocyte development to the preimplantation embryos. PMID:19712552

  9. Sirt3 prevents maternal obesity-associated oxidative stress and meiotic defects in mouse oocytes

    PubMed Central

    Zhang, Liang; Han, Longsen; Ma, Rujun; Hou, Xiaojing; Yu, Yang; Sun, Shaochen; Xu, Yinxue; Schedl, Tim; Moley, Kelle H; Wang, Qiang

    2015-01-01

    Maternal obese environment has been reported to induce oxidative stress and meiotic defects in oocytes, however the underlying molecular mechanism remains unclear. Here, using mice fed a high fat diet (HFD) as an obesity model, we first detected enhanced reactive oxygen species (ROS) content and reduced Sirt3 expression in HFD oocytes. We further observed that specific depletion of Sirt3 in control oocytes elevates ROS levels while Sirt3 overexpression attenuates ROS production in HFD oocytes, with significant suppression of spindle disorganization and chromosome misalignment phenotypes that have been reported in the obesity model. Candidate screening revealed that the acetylation status of lysine 68 on superoxide dismutase (SOD2K68) is dependent on Sirt3 deacetylase activity in oocytes, and acetylation-mimetic mutant SOD2K68Q results in almost threefold increase in intracellular ROS. Moreover, we found that acetylation levels of SOD2K68 are increased by ∼80% in HFD oocytes and importantly, that the non-acetylatable-mimetic mutant SOD2K68R is capable of partially rescuing their deficient phenotypes. Together, our data identify Sirt3 as an important player in modulating ROS homeostasis during oocyte development, and indicate that Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress and meiotic defects in oocytes under maternal obese conditions. PMID:25790176

  10. Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation.

    PubMed

    Sriraman, Kalpana; Bhartiya, Deepa; Anand, Sandhya; Bhutda, Smita

    2015-07-01

    This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin-/CD45-, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy. PMID:25779995

  11. Proteomic Analysis of Mouse Oocytes Identifies PRMT7 as a Reprogramming Factor that Replaces SOX2 in the Induction of Pluripotent Stem Cells.

    PubMed

    Wang, Bingyuan; Pfeiffer, Martin J; Drexler, Hannes C A; Fuellen, Georg; Boiani, Michele

    2016-08-01

    The reprogramming process that leads to induced pluripotent stem cells (iPSCs) may benefit from adding oocyte factors to Yamanaka's reprogramming cocktail (OCT4, SOX2, KLF4, with or without MYC; OSK(M)). We previously searched for such facilitators of reprogramming (the reprogrammome) by applying label-free LC-MS/MS analysis to mouse oocytes, producing a catalog of 28 candidates that are (i) able to robustly access the cell nucleus and (ii) shared between mature mouse oocytes and pluripotent embryonic stem cells. In the present study, we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature oocytes, (iv) depleted after the oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors. Using LC-MS/MS and isotopic labeling methods, we found that the abundance profiles of the 28 proteins were below those of known oocyte-specific and housekeeping proteins. Of the 28 proteins, only arginine methyltransferase 7 (PRMT7) changed substantially during mouse embryogenesis and promoted the conversion of mouse fibroblasts into iPSCs. Specifically, PRMT7 replaced SOX2 in a factor-substitution assay, yielding iPSCs. These findings exemplify how proteomics can be used to prioritize the functional analysis of reprogrammome candidates. The LC-MS/MS data are available via ProteomeXchange with identifier PXD003093. PMID:27225728

  12. Age-Dependent Susceptibility of Chromosome Cohesion to Premature Separase Activation in Mouse Oocytes1

    PubMed Central

    Chiang, Teresa; Schultz, Richard M.; Lampson, Michael A.

    2011-01-01

    ABSTRACT A hypothesis to explain the maternal age-dependent increase in formation of aneuploid eggs is deterioration of chromosome cohesion. Although several lines of evidence are consistent with this hypothesis, whether cohesion is actually reduced in naturally aged oocytes has not been directly tested by any experimental perturbation. To directly target cohesion, we increased the activity of separase, the protease that cleaves the meiotic cohesin REC8, in oocytes. We show that cohesion is more susceptible to premature separase activation in old oocytes than in young oocytes, demonstrating that cohesion is significantly reduced. Furthermore, cohesion is protected by two independent mechanisms that inhibit separase, securin and an inhibitory phosphorylation of separase by CDK1; both mechanisms must be disrupted to prematurely activate separase. With the continual loss of cohesins from chromosomes that occurs throughout the natural reproductive lifespan, tight regulation of separase in oocytes may be particularly important to maintain cohesion and prevent aneuploidy. PMID:21865557

  13. Shugoshin1 May Play Important Roles in Separation of Homologous Chromosomes and Sister Chromatids during Mouse Oocyte Meiosis

    PubMed Central

    Yin, Shen; Ai, Jun-Shu; Shi, Li-Hong; Wei, Liang; Yuan, Ju; Ouyang, Ying-Chun; Hou, Yi; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2008-01-01

    Background Homologous chromosomes separate in meiosis I and sister chromatids separate in meiosis II, generating haploid gametes. To address the question why sister chromatids do not separate in meiosis I, we explored the roles of Shogoshin1 (Sgo1) in chromosome separation during oocyte meiosis. Methodology/Principal Findings Sgo1 function was evaluated by exogenous overexpression to enhance its roles and RNAi to suppress its roles during two meioses of mouse oocytes. Immunocytochemistry and chromosome spread were used to evaluate phenotypes. The exogenous Sgo1 overexpression kept homologous chromosomes and sister chromatids not to separate in meiosis I and meiosis II, respectively, while the Sgo1 RNAi promoted premature separation of sister chromatids. Conclusions Our results reveal that prevention of premature separation of sister chromatids in meiosis I requires the retention of centromeric Sgo1, while normal separation of sister chromatids in meiosis II requires loss of centromeric Sgo1. PMID:18949044

  14. Quantitative Microinjection of Morpholino Antisense Oligonucleotides into Mouse Oocytes to Examine Gene Function in Meiosis-I.

    PubMed

    Nakagawa, Shoma; FitzHarris, Greg

    2016-01-01

    Specific protein depletion is a powerful approach for assessing individual gene function in cellular processes, and has been extensively employed in recent years in mammalian oocyte meiosis-I. Conditional knockout mice and RNA interference (RNAi) methods such as siRNA or dsRNA microinjection are among several approaches to have been applied in this system over the past decade. RNAi by microinjection of Morpholino antisense Oligonucleotides (MO), in particular, has proven highly popular and tractable in many studies, since MOs have high specificity of interaction, low cell toxicity, and are more stable than other microinjected RNAi molecules. Here, we describe a method of MO microinjection into the mouse germinal vesicle-stage (GV) oocyte followed by a simple immunofluorescence approach for examination of gene function in meiosis-I. PMID:27557584

  15. Altered hepatic clearance and killing of Candida albicans in the isolated perfused mouse liver model.

    PubMed Central

    Sawyer, R T; Horst, M N; Garner, R E; Hudson, J; Jenkins, P R; Richardson, A L

    1990-01-01

    The adherence of Candida albicans was studied in situ by using the perfused mouse liver model. After exhaustive washing, 10(6) C. albicans were infused into mouse livers. At the time of recovery, 62 +/- 5% (mean +/- standard error of the mean) of the infused C. albicans were recovered from the liver and 14 +/- 3% were recovered from the effluent for a total recovery of 76 +/- 4%. This indicates that 86 +/- 3% of the original inoculum was trapped by the liver and that 24 +/- 4% was killed within the liver. Chemical pretreatment of C. albicans with 8 M urea, 12 mM dithiothreitol, 2% beta-mercaptoethanol, 1% sodium dodecyl sulfate, 10% Triton X-100, or 3 M potassium chloride or enzyme pretreatment with alpha-mannosidase, alpha-chymotrypsin, subtilisin, beta-N-acetyl-glucosaminidase, pronase, trypsin, papain, or lipase did not alter adherence of C. albicans to hepatic tissue. By contrast, pepsin pretreatment significantly decreased hepatic trapping. Simultaneous perfusion with either 100 mg of C. albicans glycoprotein per liter or 100 mg of C. albicans mannan per liter also decreased trapping. Furthermore, both substances eluted previously trapped C. albicans from hepatic tissue. Chemical pretreatment with 8 M urea, 12 mM dithiothreitol, or 3 M KCI or enzymatic pretreatment with alpha-mannosidase, subtilisin, alpha-chymotrypsin, or papain increased killing of C. albicans three- to fivefold within hepatic tissue. The data suggest that mannose-containing structures on the surface of C. albicans, for example. mannans or glucomannoproteins, mediate adherence of C. albicans within the liver. Indirectly, chemical and enzymatic pretreatment renders C. albicans more susceptible to hepatic killing. PMID:2117571

  16. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes.

    PubMed

    Larman, Mark G; Sheehan, Courtney B; Gardner, David K

    2006-01-01

    Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique. PMID:16388009

  17. The Src Homology 2 Domain-Containing Adapter Protein B (SHB) Regulates Mouse Oocyte Maturation

    PubMed Central

    Calounova, Gabriela; Livera, Gabriel; Zhang, Xiao-Qun; Liu, Kui; Gosden, Roger G.; Welsh, Michael

    2010-01-01

    SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb −/− mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/− and −/− mice revealed impaired early embryo development in the −/− embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development. PMID:20585392

  18. Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis.

    PubMed

    Hoffmann, Steffen; Tsurumi, Chizuko; Kubiak, Jacek Z; Polanski, Zbigniew

    2006-04-01

    We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material. PMID:16490186

  19. Thioglycolic acid inhibits mouse oocyte maturation and affects chromosomal arrangement and spindle configuration.

    PubMed

    Hou, S Y; Zhang, L; Wu, K; Xia, L

    2008-05-01

    Previous studies have shown that thioglycolic acid (TGA) leads to potential reproductive toxicology. To clarify the exact effects of this compound on reproduction, mice oocytes were treated with different TGA doses. At the end of the culture period, the nuclear status of mice oocytes was assessed under an inverted microscope. After immunofluorescence staining, the chromosomal arrangement and spindle configuration of oocytes were evaluated. The results indicated that TGA decreases the percentage of first polar body formation but does not influence that of germinal vesicle breakdown. TGA induces abnormal chromosomal arrangement and spindle elongation. In conclusion, TGA inhibits in-vitro maturation of mice oocytes and affects chromosomal arrangement and spindle configuration. Furthermore, it probably interferes with biochemical changes that occur during meiosis, resulting in aberrant development. PMID:19022875

  20. Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes

    PubMed Central

    Nguyen, Alexandra L.; Gentilello, Amanda S.; Balboula, Ahmed Z.; Shrivastava, Vibha; Ohring, Jacob; Schindler, Karen

    2014-01-01

    ABSTRACT Meiosis I (MI), the division that generates haploids, is prone to errors that lead to aneuploidy in females. Haspin is a kinase that phosphorylates histone H3 on threonine 3, thereby recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an AURKB homolog, are enriched in germ cells, yet their significance in regulating MI is not fully understood. Using inhibitors and overexpression approaches, we show a role for haspin during MI in mouse oocytes. Haspin-perturbed oocytes display abnormalities in chromosome morphology and alignment, improper kinetochore–microtubule attachments at metaphase I and aneuploidy at metaphase II. Unlike in mitosis, kinetochore localization remained intact, whereas the distribution of the CPC along chromosomes was absent. The meiotic defects following haspin inhibition were similar to those observed in oocytes where AURKC was inhibited, suggesting that the correction of microtubule attachments during MI requires AURKC along chromosome arms rather than at kinetochores. Our data implicate haspin as a regulator of the CPC and chromosome segregation during MI, while highlighting important differences in how chromosome segregation is regulated between MI and mitosis. PMID:25315835

  1. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling✧

    PubMed Central

    Seki, Shinsuke; Mazur, Peter

    2009-01-01

    Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40°C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [Cryobiol. 55 (2007) 158] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25°C, holding them 10 min, rapidly cooling them to −70°C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56°C during warming and was complete by −46°C. The present study differs in that the oocytes were warmed rapidly from −70°C to temperatures between −65°C and −50°C and held for 3 to 60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (Ea) for the blackening process; namely, 27.5 kcal/mole. This translates to about a quadrupling of the blackening rate for every 5° rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000°C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8°C. These findings have significant implications both for cryobiology and cryo-electron microscopy. PMID:18359013

  2. Perturbing microtubule integrity blocks AMP-activated protein kinase-induced meiotic resumption in cultured mouse oocytes.

    PubMed

    Ya, Ru; Downs, Stephen M

    2014-02-01

    The oocyte meiotic spindle is comprised of microtubules (MT) that bind chromatin and regulate both metaphase plate formation and karyokinesis during meiotic maturation; however, little information is known about their role in meiosis reinitiation. This study was conducted to determine if microtubule integrity is required for meiotic induction and to ascertain how it affects activation of AMP-activated protein kinase (AMPK), an important participant in the meiotic induction process. Treatment with microtubule-disrupting agents nocodazole and vinblastine suppressed meiotic resumption in a dose-dependent manner in both arrested cumulus cell-enclosed oocytes (CEO) stimulated with follicle-stimulating hormone (FSH) and arrested denuded oocytes (DO) stimulated with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). This effect coincided with suppression of AMPK activation as determined by western blotting and germinal vesicle immunostaining. Treatment with the MT stabilizer paclitaxel also suppressed meiotic induction. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Immunolocalization experiments revealed that active AMPK colocalized with γ-tubulin during metaphase I and II stages, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole led to disruption of proper spindle pole localization of active AMPK, while paclitaxel induced excessive polymerization of spindle MT and formation of ectopic asters with accentuated AMPK colocalization. Although stimulation of AMPK increased the rate of germinal vesicle breakdown (GVB), spindle formation and polar body (PB) extrusion, the kinase had no effect on peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation. PMID:23199370

  3. Stability of mouse oocytes at -80 °C: the role of the recrystallization of intracellular ice.

    PubMed

    Seki, Shinsuke; Mazur, Peter

    2011-04-01

    The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities worldwide. Their transport to and from such facilities should be easy and inexpensive with dry ice at -79 °C. The purpose of our study was to determine the stability of mouse oocytes with time at that temperature. The metaphase II oocytes were cryopreserved with a vitrification solution (EAFS10/10) developed by M Kasai and colleagues. Two procedures were followed. In one, the samples were cooled at 187 °C/min to -196 °C, warmed to -80 °C, held at -80 °C for 1 h to 3 months, and warmed to 25 °C at one of three rates. With the highest warming rate (2950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next 2 months. With the slowest warming (139 °C/min), survival was only ∼ 5% even at 0 time at -80 °C. In the second procedure, the samples were cooled at 294 °C/min to -80 °C (without cooling to -196 °C) and held for up to 3 months before warming at 2950 °C/min. Survival was ∼ 90% after 7 days and dropped slowly to 35% after 3 months. We believe that small non-lethal quantities of intracellular ice formed during the cooling and that the intracellular crystals increased to a damaging size by recrystallization during the 3 month's storage at -80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes worldwide in dry ice. PMID:21239524

  4. Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

    PubMed

    Meredith, Megan; MacNeil, Allison H; Trasler, Jacquetta M; Baltz, Jay M

    2016-06-01

    The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis. PMID:27122634

  5. Acrylamide toxic effects on mouse oocyte quality and fertility in vivo

    PubMed Central

    Duan, Xing; Wang, Qiao-Chu; Chen, Kun-Lin; Zhu, Cheng-Cheng; Liu, Jun; Sun, Shao-Chen

    2015-01-01

    Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications. PMID:26108138

  6. SLX2 interacting with BLOS2 is differentially expressed during mouse oocyte meiotic maturation.

    PubMed

    Zhuang, Xin-Jie; Shi, Yu-Qiang; Xu, Bo; Chen, Lei; Tang, Wen-Hao; Huang, Jin; Lian, Ying; Liu, Ping; Qiao, Jie

    2014-01-01

    Gametogenesis is a complex biological process of producing cells for sexual reproduction. Xlr super family members containing a conserved COR1 domain play essential roles in gametogenesis. In the present study, we identified that Slx2, a novel member of Xlr super family, is specifically expressed in the meiotic oocytes, which is demonstrated by western blotting and immunohistochemistry studies. In the first meiotic prophase, SLX2 is unevenly distributed in the nuclei of oocytes, during which phase SLX2 is partly co-localized with SYCP3 in synaptonemal complex and γH2AX in the nucleus of oocytes. Interestingly, the localization of SLX2 was found to be switched into the cytoplasm of oocytes after prometaphase I during oocyte maturation. Furthermore, yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLX2 interacts with BLOS2, which is a novel centrosome-associated protein, and co-localized with γ-Tubulin, which is a protein marker of chromosome segregation in meiosis. These results indicated that SLX2 might get involved in chromosomes segregation during meiosis by interaction with BLOS2. In conclusion, SLX2 might be a novel gametogenesis-related protein that could play multiple roles in regulation of meiotic processes including synaptonemal complex assembly and chromosome segregation. PMID:24870619

  7. Acrylamide toxic effects on mouse oocyte quality and fertility in vivo.

    PubMed

    Duan, Xing; Wang, Qiao-Chu; Chen, Kun-Lin; Zhu, Cheng-Cheng; Liu, Jun; Sun, Shao-Chen

    2015-01-01

    Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications. PMID:26108138

  8. ROLE OF AMPK THOUGHOUT MEIOTIC MATURATION IN THE MOUSE OOCYTE: EVIDENCE FOR PROMOTION OF POLAR BODY FORMATION AND SUPPRESSION OF PREMATURE ACTIVATION

    PubMed Central

    Downs, Stephen M.; Ya, Ru; Davis, Christopher

    2014-01-01

    This study was conducted to assess the role of AMPK in regulating meiosis in mouse oocytes from the germinal vesicle stage to metaphase II. Exposure of mouse cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) during spontaneous maturation in vitro to AMPK-activating agents resulted in augmentation of the rate and frequency of polar body formation. Inhibitors of AMPK had an opposite, inhibitory effect. In addition, the AMPK inhibitor, compound C (Cmpd C) increased the frequency of oocyte activation. The stimulatory action of the AMPK-activating agent, AICAR, and the inhibitory action of Cmpd C were diminished if exposure was delayed, indicating an early action of AMPK on polar body formation. The frequency of spontaneous and Cmpd C-induced activation in CEO was reduced as the period of hormonal priming was increased, and AMPK stimulation eliminated the activation response. Immunostaining of oocytes with antibody to active AMPK revealed an association of active kinase with chromatin, spindle poles and midbody during maturation. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle, but not in the spindle poles or midbody; α2 stained only diffusely throughout the oocyte. These data suggest that AMPK is involved in a regulatory capacity throughout maturation and helps promote the completion of meiosis while suppressing premature activation. PMID:20830737

  9. HDAC1 and HDAC2 in mouse oocytes and preimplantation embryos: Specificity versus compensation.

    PubMed

    Ma, P; Schultz, R M

    2016-07-01

    Oocyte and preimplantation embryo development entail dynamic changes in chromatin structure and gene expression, which are regulated by a number of maternal and zygotic epigenetic factors. Histone deacetylases (HDACs), which tighten chromatin structure, repress transcription and gene expression by removing acetyl groups from histone or non-histone proteins. HDAC1 and HDAC2 are two highly homologous Class I HDACs and display compensatory or specific roles in different cell types or in response to different stimuli and signaling pathways. We summarize here the current knowledge about the functions of HDAC1 and HDAC2 in regulating histone modifications, transcription, DNA methylation, chromosome segregation, and cell cycle during oocyte and preimplantation embryo development. What emerges from these studies is that although HDAC1 and HDAC2 are highly homologous, HDAC2 is more critical than HDAC1 for oocyte development and reciprocally, HDAC1 is more critical than HDAC2 for preimplantation development. PMID:27082454

  10. Embryonic development after exposure of mouse oocyte to various amount of ovarian endometriotic fluid

    PubMed Central

    Kim, Hashin; Jeong, Mina; Kim, Seul Ki

    2016-01-01

    This study assesses the fertilization and blastocyst-forming rate in mice cumulus-oocyte complexes (COCs) after the exposure of human ovarian endometriotic fluid. Endometriotic fluid was obtained from a single patient by aspiration at the time of a laparoscopic cystectomy and serially diluted. COCs were obtained from 46-week-old female BDF1 mice. After exposure to ovarian endometriotic fluid for five minutes, the COCs were washed three times and the oocytes were then fertilized by mice sperm. The fertilization and blastocyst formation rate and the proportion of hatching/hatched blastocyst in the four treatment groups were not inferior to those in non-exposure group. PMID:27462598

  11. MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation.

    PubMed

    Luo, Yi-Bo; Ma, Jun-Yu; Zhang, Qing-Hua; Lin, Fei; Wang, Zhong-Wei; Huang, Lin; Schatten, Heide; Sun, Qing-Yuan

    2013-04-01

    H4K20me1 is a critical histone lysine methyl modification in eukaryotes. It is recognized and "read" by various histone lysine methyl modification binding proteins. In this study, the function of MBTD1, a member of the Polycomb protein family containing four MBT domains, was comprehensively studied in mouse oocyte meiotic maturation. The results showed that depletion of MBTD1 caused reduced expression of histone lysine methyl transferase Pr-Set7 and H4K20me1 as well as increased oocyte arrest at the GV stage. Increased γH2AX foci were formed, and DNA damage repair checkpoint protein 53BP1 was downregulated. Furthermore, depletion of MBTD1 activated the cell cycle checkpoint protein Chk1 and downregulated the expression of cyclin B1 and cdc2. MBTD1 knockdown also affected chromosome configuration in GV stage oocytes and chromosome alignment at the MII stage. All these phenotypes were reproduced when the H4K20 methyl transferase Pr-Set7 was depleted. Co-IP demonstrated that MBTD1 was correlated with Pr-Set7 in mouse oocytes. Our results demonstrate that MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation. PMID:23475131

  12. Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse.

    PubMed

    Jin, Bo; Kleinhans, F W; Mazur, Peter

    2014-06-01

    Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below -100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard. PMID:24662030

  13. Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse✰

    PubMed Central

    Jin, Bo; Kleinhans, F.W.; Mazur, Peter

    2014-01-01

    Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below − 100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard. PMID:24662030

  14. Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA.

    PubMed

    Shishova, Kseniya V; Lavrentyeva, Elena A; Dobrucki, Jurek W; Zatsepina, Olga V

    2015-01-15

    It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called "nucleolus-like bodies" (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA. PMID:25481757

  15. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes

    PubMed Central

    Xie, Hong-Li; Wang, Yan-Bo; Jiao, Guang-Zhong; Kong, De-Ling; Li, Qing; Li, Hong; Zheng, Liang-Liang; Tan, Jing-He

    2016-01-01

    Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation. PMID:26857840

  16. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes.

    PubMed

    Xie, Hong-Li; Wang, Yan-Bo; Jiao, Guang-Zhong; Kong, De-Ling; Li, Qing; Li, Hong; Zheng, Liang-Liang; Tan, Jing-He

    2016-01-01

    Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation. PMID:26857840

  17. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    PubMed

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. PMID:27317807

  18. A comparison of the multiple oocyte maturation gene expression patterns between the newborn and adult mouse ovary

    PubMed Central

    Bahmanpour, Soghra; Talaei Khozani, Tahereh; Zarei fard, Nehleh; Jaberipour, Mansoureh; Hosseini, Ahmah; Esmaeilpour, Tahereh

    2013-01-01

    Background: The interaction between follicular cells and oocyte leads to a change in gene expression involved in oocyte maturation processes. Objective: The purpose of this study was to quantify the expression of more common genes involved in follicular growth and oocyte developmental competence. Materials and Methods: In this experimental study, the expression of genes was evaluated with qRT-PCR assay in female BALB/c mice pups at 3-day of pre-pubertal and 8 week old virgin adult ovaries. The tissue was prepared by H&E staining for normal morphological appearance. The data were calculated with the 2-∆Ct formula and assessed using non-parametric two-tailed Mann-Whitney test. The p<0.05 was considered as significant. Results: The data showed a significant increase in the level of Stra8 and GDF9 in adult compared with newborn mice ovaries (p=0.049). In contrast, a significant decrease in the level of Mvh, REC8, SCP1, SCP3, and ZP2 was observed in adult mice ovaries compared to those in the newborn mice ovaries (all p=0.049 except SCP1: p=0.046). There was no significant difference in the level of OCT4 and Cx37 expression between adult and newborn mice ovaries. Conclusion: The modifications in gene expression patterns coordinate the follicular developmental processes. Furthermore, the findings showed higher expression level of premeiotic gene (Stra8) and lower level of meiotic entry markers (SCP1, SCP3, and REC8) in juvenile than newborn mouse ovaries. This article extracted from Ph.D. thesis. (Nehleh Zarei fard) PMID:24639702

  19. DEHP exposure impairs mouse oocyte cyst breakdown and primordial follicle assembly through estrogen receptor-dependent and independent mechanisms.

    PubMed

    Mu, Xinyi; Liao, Xinggui; Chen, Xuemei; Li, Yanli; Wang, Meirong; Shen, Cha; Zhang, Xue; Wang, Yingxiong; Liu, Xueqing; He, Junlin

    2015-11-15

    Estrogen plays an essential role in the development of mammalian oocytes, and recent studies suggest that it also regulates primordial follicle assembly in the neonatal ovaries. During the last decade, potential exposure of humans and animals to estrogen-like endocrine disrupting chemicals has become a growing concern. In the present study, we focused on the effect of diethylhexyl phthalate (DEHP), a widespread plasticizer with estrogen-like activity, on germ-cell cyst breakdown and primordial follicle assembly in the early ovarian development of mouse. Neonatal mice injected with DEHP displayed impaired cyst breakdown. Using ovary organ cultures, we revealed that impairment was mediated through estrogen receptors (ERs), as ICI 182,780, an efficient antagonist of ER, reversed this DEHP-mediated effect. DEHP exposure reduced the expression of ERβ, progesterone receptor (PR), and Notch2 signaling components. Finally, DEHP reduced proliferation of pregranulosa precursor cells during the process of primordial folliculogenesis. Together, our results indicate that DEHP influences oocyte cyst breakdown and primordial follicle formation through several mechanisms. Therefore, exposure to estrogen-like chemicals during fetal or neonatal development may adversely influence early ovarian development. PMID:26073378

  20. Temporary developmental arrest after storage of fertilized mouse oocytes at 4 degrees C: effects on embryonic development, maternal mRNA processing and cell cycle.

    PubMed

    Sakurai, Takayuki; Kimura, Minoru; Sato, Masahiro

    2005-05-01

    The aim of this study was to examine whether fertilized mouse oocytes can survive after short-term incubation (for 6-48 h) at 4 degrees C. When fertilized oocytes of ICR and C57BL/6 (B6) strain were incubated at 4 degrees C and returned to normal culture conditions (37 degrees C), development of these 4 degrees C-treated embryos for up to 12 h (for ICR) to blastocyst stage did not differ from that of untreated oocytes. Even 4 degrees C-treated embryos for 48 h developed to blastocysts at relatively good rates (33.3% for ICR and 50.8% for B6). The in vivo development of 4 degrees C-treated embryos for 12, 24 and 36 h to fetal stage was similar to that of untreated ones. BrdU labelling assay revealed temporary cessation of DNA replication in 4 degrees C-treated fertilized oocytes. Post-fertilization events including cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene and elevated mRNA expression of zygotic gene activation-related genes were temporarily suppressed in 4 degrees C-treated embryos. These findings indicate that 4 degrees C-treatment of fertilized murine oocytes results in temporary cessation of molecular events. We also show that 4 degrees C-treated fertilized oocytes for 12 h can be used for preparation of transgenic mice. PMID:15805144

  1. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

    PubMed Central

    Choi, T; Fukasawa, K; Zhou, R; Tessarollo, L; Borror, K; Resau, J; Vande Woude, G F

    1996-01-01

    Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692939

  2. [Influence of Acetylcysteine on Cytogenetic Effects of Etoposide in Mouse Oocytes].

    PubMed

    Pligina, K L; Zhanataev, A K; Kulakova, A V; Chaika, Z V; Durnev, A D

    2016-02-01

    The influence of N-acetylcysteine (ACC) on the cytogenetic effects of etoposide in F1 CBA x C57BL/6 mice was studied. Etoposide introduced intraperitoneally in doses of 10, 20, 40, and 60 mg/kg has a dose-dependent clastogenic activity and has an aneugenic effect with the induction of mainly hypohaploid oocytes. ACC significantly decreases the aneugenic and clastogenic activity of etoposide (20 mg/kg) in oocytes of 6-, 9-, and 12-week-old mice during triple introduction at a dose 200 mg/kg per os. The most pronounced anticlastogenic ACC activity (an 80% decrease) was registered in 9-week-old females; a 100% decrease in aneugenesis was detected in 6-week-old female mice. PMID:27215036

  3. DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint

    PubMed Central

    Collins, Josie K.; Lane, Simon I. R.; Merriman, Julie A.; Jones, Keith T.

    2015-01-01

    Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis. PMID:26522232

  4. DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint.

    PubMed

    Collins, Josie K; Lane, Simon I R; Merriman, Julie A; Jones, Keith T

    2015-01-01

    Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule-kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis. PMID:26522232

  5. Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.

    PubMed

    Corbett, Hannah E; Dubé, Chantal D; Slow, Sandy; Lever, Michael; Trasler, Jacquetta M; Baltz, Jay M

    2014-04-01

    Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization. PMID:24599290

  6. Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

    PubMed

    Morohaku, Kanako; Tanimoto, Ren; Sasaki, Keisuke; Kawahara-Miki, Ryouka; Kono, Tomohiro; Hayashi, Katsuhiko; Hirao, Yuji; Obata, Yayoi

    2016-08-01

    Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells. PMID:27457928

  7. Complete in vitro generation of fertile oocytes from mouse primordial germ cells

    PubMed Central

    Morohaku, Kanako; Tanimoto, Ren; Sasaki, Keisuke; Kawahara-Miki, Ryouka; Kono, Tomohiro; Hayashi, Katsuhiko; Hirao, Yuji; Obata, Yayoi

    2016-01-01

    Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells. PMID:27457928

  8. Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation.

    PubMed

    Seki, Shinsuke; Edashige, Keisuke; Wada, Sakiko; Mazur, Peter

    2011-10-01

    The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0  M ethylene glycol in PBS for 15  min, cooled in a Linkam cryostage to -7.0  ° C, induced to freeze externally, and finally cooled at 20  ° C/min to -70  ° C. IIF that occurred during the 20  ° C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were -34, -40, -35, and -25  ° C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10-15  ° C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded Aqp3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested. PMID:21734033

  9. Effect of lectins on hepatic clearance and killing of Candida albicans by the isolated perfused mouse liver.

    PubMed Central

    Sawyer, R T; Garner, R E; Hudson, J A

    1992-01-01

    The isolated perfused mouse liver model was used to study the effects of various lectins on hepatic trapping and killing of Candida albicans. After mouse livers were washed with 20 to 30 ml of perfusion buffer, 10(6) C. albicans CFU were infused into the livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicated that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. When included in both preperfusion and postperfusion buffers (0.2 mg of lectin per ml), Ulex europeaus lectin (binding specificity for fucose) decreased hepatic trapping of C. albicans by 37% and eluted trapped C. albicans from the liver only when included in postperfusion buffer. By comparison, treatment of C. albicans with U. europeaus lectin before infusion had no effect on the trapping or killing of yeast cells. When Lens culinaris lectin (binding specificity for mannose) was included in the perfusion buffers, hepatic killing of C. albicans increased by 16% with no significant effect on hepatic killing when yeast cells were treated with L. culinaris lectin before infusion. Forty to 55% of the infused C. albicans were killed when concanavalin A (binding specificities for mannose and glucose), Glycine max (binding specificity for N-acetylgalactosamine), or Arachis hypogea (binding specificity for galactose) lectin was included in the perfusion buffer or when yeast cells were treated with these lectins before their infusion. When C. albicans was treated with concanavalin A at a concentration of less than 0.02 mg/ml, hepatic killing of yeast cells was not significantly increased. The data suggest that a fucose-containing receptor on the surface of either sinusoidal endothelial cells or Kupffer cells is involved in the trapping of C. albicans by the perfused mouse

  10. Effects of simulated weightlessness on mammalian development. Part 1: Development of clinostat for mammalian tissue culture and use in studies on meiotic maturation of mouse oocytes

    NASA Technical Reports Server (NTRS)

    Wolegemuth, D. J.; Grills, G. S.

    1984-01-01

    The effects of weightlessness on three aspects of mammalian reproduction: oocyte development, fertilization, and early embryogenesis was studied. Zero-gravity conditions within the laboratory by construction of a clinostat designed to support in vitro tissue culture were simulated and the effects of simulated weightlessness on meiotic maturation in mammalian oocytes using mouse as the model system were studied. The timing and frequency of germinal vesicule breakdown and polar body extrusion, and the structural and numerical properties of meiotic chromosomes at Metaphase and Metaphase of meiosis are assessed.

  11. [Visualization of the age-related changes in expressions of DNA methyltransferases in mouse oocytes using two-photon imaging system].

    PubMed

    Tian, Ning; Zhang, Lu; Zheng, Jing-Hao; Li, Ying; Ma, Wan-Yun

    2012-12-01

    Quantum dot (QD) is widely used as fluorescent labeling dye for its strong anti-fluorescence quenching, high quantum yield, wide absorption spectra, and narrow emission spectra. In the present paper, QD 585-labeled DNA methyltransferases (Dnmts) and Hoechst 33342-labeled chromosomes were imaged simultaneously using two-photon imaging system. The authors' results showed that aging mouse oocytes may be not suitable for in-vitro maturation: both the localizations and expression levels of Dnmts in in-vitro matured oocytes of aging mice were changed, and these changes may be related to the abnormal DNA methylation modification. PMID:23427529

  12. A requirement for fatty acid oxidation in the hormone-induced meiotic maturation of mouse oocytes.

    PubMed

    Valsangkar, Deepa; Downs, Stephen M

    2013-08-01

    We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with (3)H-palmitic acid as the substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into the mitochondria. The carnitine palmitoyltransferase 1 activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor, and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated human chorionic gonadotropin (hCG)-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO. PMID:23863407

  13. Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

    SciTech Connect

    Ganusov, Vitaly V

    2009-01-01

    In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targets with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the

  14. The Effects of cAMP-elevating Agents and Alpha Lipoic Acid on In Vitro Maturation of Mouse Germinal Vesicle Oocytes

    PubMed Central

    Rahnama, Ali; Zavareh, Saeed; Ghorbanian, Mohammad Taghi; Karimi, Isaac

    2013-01-01

    Background In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid (ALA) on in vitro oocyte maturation and fertilization. Methods Mouse germinal vesicle (GV) oocytes were categorized into cumulus denuded oocytes (DOs; n=896) and cumulus oocyte complexes (COCs; n=1077) groups. GV oocytes were matured in vitro with or without ALA; (I) without the meiotic inhibitors; (II) supplemented with cilostamide; (III) supplemented with forskolin and (IV) supplemented with Forskolin plus cilostamide. The obtained metaphase II (MII) oocytes were subjected to in vitro fertilization. Independent-samples t-testand ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. Results The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. Conclusion cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner. PMID:24551571

  15. Fertilization of C57BL/6 mouse sperm collected from cauda epididymides after preservation or transportation at 4 degrees C using laser-microdissected oocytes.

    PubMed

    Kaneko, Takehito; Fukumoto, Kiyoko; Haruguchi, Yukie; Kondo, Tomoko; Machida, Hiromi; Koga, Mika; Nakagawa, Yoshiko; Tsuchiyama, Shuuji; Saiki, Kiyora; Noshiba, Shiho; Nakagata, Naomi

    2009-08-01

    The C57BL/6 mouse is commonly used to produce transgenic and knockout strains for biomedical research. However, the motility and fertility of its sperm decrease markedly with freezing. Short-term preservation of sperm without freezing can avoid this. Furthermore, such samples can be transported safety without the special skills or equipment needed for the transportation of live animals or frozen products. We evaluated the motility and fertility of sperm collected from cauda epididymides after preservation or transportation at 4 degrees C. Oocytes with the zona pellucida subjected to laser-microdissection were used to assist fertilization in vitro. Although the motility of sperm gradually decreased with storage (P<0.05), no disruption of the sperm plasma membrane was seen. The proportion of zona-intact oocytes fertilized with sperm preserved for 0, 24, 48 and 72h were 70, 14, 5 and 1%, respectively. On the other hand, 45, 20 and 14% of laser-microdissected oocytes were fertilized by sperm preserved for 24, 48 and 72h, respectively (P<0.05). The fertility of sperm collected from cauda epididymides of two transgenic strains after transportation at 4 degrees C were also significantly increased using laser-microdissected oocytes rather than zona-intact oocytes (57 and 68% vs. 5%, P<0.05). Efficient production of offspring from sperm preserved or transported at 4 degrees C was achieved using laser-microdissected oocytes. Thus the fertility of sperm preserved or transported at 4 degrees C could be maintained, although motility gradually decreased with storage. Laser-microdissected oocytes will contribute to the efficient production of embryos and offspring using such preserved sperm samples. PMID:19394323

  16. The behavior of the X- and Y-chromosomes in the oocyte during meiotic prophase in the B6.Y(TIR)sex-reversed mouse ovary.

    PubMed

    Alton, Michelle; Lau, Mau Pan; Villemure, Michele; Taketo, Teruko

    2008-02-01

    Sexual differentiation of the germ cells follows gonadal differentiation, which is determined by the presence or the absence of the Y-chromosome. Consequently, oogenesis and spermatogenesis take place in the germ cells with XX and XY sex chromosomal compositions respectively. It is unclear how sexual dimorphic regulation of meiosis is associated with the sex-chromosomal composition. In the present study, we examined the behavior of the sex chromosomes in the oocytes of the B6.Y(TIR) sex-reversed female mouse, in comparison with XO and XX females. As the sex chromosomes fail to pair in both XY and XO oocytes during meiotic prophase, we anticipated that the pairing failure may lead to excessive oocyte loss. However, the total number of germ cells, identified by immunolabeling of germ cell nuclear antigen 1 (GCNA1), did not differ between XY and XX ovaries or XO and XX ovaries up to the day of delivery. The progression of meiotic prophase, assessed by immunolabeling of synaptonemal complex components, was also similar between the two genotypes of ovaries. These observations suggest that the failure in sex-chromosome pairing is not sufficient to cause oocyte loss. On the other hand, labeling of phosphorylated histone gammaH2AX, known to be associated with asynapsis and transcriptional repression, was seen over the X-chromosome but not over the Y-chromosome in the majority of XY oocytes at the pachytene stage. For comparison, gammaH2AX labeling was seen only in the minority of XX oocytes at the same stage. We speculate that the transcriptional activity of sex chromosomes in the XY oocyte may be incompatible with ooplasmic maturation. PMID:18239052

  17. Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogencially activated and fertilized embryos.

    PubMed

    Martin, Jacinta H; Bromfield, Elizabeth G; Aitken, R John; Lord, Tessa; Nixon, Brett

    2016-09-01

    These data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogencially activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article "Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes" (Martin et al., in press) [1]. PMID:27547800

  18. Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro.

    PubMed

    Leonardsen, L; Strömstedt, M; Jacobsen, D; Kristensen, K S; Baltsen, M; Andersen, C Y; Byskov, A G

    2000-01-01

    Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in

  19. Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution.

    PubMed

    Seki, Shinsuke; Mazur, Peter

    2012-01-01

    Intracellular ice is generally lethal. One way to avoid it is to vitrify cells; that is, to convert cell water to a glass rather than to ice. The belief has been that this requires both the cooling rate and the concentration of glass-inducing solutes be very high. But high solute concentrations can themselves be damaging. However, the findings we report here on the vitrification of mouse oocytes are not in accord with the first belief that cooling needs to be extremely rapid. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad range of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000°C/min) even when the prior cooling rate was as low as 880°C/min. Functional survival was >81% and 54% with the highest warming rate after cooling at 69,000 and 880°C/min, respectively. Our findings are also contrary to the second belief. We show that a high percentage of mouse oocytes survive vitrification in media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; that is, >100,000°C/min. Again, the cooling rate is of less consequence. PMID:22558325

  20. Ultra-Rapid Warming Yields High Survival of Mouse Oocytes Cooled to −196°C in Dilutions of a Standard Vitrification Solution

    PubMed Central

    Seki, Shinsuke; Mazur, Peter

    2012-01-01

    Intracellular ice is generally lethal. One way to avoid it is to vitrify cells; that is, to convert cell water to a glass rather than to ice. The belief has been that this requires both the cooling rate and the concentration of glass-inducing solutes be very high. But high solute concentrations can themselves be damaging. However, the findings we report here on the vitrification of mouse oocytes are not in accord with the first belief that cooling needs to be extremely rapid. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad range of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000°C/min) even when the prior cooling rate was as low as 880°C/min. Functional survival was >81% and 54% with the highest warming rate after cooling at 69,000 and 880°C/min, respectively. Our findings are also contrary to the second belief. We show that a high percentage of mouse oocytes survive vitrification in media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; that is, >100,000°C/min. Again, the cooling rate is of less consequence. PMID:22558325

  1. Oocyte-Specific Expression of Mouse MEX3C652AA in the Ovary and Its Potential Role in Regulating Maternal Fos mRNA.

    PubMed

    Li, Xue; Li, Yan; Liu, Chunlian; Jin, Mulan; Lu, Baisong

    2016-05-01

    Currently, the human MEX3C gene is known to encode an RNA-binding protein of 659 amino acid residues. Here we show that the MEX3C gene has alternative splicing forms giving rise to multiple MEX3C variants, and some cells express MEX3C transcripts coding for short MEX3C isoforms but not transcripts for MEX3C(659AA) MEX3C(659AA) functions as an adaptor protein for Exportin 1 (XPO1)-mediated nuclear export since it increases the cytoplasmic distribution of poly(A)(+) RNA and since addition of the nuclear export signal (NES) sequence to a short MEX3C isoform MEX3C(464AA) confers similar cytoplasmic poly(A)(+) RNA accumulation activity as MEX3C(659AA) FOS mRNA is a potential MEX3C target mRNA. One mechanism by which MEX3C(659AA) could regulate FOS mRNA is by promoting its nuclear export. Overexpressing MEX3C(659AA) significantly increased FOS mRNA expression, whereas mutating the NES of MEX3C(659AA) and treating cells with leptomycin B to inhibit XPO1-mediated nuclear export attenuated FOS upregulation. FOS mRNA is unstable in somatic cells but less so in oocytes; how it is stabilized in the oocytes is unknown. Transcripts for the mouse counterpart of human MEX3C(659AA) (MEX3C(652AA)) are specifically expressed in developing oocytes in the ovary, although total Mex3c transcripts are expressed in both granulosa cells and oocytes. The specific expression of this long MEX3C isoform in oocytes and its ability to enhance FOS mRNA nuclear export and stability all suggest that MEX3C(659AA) is an RNA-binding protein that preserves maternal FOS mRNA in oocytes. PMID:27053362

  2. Oocyte-expressed yes-associated protein is a key activator of the early zygotic genome in mouse

    PubMed Central

    Yu, Chao; Ji, Shu-Yan; Dang, Yu-Jiao; Sha, Qian-Qian; Yuan, Yi-Feng; Zhou, Jian-Jie; Yan, Li-Ying; Qiao, Jie; Tang, Fuchou; Fan, Heng-Yu

    2016-01-01

    In early mammalian embryos, the genome is transcriptionally quiescent until the zygotic genome activation (ZGA) which occurs 2-3 days after fertilization. Despite a long-standing effort, maternal transcription factors regulating this crucial developmental event remain largely elusive. Here, using maternal and paternal mouse models of Yap1 deletion, we show that maternally accumulated yes-associated protein (YAP) in oocyte is essential for ZGA. Maternal Yap1-knockout embryos exhibit a prolonged two-cell stage and develop into the four-cell stage at a much slower pace than the wild-type controls. Transcriptome analyses identify YAP target genes in early blastomeres; two of which, Rpl13 and Rrm2, are required to mediate maternal YAP's effect in conferring developmental competence on preimplantation embryos. Furthermore, the physiological YAP activator, lysophosphatidic acid, can substantially improve early development of wild-type, but not maternal Yap1-knockout embryos in both oviduct and culture. These observations provide insights into the mechanisms of ZGA, and suggest potentials of YAP activators in improving the developmental competence of cultured embryos in assisted human reproduction and animal biotechnology. PMID:26902285

  3. High survival of mouse oocytes/embryos after vitrification without permeating cryoprotectants followed by ultra-rapid warming with an IR laser pulse.

    PubMed

    Jin, Bo; Mazur, Peter

    2015-01-01

    Vitrification is now the main route to the cryopreservation of human and animal oocytes and preimplantation embryos. A central belief is that for success, the cells must be placed in very high concentrations of cryoprotective solutes and must be cooled extremely rapidly. We have shown recently that these beliefs are incorrect. Over 90% of mouse oocytes and embryos survive being cooled relatively slowly even in solutions containing only 1/3(rd) the normal solute concentrations, provided that they are warmed ultra-rapidly at 10(7)°C/min by a laser pulse. Nearly all vitrification solutions contain both permeating and non-permeating solutes, and an important question is whether the former protect because they permeate the cells and promote intracellular vitrification (as is almost universally believed), or because they osmotically withdraw a large fraction of intracellular water prior to cooling. The answer for the mouse system is clearly the latter. When oocytes or embryos are placed in 1 molal concentrations of the impermeable solute sucrose, they osmotically lose ~85% of their cellular water in less than 2 minutes. If the cells are then cooled rapidly to -196°C, nearly 90% remain viable after warming, again provided that the warming is ultra rapid. PMID:25786677

  4. Estrogen Promotes the Development of Mouse Cumulus Cells in Coordination with Oocyte-Derived GDF9 and BMP15

    PubMed Central

    Sugiura, Koji; Su, You-Qiang; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.

    2010-01-01

    The differentiation and function of cumulus cells depend upon oocyte-derived paracrine factors, but studies on the estrogen receptor knockout mice suggested that estrogen also participates in these processes. This study investigates the possible coordination of estrogen and oocytes in the development and function of cumulus cells using cumulus expansion and the expression of transcripts required for expansion as functional endpoints. Preantral granulosa cell-oocyte complexes developed in vitro with 17β-estradiol (E2) exhibited increased levels of cumulus expansion and Has2 transcripts, encoding hyaluronan synthase 2, compared with those developed without E2. Moreover, cumulus cell-oocyte complexes (COCs) isolated from antral follicles and maintained in culture without E2 exhibited reduced cumulus expansion and Has2 mRNA levels compared with freshly isolated COCs. Exogenous E2, provided during the maintenance culture, alleviated these deficiencies. However, when oocytes were removed from COCs, E2 supplementation did not maintain competence to undergo expansion; the presence in culture of either fully grown oocytes or recombinant growth differentiation factor 9 (GDF9) was required. Recombinant bone morphogenetic protein 15, but not fibroblast growth factor 8, augmented the GDF9 effect. Oocytes or GDF9 suppressed cumulus cell levels of Nrip1 transcripts encoding nuclear receptor-interacting protein 1, a potential inhibitor of estrogen receptor signals. Therefore, E2 and oocyte-derived paracrine factors GDF9 and bone morphogenetic protein 15 coordinate to promote the development of cumulus cells and maintain their competence to undergo expansion. Furthermore, suppression of Nrip1 expression in cumulus cells by oocyte may be one mechanism mediating cross talk between oocyte and E2 signals that promotes follicular development. PMID:21047911

  5. The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

    PubMed

    Downs, S M; Dow, M P; Fagbohun, C F

    1991-06-01

    Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the

  6. Heat-killed bacteria induce genome instability in mouse small intestine, liver and spleen tissues.

    PubMed

    Koturbash, Igor; Thomas, James E; Kovalchuk, Olga; Kovalchuk, Igor

    2009-06-15

    Bacterial infection has been associated with several malignancies, yet the exact mechanism of infection-associated carcinogenesis remains obscure. Furthermore, it is still not clear whether oncontransformation requires an active infection process, or merely the presence of inactivated bacteria remnants is enough to cause deleterious effects. Here, we analyzed whether or not consumption of non-pathogenic and pathogenic heat-killed Escherichia coli leads to changes in genome stability in somatic tissues of exposed animals. For one week, mice were given to drink filtered or not-filtered water contaminated with heat-killed non-pathogenic E. coli DH5alpha or heat-killed pathogenic E. coli O157:H7 Sakai. Control animals received tap water. One week after exposure, molecular changes were analyzed in the small intestine, an organ that is in immediate contact with contaminated water. Additionally, we studied the effect in the distant spleen and liver, the organs that are involved in an immune response and detoxification, respectively. Finally, muscles were chosen as neutral tissues that were not supposed to be affected. Intestinal, liver and spleen but not muscle cells responded to all bacterial treatments with an increased level of DNA damage monitored by the induction of gammaH2AX foci. In the intestine, elevated levels of DNA damage were in parallel with an increase in Ku70 and p53 expression. We have also found an elevated level of cellular proliferation in the intestine, liver and spleen but not in muscle tissues of all exposed animals as measured by increase in PCNA levels. Our data suggest that exposure to heat-killed filtered bacteria can trigger substantial molecular responses and cause genomic instability in target and distant organs. Even though bacteria were non-pathogenic and unable to cause infection, their remnants still caused a profound effect on exposed animals. PMID:19440049

  7. In Vitro Maturation of Mouse Oocytes Increases the Level of Kif11/Eg5 on Meiosis II Spindles.

    PubMed

    Kovacovicova, Kristina; Awadova, Thuraya; Mikel, Pavel; Anger, Martin

    2016-07-01

    Although in vitro maturation (IVM) of oocytes has been used for a relatively long time, during which the culture conditions have improved remarkably, the resulting germ cells are still not fully comparable to the cells obtained from the ovary in many important aspects, namely in fertilization rate and subsequent embryonic development. Some of the differences between IVM and in vivo maturation (IVV) oocytes were already discovered, including variability in spindle assembly and morphology. In this study we focused on a role of molecular motor Kif11 (hereafter referred to as Eg5) in maintaining bipolar spindle structure in IVM and IVV oocytes. Our experiments revealed that in IVM oocytes, Eg5 is abundant on meiosis II spindle, which makes these cells more sensitive to Eg5 inhibition than IVV oocytes. We further demonstrate that this sensitivity is acquired gradually with exposure to the in vitro conditions. This is a remarkable difference in function of spindle apparatus between IVM and IVV oocytes, and we believe our results are important not only for understanding of the chromosome segregation in mammalian oocytes but also because they indicate cells are using alternative pathways to achieve the same function when exposed to different conditions. PMID:27146033

  8. Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes.

    PubMed

    Marh, Joel; Tres, Laura L; Yamazaki, Yukiko; Yanagimachi, Ryuzo; Kierszenbaum, Abraham L

    2003-07-01

    It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (gamma-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/ approximately kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins. PMID:12620938

  9. Targeting and Killing of Metastatic Cells in the Transgenic Adenocarcinoma of Mouse Prostate Model With Vesicular Stomatitis Virus

    PubMed Central

    Moussavi, Maryam; Tearle, Howard; Fazli, Ladan; Bell, John C; Jia, William; Rennie, Paul S

    2013-01-01

    Vesicular stomatitis virus (VSV) is an oncolytic virus which selectively infects and kills cancer cells. The goal of the present study was to determine whether VSV is capable of targeting metastatic lesions that arise in situ in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The interferon (IFN)-responsive luciferase containing VSV(AV3) strain was injected intraprostatically into both control and TRAMP mice. Distribution, infectivity, apoptosis, and status of the IFN response were evaluated at the site of viral injection (prostate), as well as in metastatic lesions (lymph nodes), through plaque, polymerase chain reaction (PCR), and immunohistochemical analysis. Bioluminescence analyses demonstrated that VSV(AV3) persisted at high levels in the prostate region of TRAMP mice for up to 96 hours, but at relatively low levels and for only 48 hours in control mice. Live virus was discovered in the lymph nodes of TRAMP mice, but not in control mice. TUNEL staining revealed increased cell death in VSV(AV3) infected metastatic cells present in the lymph nodes of TRAMP mice. There was an evidence of IFN activation in lymph nodes containing metastatic cells. Our results indicate that intraprostatic injections of VSV(AV3) can be used as a means to infect and kill metastatic lesions associated with advanced prostate cancer. PMID:23337981

  10. Association of Maternal mRNA and Phosphorylated EIF4EBP1 Variants With the Spindle in Mouse Oocytes: Localized Translational Control Supporting Female Meiosis in Mammals

    PubMed Central

    Romasko, Edward J.; Amarnath, Dasari; Midic, Uros; Latham, Keith E.

    2013-01-01

    In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. We find by cDNA microarray analysis enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. We also find that the key translational regulator, EIF4EBP1, undergoes a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appear at different positions along the spindle at different stages of meiosis. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types. PMID:23852387

  11. Effect of recombinant-LH and hCG in the absence of FSH on in vitro maturation (IVM) fertilization and early embryonic development of mouse germinal vesicle (GV)-stage oocytes.

    PubMed

    Dinopoulou, Vasiliki; Drakakis, Peter; Kefala, Stella; Kiapekou, Erasmia; Bletsa, Ritsa; Anagnostou, Elli; Kallianidis, Konstantinos; Loutradis, Dimitrios

    2016-06-01

    During in vitro maturation (IVM), intrinsic and extrinsic factors must co-operate properly in order to ensure cytoplasmic and nuclear maturation. We examined the possible effect of LH/hCG in the process of oocyte maturation in mice with the addition of recombinant LH (r-LH) and hCG in our IVM cultures of mouse germinal vesicle (GV)-stage oocytes. Moreover, the effects of these hormones on fertilization, early embryonic development and the expression of LH/hCG receptor were examined. Nuclear maturation of GV-stage oocytes was evaluated after culture in the presence of r-LH or hCG. Fertilization rates and embryonic development were assessed after 24h. Total RNA was isolated from oocytes of different stages of maturation and from zygotes and embryos of different stages of development in order to examine the expression of LH/hCG receptor, using RT-PCR. The in vitro nuclear maturation rate of GV-stage oocytes that received hCG was significantly higher compared to the control group. Early embryonic development was increased in the hCG and LH cultures of GV oocytes when LH was further added. The LH/hCG receptor was expressed in all stages of in vitro matured mouse oocytes and in every stage of early embryonic development. Addition of hCG in IVM cultures of mouse GV oocytes increased maturation rates significantly. LH, however, was more beneficial to early embryonic development than hCG. This suggests a promising new technique in basic science research or in clinical reproductive medicine. PMID:27288338

  12. The Testicular and Epididymal Expression Profile of PLCζ in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor

    PubMed Central

    Aarabi, Mahmoud; Yu, Yang; Xu, Wei; Tse, Man Y.; Pang, Stephen C.; Yi, Young-Joo; Sutovsky, Peter; Oko, Richard

    2012-01-01

    Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides. PMID:22428063

  13. The GTPase SPAG-1 orchestrates meiotic program by dictating meiotic resumption and cytoskeleton architecture in mouse oocytes.

    PubMed

    Huang, Chunjie; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiaofei; Guan, Kaifeng; Huo, Lijun

    2016-06-01

    In mammals, a finite population of oocytes is generated during embryogenesis, and proper oocyte meiotic divisions are crucial for fertility. Sperm-associated antigen 1 (SPAG-1) has been implicated in infertility and tumorigenesis; however, its relevance in cell cycle programs remains rudimentary. Here we explore a novel role of SPAG-1 during oocyte meiotic progression. SPAG-1 associated with meiotic spindles and its depletion severely compromised M-phase entry (germinal vesicle breakdown [GVBD]) and polar body extrusion. The GVBD defect observed was due to an increase in intraoocyte cAMP abundance and decrease in ATP production, as confirmed by the activation of AMP-dependent kinase (AMPK). SPAG-1 RNA interference (RNAi)-elicited defective spindle morphogenesis was evidenced by the dysfunction of γ-tubulin, which resulted from substantially reduced phosphorylation of MAPK and irregularly dispersed distribution of phospho-MAPK around spindles instead of concentration at spindle poles. Significantly, actin expression abruptly decreased and formation of cortical granule-free domains, actin caps, and contractile ring disrupted by SPAG-1 RNAi. In addition, the spindle assembly checkpoint remained functional upon SPAG-1 depletion. The findings broaden our knowledge of SPAG-1, showing that it exerts a role in oocyte meiotic execution via its involvement in AMPK and MAPK signaling pathways. PMID:27053660

  14. Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction.

    PubMed

    Gordon, J W

    2001-04-01

    The risk of insertion of adenovirus gene therapy DNA into female germ cells during the course of somatic gene therapy was stringently tested in the mouse by injecting up to 10(10) infectious particles directly into the ovary and by incubating naked oocytes in a solution of 2 x 10(8) particles/ml for 1 h prior to in vitro fertilization (IVF). The vector used was a recombinant adenovirus carrying the bacterial lacZ gene driven by the cytomegalovirus promoter (Adbeta-gal). Ovaries were stained for LacZ activity, or immunochemically for LacZ, 5-7 days after injection. Although very large amounts of LacZ activity and protein were detected, all positive staining was in the thecal portion of the ovary, with no staining seen in oocytes. In another series of experiments, mice with injected ovaries were mated, and preimplantation embryos or fetuses were analyzed either for LacZ expression or by PCR for lacZ DNA. None of 202 preimplantation embryos stained positively for LacZ and none of 58 fetuses were positive for DNA by PCR analysis. Finally, more than 1400 eggs were fertilized after exposure to the vector prior to IVF and stained as morulae for LacZ activity. Fewer than 2% of the embryos stained positively for LacZ, and experiments indicated that the staining was due to incomplete washing of the eggs prior to IVF. These data provide strong evidence that adenoviruses cannot infect oocytes and that the risk of female germ-line transduction with such vectors is very low. PMID:11319918

  15. Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

    NASA Technical Reports Server (NTRS)

    Tombes, R. M.; Simerly, C.; Borisy, G. G.; Schatten, G.

    1992-01-01

    During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

  16. Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

    SciTech Connect

    Jones, J.; Schultz, R.M. )

    1990-06-01

    G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

  17. The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes.

    PubMed

    Sharif, Bedra; Na, Jie; Lykke-Hartmann, Karin; McLaughlin, Stephen H; Laue, Ernest; Glover, David M; Zernicka-Goetz, Magdalena

    2010-12-15

    The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I. PMID:21123620

  18. Intrinsic and extrinsic mechanisms of oocyte loss.

    PubMed

    Thomson, Travis C; Fitzpatrick, Katherine E; Johnson, Joshua

    2010-12-01

    A great deal of evolutionary conservation has been found in the control of oocyte development, from invertebrates to women. However, little is known of mechanisms that control oocyte loss over time. Oocyte loss is often assumed to be a result of oocyte-intrinsic deficiencies or damage. In fruit flies, starvation results in halted oocyte production by germline stem cells and induces oocyte loss midway through development. When we fed wild-type flies the bacterial compound Rapamycin (RAP) to mimic starvation, production of new oocytes continued, but mid-stage loss sterilized the animals. Surprisingly, follicle cell invasion and phagocytosis of the oocyte preceded any signs of germ cell death. RAP-induced egg chamber loss was prevented when RAP receptor FKBP12 was knocked down specifically in follicle cells. Oogenesis continued past the mid-stages, and these mutants continued to lay embryos that could develop into normal adults. Hence, intact healthy oocytes can be destroyed by somatic cells responding to extrinsic stimuli. We termed this process inducible somatic oocyte destruction. RAP treatment of mouse follicles in vitro resulted in phagocytic uptake of the oocyte by granulosa cells as seen in flies. We hypothesize that extrinsic modes of oocyte loss occur in mammals. PMID:20651035

  19. Antimicrobial photodynamic therapy with RLP068 kills methicillin-resistant Staphylococcus aureus and improves wound healing in a mouse model of infected skin abrasion PDT with RLP068/Cl in infected mouse skin abrasion.

    PubMed

    Vecchio, Daniela; Dai, Tianhong; Huang, Liyi; Fantetti, Lia; Roncucci, Gabrio; Hamblin, Michael R

    2013-09-01

    Photodynamic therapy (PDT) is an alternative treatment for infections that can kill drug resistant bacteria without damaging host-tissue. In this study we used bioluminescent methicillin-resistant Staphylococcus aureus, in a mouse skin abrasion model, to investigate the effect of PDT on bacterial inactivation and wound healing. RLP068/Cl, a tetracationic Zn(II)phthalocyanine derivative and toluidine blue (TBO) were used. The light-dose response of PDT to kill bacteria in vivo and the possible recurrence in the days post-treatment were monitored by real-time bioluminescence imaging, and wound healing by digital photography. The results showed PDT with RLP068/Cl (but not TBO) was able to kill bacteria, to inhibit bacterial re-growth after the treatment and to significantly accelerate the wound healing process (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). PMID:22987338

  20. Enhanced killing effects of caffein post-treatment in ultraviolet-light irradiated mouse lymphoma cells: is cAMP a mediator of the effects?

    PubMed

    Kuwashima, Y; Miyachi, Y; Okada, S; Iio, M; Nakamura, N

    1983-01-01

    Effects of post-treatment with caffein, cyclic adenosine 3':5'-monophosphate (cAMP) and N6, O2-dibutyryl cAMP (dbcAMP) were investigated in ultraviolet light (UV)-irradiated mouse lymphoma L5178Y cells. Under conditions where UV or each chemical alone caused only slight cytotoxic effects, caffein post-treatment showed clear synergistic effects in cell killing but not for cAMP or dbcAMP. Subsequently, a mutant clone resistant to cAMP was isolated. This mutant was supposed to be deficient in cAMP-mediated cellular functions. Using the mutant cells, it was found that these cells were also sensitive to caffein post-treatment as wild cells after UV-irradiation. The results imply that the enhanced killing effects by caffein post-treatment in UV irradiated cells is not mediated by cAMP. PMID:6093199

  1. Proteomes of Animal Oocytes: What Can We Learn for Human Oocytes in the In Vitro Fertilization Programme?

    PubMed Central

    Virant-Klun, Irma; Krijgsveld, Jeroen

    2014-01-01

    Oocytes are crucial cells for mammalian reproduction, yet the molecular principles underlying oocyte development are only partially understood. Therefore, contemporary proteomic approaches have been used increasingly to provide new insights into oocyte quality and maturation in various species such as mouse, pig, and cow. Especially, animal studies have helped in elucidating the molecular status of oocytes during in vitro maturation and other procedures of assisted reproduction. The aim of this review is to summarize the literature on mammalian oocyte proteome and secretome research in the light of natural and assisted reproduction and on lessons to be learned for human oocytes, which have so far remained inaccessible for proteome analysis. PMID:24804254

  2. Phagocytic and chemiluminescent responses of mouse peritoneal macrophages to living and killed Salmonella typhimurium and other bacteria.

    PubMed Central

    Tomita, T; Blumenstock, E; Kanegasaki, S

    1981-01-01

    In the presence of luminol, resident as well as thioglycolate-induced and immunized macrophages emitted chemiluminescence more efficiently when the cells were exposed to living Salmonella typhimurium than when they were exposed to the same bacterium killed by ultraviolet light or heat. This phenomenon was observed whether or not the bacterium was opsonized. The different response to living and killed bacteria was also found with Escherichia coli, Pseudomonas aeruginosa, Proteus morganii, and Enterobacter aerogenes, but not with Shigella sonnei, Klebsiella pneumoniae, and Propionibacterium acnes. The results suggest that macrophages respond better to living, motile bacteria than to nonmotile or killed bacteria. The experimental results obtained with motility mutants of S. typhimurium, E. coli, and P. aeruginosa confirm that macrophages exposed to the motile bacteria emit chemiluminescence more efficiently and ingest the motile bacteria at a much faster rate than the nonmotile bacteria. Images PMID:6788707

  3. Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing.

    PubMed Central

    Engelward, B P; Dreslin, A; Christensen, J; Huszar, D; Kurahara, C; Samson, L

    1996-01-01

    In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity. Images PMID:8631315

  4. How neutrophils kill fungi.

    PubMed

    Gazendam, Roel P; van de Geer, Annemarie; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2016-09-01

    Neutrophils play a critical role in the prevention of invasive fungal infections. Whereas mouse studies have demonstrated the role of various neutrophil pathogen recognition receptors (PRRs), signal transduction pathways, and cytotoxicity in the murine antifungal immune response, much less is known about the killing of fungi by human neutrophils. Recently, novel primary immunodeficiencies have been identified in patients with a susceptibility to fungal infections. These human 'knock-out' neutrophils expand our knowledge to understand the role of PRRs and signaling in human fungal killing. From the studies with these patients it is becoming clear that neutrophils employ fundamentally distinct mechanisms to kill Candida albicans or Aspergillus fumigatus. PMID:27558342

  5. MULTIDRUG RESISTANT TRANSPORT ACTIVITY PROTECTS OOCYTES FROM CHEMOTHERAPEUTIC AGENTS AND CHANGES DURING OOCYTE MATURATION

    PubMed Central

    Brayboy, Lynae M.; Oulhen, Nathalie; Witmyer, Jeannine; Robins, Jared; Carson, Sandra; Wessel, Gary M.

    2013-01-01

    Objective To determine the multidrug resistant (MDR) transporter activity in oocytes and their potential role in oocyte susceptibility to chemotherapy. Design Experimental laboratory study Setting University and Academic Center for reproductive medicine. Patients/Animals Women with eggs retrieved for ICSI cycles and adult female FVBN and B6C3F1 mouse strains. Intervention Inhibition of MDR activity in oocytes. Main Outcome measure(s) Efflux activity of MDRs using quantitative fluorescent dye efflux and oocyte cell death when exposed to chemotherapy. Results Oocytes effluxed fluorescent reporters and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. GV oocytes are more efficient at efflux compared to M2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death using two different viability assays compared to controls and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes with the greatest accumulation in the GV stage. Conclusions Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis, and these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up regulation of these transporters during gonadotoxic therapy. PMID:23953328

  6. Oocyte and cumulus cell transcripts from cultured mouse follicles are induced to deviate from normal in vivo conditions by combinations of insulin, follicle-stimulating hormone, and human chorionic gonadotropin.

    PubMed

    Sánchez, Flor; Romero, Sergio; Smitz, Johan

    2011-09-01

    Gonadotropins and insulin are major regulators of cell proliferation, differentiation, and survival in cultured mouse ovarian follicles. Applications of variable doses of insulin in combination with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were studied at the gene expression level in oocytes and cumulus cells. Early preantral follicles grown over 9 days were sequentially exposed to combinations of doses of insulin, FSH, and human chorionic gonadotropin (hCG). From culture Day 1 to 6 (preantral stage), two insulin concentrations (5 ng/ml and 5 μg/ml) were combined with 10 mIU/ml FSH. From Days 6 to 9 (antral stage), the three variable gonadotropin treatments set under each insulin condition were 10 mIU/ml FSH, 25 mIU/ml FSH, and 25 mIU/ml FSH plus 3 mIU/ml hCG. The Gdf9, Bmp15, Fgf8, Dazl, Pou5f1, and Pik3ca mRNA transcripts were quantified in oocytes, and the Amh, Lhcgr, Hsd3b1, Vegfa, and Insig1 mRNA transcripts were quantified in cumulus cells. In vivo controls were unprimed and eCG (equine chorionic gonadotropin)-primed prepubertal female mice. During the preantral stage, none except the Amh transcripts was regulated by insulin. Oocyte transcripts were not affected by the variable gonadotropin treatments on the last culture day but were upregulated in the combination of high insulin plus 25 mIU/ml FSH. Under low insulin conditions, high FSH levels increased levels of Lhcgr and Vegfa expression, and hCG abated this effect. However, under high insulin conditions, hCG upregulated levels of Lhcgr, Vegfa, and Insig1 mRNA. High insulin concentrations upregulated Hsd3b1 transcripts. These results demonstrate that in an in vitro follicle culture, a near physiological insulin background yields oocyte and cumulus cell transcript levels that are more similar to those in vivo. PMID:21565993

  7. Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability.

    PubMed

    Di Giacomo, Monica; Camaioni, Antonella; Klinger, Francesca G; Bonfiglio, Rita; Salustri, Antonietta

    2016-02-19

    Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability. PMID:26694612

  8. Candida albicans killing by RAW 264.7 mouse macrophage cells: effects of Candida genotype, infection ratios, and gamma interferon treatment.

    PubMed

    Marcil, A; Harcus, D; Thomas, D Y; Whiteway, M

    2002-11-01

    Phagocytic cells such as neutrophils and macrophages are potential components of the immune defense that protects mammals against Candida albicans infection. We have tested the interaction between the mouse macrophage cell line RAW 264.7 and a variety of mutant strains of C. albicans. We used an end point dilution assay to monitor the killing of C. albicans at low multiplicities of infection (MOIs). Several mutants that show reduced virulence in mouse systemic-infection models show reduced colony formation in the presence of macrophage cells. To permit analysis of the macrophage-Candida interaction at higher MOIs, we introduced a luciferase reporter gene into wild-type and mutant Candida cells and used loss of the luminescence signal to quantify proliferation. This assay gave results similar to those for the end point dilution assay. Activation of the macrophages with mouse gamma interferon did not enhance anti-Candida activity. Continued coculture of the Candida and macrophage cells eventually led to death of the macrophages, but for the RAW 264.7 cell line this was not due to apoptotic pathways involving caspase-8 or -9 activation. In general Candida cells defective in the formation of hyphae were both less virulent in animal models and more sensitive to macrophage engulfment and growth inhibition. However the nonvirulent, hypha-defective cla4 mutant line was considerably more resistant to macrophage-mediated inhibition than the wild-type strain. Thus although mutants sensitive to engulfment are typically less virulent in systemic-infection models, sensitivity to phagocytic macrophage cells is not the unique determinant of C. albicans virulence. PMID:12379711

  9. Exposing cultured mouse ovarian follicles under increased gonadotropin tonus to aromatizable androgens influences the steroid balance and reduces oocyte meiotic capacity.

    PubMed

    Romero, Sergio; Smitz, Johan

    2010-10-01

    Acquisition of oocyte developmental competence relies on the well-controlled events accompanying antral follicular development. Elevated basal androgen levels, as in PCOS, potentially affect oocyte quality. Current experiments in an in vitro follicle bioassay studied dose-effects of androstenedione and testosterone on FSH and hCG stimulated antral follicle growth and meiotic maturation. The addition of either androgens altered follicle's endogenous production of androstenedione, testosterone, estradiol, and progesterone and affected the oocyte's capacity to resume meiosis. Exposure to 200 nM androstenedione induced an increased production of testosterone and estradiol. Exposure to a concentration of ≥200 nM testosterone induced elevated levels of estradiol and progesterone. Significant dose-dependent negative effects on polar body extrusion were seen at concentrations of ≥200 nM of either androgen. In addition, chromosome displacement on the metaphase plate was observed in oocytes obtained from androstenedione-treated follicles. Follicles exposed to a combination of 25 mIU/ml FSH and 3 mIU/ml hCG and elevated aromatizable androgens altered the steroid production profile, affected the follicular development and impaired oocyte meiotic competence. PMID:21046485

  10. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

    PubMed

    Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  11. A novel chemosynthetic peptide with β-sheet motif efficiently kills Klebsiella pneumoniae in a mouse model

    PubMed Central

    Tan, Shirui; Gan, Changpei; Li, Rongpeng; Ye, Yan; Zhang, Shuang; Wu, Xu; Yang, Yi Yan; Fan, Weimin; Wu, Min

    2015-01-01

    Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is increasingly becoming multiple drug resistant. However, the molecular pathogenesis of Kp in causing tissue injury and dysregulated host defense remains elusive, further dampening the development of novel therapeutic measures. We have previously screened a series of synthetic antimicrobial beta-sheet forming peptides and identified a peptide (IRIKIRIK; ie, IK8L) with a broad range of bactericidal activity and low cytotoxicity in vitro. Here, employing an animal model, we investigated the antibacterial effects of IK8L in acute infection and demonstrated that peritoneal injection of IK8L to mice down-regulated inflammatory cytokines, alleviated lung injury, and importantly, decreased mortality compared to sham-injected controls. In addition, a math model was used to evaluate in vivo imaging data and predict infection progression in infected live animals. Mechanistically, IK8L can kill Kp by inhibiting biofilm formation and modulating production of inflammatory cytokines through the STAT3/JAK signaling both in vitro and in vivo. Collectively, these findings reveal that IK8L may have potential for preventing or treating Kp infection. PMID:25709431

  12. A novel chemosynthetic peptide with β-sheet motif efficiently kills Klebsiella pneumoniae in a mouse model.

    PubMed

    Tan, Shirui; Gan, Changpei; Li, Rongpeng; Ye, Yan; Zhang, Shuang; Wu, Xu; Yang, Yi Yan; Fan, Weimin; Wu, Min

    2015-01-01

    Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is increasingly becoming multiple drug resistant. However, the molecular pathogenesis of Kp in causing tissue injury and dysregulated host defense remains elusive, further dampening the development of novel therapeutic measures. We have previously screened a series of synthetic antimicrobial beta-sheet forming peptides and identified a peptide (IRIKIRIK; ie, IK8L) with a broad range of bactericidal activity and low cytotoxicity in vitro. Here, employing an animal model, we investigated the antibacterial effects of IK8L in acute infection and demonstrated that peritoneal injection of IK8L to mice down-regulated inflammatory cytokines, alleviated lung injury, and importantly, decreased mortality compared to sham-injected controls. In addition, a math model was used to evaluate in vivo imaging data and predict infection progression in infected live animals. Mechanistically, IK8L can kill Kp by inhibiting biofilm formation and modulating production of inflammatory cytokines through the STAT3/JAK signaling both in vitro and in vivo. Collectively, these findings reveal that IK8L may have potential for preventing or treating Kp infection. PMID:25709431

  13. Killing and conformal Killing tensors

    NASA Astrophysics Data System (ADS)

    Heil, Konstantin; Moroianu, Andrei; Semmelmann, Uwe

    2016-08-01

    We introduce an appropriate formalism in order to study conformal Killing (symmetric) tensors on Riemannian manifolds. We reprove in a simple way some known results in the field and obtain several new results, like the classification of conformal Killing 2-tensors on Riemannian products of compact manifolds, Weitzenböck formulas leading to non-existence results, and construct various examples of manifolds with conformal Killing tensors.

  14. Luteinizing Hormone Reduces the Activity of the NPR2 Guanylyl Cyclase in Mouse Ovarian Follicles, Contributing to the Cyclic GMP Decrease that Promotes Resumption of Meiosis in Oocytes

    PubMed Central

    Robinson, Jerid W.; Zhang, Meijia; Shuhaibar, Leia C.; Norris, Rachael P.; Geerts, Andreas; Wunder, Frank; Eppig, John J.; Potter, Lincoln R.; Jaffe, Laurinda A.

    2012-01-01

    In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 minutes, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2 hours, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte. PMID:22546688

  15. Killing Coyotes.

    ERIC Educational Resources Information Center

    Beasley, Conger, Jr.

    1993-01-01

    Presents different viewpoints concerning the federal government's Animal Damage Control (ADC) Program cited as responsible for killing millions of predators. Critics provide evidence of outdated and inhumane methods exemplified in the coyote killings. The ADC emphasizes new, nonlethal methods of controlling animals cited as "noxious." (MCO)

  16. Technical aspects of the piezo, laser-assisted, and conventional methods for nuclear transfer of mouse oocytes and their efficiency and efficacy: Piezo minimizes damage of the ooplasmic membrane at injection.

    PubMed

    Chen, Shee-Uan; Chao, Kuang-Han; Chang, Chia-Yi; Hsieh, Fon-Jou; Ho, Hong-Nerng; Yang, Yu-Shih

    2004-04-01

    Assessment of the advantages and disadvantages of the piezo, laser, and conventional methods for nuclear transfer has remained elusive. Furthermore, although the piezo method had been used by some investigators for research of sperm injection and nuclear transfer for several years, many researchers have failed to operate the technique smoothly and achieve reproducible results. The procedures of nuclear transfer using piezo were ascertained and described in detail. Mouse oocytes were enucleated, and injected with cumulus cells using the piezo, laser, or conventional methods. We investigated the time needed and survival of nuclear transfer. Development was compared among the three methods and parthenogenetic control specimens. The average time of nuclear transfer for each oocyte was significantly shorter using the piezo (118 +/- 9 s) and laser methods (120 +/- 11 s) than using the conventional method (170 +/- 11 s). The damage rate was smaller for the piezo group (10%) than the laser (37%) and conventional (40%) groups. The percentages of blastocyst formation (14%, 12%, and 11%) and the number of nuclei of blastocysts (54 +/- 13, 51 +/- 11, and 52 +/- 12) were similar among the piezo, laser, and conventional groups, but significantly lower than for the control group (83%, 105 +/- 14). The piezo technique is more efficient than the conventional method for nuclear transfer. The laser method is easy to operate, but the equipment is expensive. In addition, piezo induced fewer traumas while breaking the membrane than the aspiration techniques used in the laser and conventional methods. PMID:15039993

  17. Inhibition of GABAA receptor-mediated current responses by enoxacin (new quinolone) and felbinac (non-steroidal anti-inflammatory drug) in Xenopus oocytes injected with mouse-brain messenger RNA.

    PubMed

    Kawakami, J; Shimokawa, M; Yamamoto, K; Sawada, Y; Asanuma, A; Yanagisawa, K; Iga, T

    1993-07-01

    The convulsant interaction between enoxacin (ENX), a new quinolone antibacterial agent (NQ), and felbinac (FLB), a non-steroidal anti-inflammatory drug (NSAID), in vivo was reproduced as the change of GABA-induced current response in Xenopus oocytes injected with mouse brain mRNA. GABA (10 microM) response was inhibited by ENX in a dose-dependent manner, and IC50 of ENX was 96 microM. Moreover, the inhibitory effect of ENX was 80-fold potentiated in the presence of 10 microM FLB. The GABAA-antagonistic interaction between these two drugs in vitro was considered a possible mechanism of convulsant reaction after concomitant administration of NQs and NSAIDs in vivo. PMID:7691340

  18. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

    PubMed Central

    Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang

    2016-01-01

    Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods. PMID:26966678

  19. Phenotypes of Aging Postovulatory Oocytes After Somatic Cell Nuclear Transfer in Mice.

    PubMed

    Lee, Ah Reum; Shimoike, Takashi; Wakayama, Teruhiko; Kishigami, Satoshi

    2016-06-01

    Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-derived nuclei on oocyte physiology during aging. Normal oocytes started undergoing cytofragmentation 24 hours after oocyte collection; however, this occurred earlier in SCNT oocytes and was more severe at 48 hours, suggesting that the transferred somatic cell nuclei affected oocyte physiology. We found no difference in the status of acetylated α-tubulin (Ac-Tub) and α-tubulin (Tub) between normal and SCNT aging oocytes, but unlike normal oocytes, aging SCNT oocytes did not have astral microtubules. Interestingly, aging SCNT oocytes displayed more severely scattered chromosomes or irregularly shaped spindles. Observations of the microfilaments showed that, in normal oocytes, there was a clear actin ring beneath the plasma membrane and condensed microfilaments around the spindle (the actin cap) at 0 hours, and the actin filaments started degenerating at 1 hour, becoming completely disrupted and distributed to the cytoplasm at 24 hours. By contrast, in SCNT oocytes, an actin cap formed around the transplanted nuclei within 1 hour of SCNT, which was still present at 24 hours. Thus, SCNT oocytes age in a similar but distinct way, suggesting that they not only contain nuclei with abnormal epigenetics but are also physiologically different. PMID:27253626

  20. A comparison of cell killing by heat and/or x rays in Chinese hamster V79 cells, Friend erythroleukemia mouse cells, and human thymocyte MOLT-4 cells

    SciTech Connect

    Raaphorst, G.P.; Szekely, J.; Lobreau, A.; Azzam, E.I.

    1983-05-01

    The radiation and/or heat sensitivity of Chinese hamster V79 cells, Friend erythroleukemia (FELC) mouse cells, and MOLT-4 human transformed thymocytes were compared. MOLT-4 cells were more radiosensitive (D/sub o/=0.50 Gy) than FELC (D/sub o/ = 0.65 Gy) and V79 cells (D/sub o/ = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0/sup 0/C, but more heat resistant at higher temperatures. In addition, the MOLT-4 cells showed a single-heat inactivation energy between 41.0 and 45.0/sup 0/C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5/sup 0/C, respectively. These differences may be related to the fact that the upper temperature limit for the development of thermal tolerance during continuous heating was lower for MOLT-4 cells than for FELC or V79 cells. Killing of FELC and V79 cells was dependent on the sequence in which heat and X rays were applied, but the greatest effect was obtained when both treatments were given simultaneously. Recovery occurred when treatments were separated by incubation at 37.0/sup 0/C. The MOLT-4 cells did not show a sequence dependence for heating and irradiation. Survival of MOLT-4 cells after heating and/or irradiation was compared using trypan blue dye exclusion or colony formation. Both assays showed similar qualitative responses, but survival levels measured by the trypan blue assay were much higher than those determined from the colony-forming assay.

  1. Molecular control of the oocyte to embryo transition.

    PubMed Central

    Knowles, Barbara B; Evsikov, Alexei V; de Vries, Wilhelmine N; Peaston, Anne E; Solter, Davor

    2003-01-01

    The elucidation of the molecular control of the initiation of mammalian embryogenesis is possible now that the transcriptomes of the full-grown oocyte and two-cell stage embryo have been prepared and analysed. Functional annotation of the transcriptomes using gene ontology vocabularies, allows comparison of the oocyte and two-cell stage embryo between themselves, and with all known mouse genes in the Mouse Genome Database. Using this methodology one can outline the general distinguishing features of the oocyte and the two-cell stage embryo. This, when combined with oocyte-specific targeted deletion of genes, allows us to dissect the molecular networks at play as the differentiated oocyte and sperm transit into blastomeres with unlimited developmental potential. PMID:14511485

  2. Functional expression of murine multidrug resistance in Xenopus laevis oocytes

    SciTech Connect

    Castillo, G.; Vera, J.C.; Rosen, O.M. ); Yang, Chiaping Huang; Horwitz, S.B. )

    1990-06-01

    The development of multidrug resistance (MDR) is associated with the overproduction of a plasma membrane glycoprotein, P glycoprotein. Here the authors report the functional expression of a member of the murine MDR family of proteins and show that Xenopus oocytes injected with RNA encoding the mouse mdr1b P glycoprotein develop a MDR-like phenotype. Immunological analysis indicated that oocytes injected with the mdr1b RNA synthesized a protein with the size and immunological characteristics of the mouse mdr1b P glycoprotein. These oocytes exhibited a decreased accumulation of ({sup 3}H)vinblastine and showed an increased capacity to extrude the drug compared to control oocytes not expressing the P glycoprotein. In addition, competition experiments indicated that verapamil, vincristine, daunomycin, and quinidine, but not colchicine, can overcome the rapid drug efflux conferred by the expression of the mouse P glycoprotein.

  3. Killing Range

    PubMed Central

    Asal, Victor; Rethemeyer, R. Karl; Horgan, John

    2015-01-01

    This paper presents an analysis of the Provisional Irish Republican Army's (PIRA) brigade level behavior during the Northern Ireland Conflict (1970-1998) and identifies the organizational factors that impact a brigade's lethality as measured via terrorist attacks. Key independent variables include levels of technical expertise, cadre age, counter-terrorism policies experienced, brigade size, and IED components and delivery methods. We find that technical expertise within a brigade allows for careful IED usage, which significantly minimizes civilian casualties (a specific strategic goal of PIRA) while increasing the ability to kill more high value targets with IEDs. Lethal counter-terrorism events also significantly affect a brigade's likelihood of killing both civilians and high-value targets but in different ways. Killing PIRA members significantly decreases IED fatalities but also significantly decreases the possibility of zero civilian IED-related deaths in a given year. Killing innocent Catholics in a Brigade's county significantly increases total and civilian IED fatalities. Together the results suggest the necessity to analyze dynamic situational variables that impact terrorist group behavior at the sub-unit level. PMID:25838603

  4. Follicle environment and quality of in vitro matured oocytes.

    PubMed

    Sirard, Marc-André

    2011-06-01

    In mammalian reproduction, the oocyte depends on the ovarian follicle for most of its growth. They form a bipolar partnership and the status of one will impact the functioning of the other. When oocytes are removed from their follicle by ovulation, they have normally completed all the steps required to begin their journey into the oviduct and drive the early embryonic development. When oocytes are removed from their follicle before natural ovulation, the process by which they acquire all the important components for their journey might not be completed and their ability to mature, fertilize or develop into embryos or to term might be compromised. Animal models have been useful to define the important steps required for the oocyte's growth phase, and in the mouse, when the oocyte has reached its full size, the program is ready. This is not the case in larger mammals where the completion of growth does not ensure that the oocyte is fully capable of undergoing all the steps to the embryo and to term. The final steps of oocyte preparation also involve a progressive condensation of the chromatin that may facilitate normal maturation but may also indirectly reduce the lifespan of the oocyte. In such a scenario, the oocyte would have an expiration date when fully competent. In humans, a number of indications may justify the aspiration of oocytes from unstimulated patients and the development of an in vitro maturation (IVM) process that would allow fertilization and subsequent development. This objective could be realized by a better understanding of the essential follicular contribution required before removing the oocyte. Therefore, this review will focus on the large animal models where IVM has been used and studied for more than 25 years. The status of the follicle at the time of oocyte recovery and the status of the oocyte's chromatin will be described in detail as they have a significant impact on the outcome. PMID:21394521

  5. Tetraspanin CD9 in bovine oocytes and its role in fertilization.

    PubMed

    Zhou, Guang-Bin; Liu, Guo-Shi; Meng, Qing-Gang; Liu, Ying; Hou, Yun-Peng; Wang, Xiao-Xu; Li, Ning; Zhu, Shi-En

    2009-06-01

    This study was conducted in bovine to investigate whether CD9 (a member of the tetraspanin superfamily of proteins) is present on oocytes and whether it functions in sperm-oocyte binding and fusion. First, the presence of CD9 in bovine matured oocytes was examined by immunofluorescence with the anti-CD9 monoclonal antibody (mAb) and fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and the results showed that CD9 was expressed on the plasma membrane of matured oocytes. Sperm binding and fusion with oocytes was then examined by in vitro fertilization. When the zona pellucida-free matured oocytes were fertilized, both sperm binding to ooplasma and sperm penetrating into oocytes were significantly (P<0.01) reduced in anti-CD9 antibody-treated oocytes (6.3 +/- 0.7 per oocyte and 41.6%, respectively) compared with untreated control oocytes (19.0 +/- 0.7 per oocyte and 81.3%, respectively), indicating that the anti-CD9 mAb potentially inhibits sperm-oocyte binding and fusion. These results demonstrated that the CD9 present on bovine matured oocytes is involved in sperm-oocyte interaction during fertilization. PMID:19293563

  6. Photo activation of HPPH encapsulated in "Pocket" liposomes triggers multiple drug release and tumor cell killing in mouse breast cancer xenografts.

    PubMed

    Sine, Jessica; Urban, Cordula; Thayer, Derek; Charron, Heather; Valim, Niksa; Tata, Darrell B; Schiff, Rachel; Blumenthal, Robert; Joshi, Amit; Puri, Anu

    2015-01-01

    We recently reported laser-triggered release of photosensitive compounds from liposomes containing dipalmitoylphosphatidylcholine (DPPC) and 1,2 bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC). We hypothesized that the permeation of photoactivated compounds occurs through domains of enhanced fluidity in the liposome membrane and have thus called them "Pocket" liposomes. In this study we have encapsulated the red light activatable anticancer photodynamic therapy drug 2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) (Ex/Em410/670 nm) together with calcein (Ex/Em490/517 nm) as a marker for drug release in Pocket liposomes. A mole ratio of 7.6:1 lipid:HPPH was found to be optimal, with >80% of HPPH being included in the liposomes. Exposure of liposomes with a cw-diode 660 nm laser (90 mW, 0-5 minutes) resulted in calcein release only when HPPH was included in the liposomes. Further analysis of the quenching ratios of liposome-entrapped calcein in the laser treated samples indicated that the laser-triggered release occurred via the graded mechanism. In vitro studies with MDA-MB-231-LM2 breast cancer cell line showed significant cell killing upon treatment of cell-liposome suspensions with the laser. To assess in vivo efficacy, we implanted MDA-MB-231-LM2 cells containing the luciferase gene along the mammary fat pads on the ribcage of mice. For biodistribution experiments, trace amounts of a near infrared lipid probe DiR (Ex/Em745/840 nm) were included in the liposomes. Liposomes were injected intravenously and laser treatments (90 mW, 0.9 cm diameter, for an exposure duration ranging from 5-8 minutes) were done 4 hours postinjection (only one tumor per mouse was treated, keeping the second flank tumor as control). Calcein release occurred as indicated by an increase in calcein fluorescence from laser treated tumors only. The animals were observed for up to 15 days postinjection and tumor volume and luciferase expression was measured. A

  7. History of oocyte cryopreservation.

    PubMed

    Gook, Debra A

    2011-09-01

    The potential advantages of being able to cryopreserve oocytes have been apparent for many decades. Technical difficulties associated with the unique properties of the mammalian oocyte initially retarded rapid development in this area but recent advances have overcome many of the problems. A stage has now been reached where oocyte cryopreservation can be considered an important component of human assisted reproductive technology. The potential advantages of being able to cryopreserve oocytes have been apparent for many decades. Technical difficulties associated with the unique properties of the mammalian oocyte initially retarded rapid development in this area but recent advances have overcome many of the problems. A stage has now been reached where oocyte cryopreservation can be considered an important component of human assisted reproductive technology. PMID:21549640

  8. Cryopreservation of starfish oocytes.

    PubMed

    Hamaratoğlu, Fisun; Eroğlu, Ali; Toner, Mehmet; Sadler, Kirsten C

    2005-02-01

    Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos. PMID:15710368

  9. Isolation of Xenopus oocytes.

    PubMed

    Sive, Hazel L; Grainger, Robert M; Harland, Richard M

    2010-12-01

    Xenopus oocytes are obtained from sexually mature females by surgically removing parts of the ovary. The operation is not fatal and can be performed on an anesthetized frog several times during its lifetime. However, a recovery period of 2 wk is recommended between operations. A careful record of all operations performed, including details of oocyte quality, should be kept. A frog that produces one good batch of oocytes; e.g., those that translate injected messenger RNAs (mRNAs) efficiently, should be recorded and used again, because oocyte quality is generally frog-dependent. PMID:21123421

  10. Developmental competence of oocytes grown in vitro: Has it peaked already?

    PubMed Central

    MOROHAKU, Kanako; HIRAO, Yuji; OBATA, Yayoi

    2015-01-01

    In vitro growth of immature oocytes provides opportunities to increase gametic resources and to understand the mechanisms underlying oocyte development. Many studies on the in vitro growth of oocytes have been reported thus far; however, only a few cases have been reported, which demonstrated that oocytes can support full-term development after in vitro fertilization. Our research group recently found that culture of mouse neonatal primordial follicles increased the birthrate; however, the establishment of an in vitro system that can completely mimic follicle or oocyte growth in vivo and control oogenesis remains an ongoing challenge. PMID:26685717