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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. Distribution of mitochondria in reconstructed mouse oocytes.

    PubMed

    Fulka, Helena

    2004-02-01

    It has been suggested that nucleus replacement (transfer) may be used as an efficient oocyte therapy in order to prevent transmission of mutated mitochondrial DNA from mother to offspring in humans. The essential and not yet answered question is how mitochondria surrounding the karyoplast will be distributed in the newly reconstructed oocytes. In our model experiments, we have evaluated the distribution of mitochondria in reconstructed immature mouse oocytes when germinal vesicle karyoplasts, with labeled mitochondria, were fused to unlabeled cytoplasts. The penetration of mitochondria from karyoplasts into cytoplasts can be detected almost immediately after the beginning of fusion. In immature reconstructed oocytes, mitochondria are first located in the oocyte center but they are homogeneously distributed within the whole cytoplasm before the completion of maturation. Fusion of oocytes at different stages of maturation suggests that the speed of mitochondria distribution is cell cycle dependent.

  3. Effects of alphafetoprotein on isolated mouse oocytes.

    PubMed

    Lambert, J C; Seralini, G E; Stora, C; Vallette, G; Vranckx, R; Nunez, E A

    1986-01-01

    The supposition of an effect of alphafetoprotein (AFP) on female germinal cells is put forward. The spontaneous in vitro maturation of adult mouse oocytes is significantly inhibited when mouse AFP replaces albumin in culture medium. Furthermore, the very unusual degenerative appearance of the cells subjected to AFP seems to indicate that this meiotic inhibition is linked to a premature degeneration of the oocytes rather than to a blockage of the cells at an earlier stage of maturation. Accordingly AFP, perhaps through its ligands, may play a role in reducing the number of gonocytes during fetal and immediate post-natal life rather than in stopping oocyte meiosis at the diplotene stage.

  4. 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.

  5. Control of nucleus positioning in mouse oocytes.

    PubMed

    Almonacid, Maria; Terret, Marie-Emilie; Verlhac, Marie-Hélène

    2017-08-12

    The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos. Copyright © 2017. Published by Elsevier Ltd.

  6. Caffeine delays oocyte aging and maintains the quality of aged oocytes safely in mouse.

    PubMed

    Zhang, Xia; Liu, Xiaoyan; Chen, Li; Wu, Dan-Ya; Nie, Zheng-Wen; Gao, Ying-Ying; Miao, Yi-Liang

    2017-03-28

    Caffeine, as an oocyte aging inhibitor, was used in many different species to control or delay oocyte aging. However, the safety of caffeine and developmental competence of aged oocytes inhibited by caffeine has not been studied systematically. So we detected the spindle morphology, distribution of cortical granules, zona pellucida hardening and pronucleus formation to assess oocyte quality of caffeine treated oocytes. We found that aged oocytes treated by caffeine maintained weak susceptibility to activating stimuli and regained normal competent after aged further 6 hr. Caffeine maintained the spindle morphology, changed cortical granules distribution of aged oocytes and could not prevent zona pellucida hardening. Furthermore, caffeine increased pronucleus formation of aged oocytes and decreased fragmentation after fertilization. These results suggested that caffeine could maintain the quality of aged oocytes safely in mouse.

  7. 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

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

    PubMed

    Mok, Hyuck Jun; Shin, Hyejin; 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.

  9. 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

  10. Tristetraprolin functions in cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Liu, Xiaohui; Li, Xiaoyan; Ma, Rujun; Xiong, Bo; Sun, Shao-Chen; Liu, Honglin; Gu, Ling

    2016-01-01

    Tristetraprolin (TTP), a member of TIS11 family containing CCCH tandem zinc finger, is one of the best characterized RNA-binding proteins. However, to date, the role of TTP in mammalian oocytes remains completely unknown. In the present study, we report the altered maturational progression and cytokinesis, upon specific knockdown of TTP in mouse oocytes. Furthermore, by confocal scanning, we observe the failure to form cortical actin cap during meiosis of TTP-depleted oocytes. Loss of TTP in oocytes also results in disruption of meiotic spindle morphology and chromosome alignment. In support of these findings, incidence of aneuploidy is accordingly increased when TTP is abated in oocytes. Our results suggest that TTP as a novel cytoskeletal regulator is required for spindle morphology/chromosome alignment and actin polymerization in oocytes. PMID:27458159

  11. Asymmetries and Symmetries in the Mouse Oocyte and Zygote.

    PubMed

    Chaigne, Agathe; Terret, Marie-Emilie; Verlhac, Marie-Hélène

    2017-01-01

    Mammalian oocytes grow periodically after puberty thanks to the dialogue with their niche in the follicle. This communication between somatic and germ cells promotes the accumulation, inside the oocyte, of maternal RNAs, proteins and other molecules that will sustain the two gamete divisions and early embryo development up to its implantation. In order to preserve their stock of maternal products, oocytes from all species divide twice minimizing the volume of their daughter cells to their own benefit. For this, they undergo asymmetric divisions in size where one main objective is to locate the division spindle with its chromosomes off-centred. In this chapter, we will review how this main objective is reached with an emphasis on the role of actin microfilaments in this process in mouse oocytes, the most studied example in mammals. This chapter is subdivided into three parts: I-General features of asymmetric divisions in mouse oocytes, II-Mechanism of chromosome positioning by actin in mouse oocytes and III-Switch from asymmetric to symmetric division at the oocyte-to-embryo transition.

  12. 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.

  13. DNA damage response during mouse oocyte maturation

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  14. Free-hand bisection of mouse oocytes and embryos.

    PubMed

    Polanski, Zbigniew; Kubiak, Jacek Z

    2013-01-01

    Mouse oocytes and zygotes are semitransparent and large cells approximately 80 μm in diameter. Bisection is one of the easiest ways for performing micromanipulations on such cells. It allows living sister halves or smaller fragments to be obtained, which can be cultured and observed for long periods of time. Bisection can be used for different kinds of experiments such as analysis of nucleo-cytoplasmic interactions, the relationship between different cellular structures or between different parts of embryos, eventually for analyzing the developmental potential of embryonic fragments. Oocyte or embryo halves can be examined by immunostaining, by measuring different cellular functions and by Western blot and genetic analysis (e.g., RT-PCR). Here we describe a detailed protocol for the free-hand bisection of mouse zona pellucida-free oocytes and embryos on an agar layer using a glass needle.

  15. Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

    PubMed Central

    Buschiazzo, Jorgelina; Ialy-Radio, Come; Auer, Jana; Wolf, Jean-Philippe; Serres, Catherine

    2013-01-01

    Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. PMID:23638166

  16. [Effect of alpha-fetoprotein on isolated mouse oocytes].

    PubMed

    Lambert, J C; Vallette, G; Seralini, G E; Vranckx, R; Nunez, E; Stora, C

    1986-01-01

    Data are presented which indicate a possible action of alpha-fetoprotein (AFP) on female germinal cells. The in vitro maturation of mature mice oocytes was significantly inhibited when mouse AFP replaced albumin in the culture medium. In addition, the degenerative aspect of oocytes cultured with AFP seemed to indicate that this meïotic inhibition was caused by a premature degeneration of oocytes rather than by a blockage at a specific stage of maturation. Thus AFP, perhaps through its ligands, may play a role in the reduction of germinal cells during fetal and immediate post-natal life rather than in the arrest of meïosis at the diplotene stage.

  17. Vitrification of mouse MII oocytes: Developmental competency using paclitaxel.

    PubMed

    Fesahat, Farzaneh; Faramarzi, Azita; Khoradmehr, Arezoo; Omidi, Marjan; Anbari, Fatemeh; Khalili, Mohammad Ali

    2016-12-01

    Oocyte cryopreservation provides an important alternative for fertility preservation for women who will be treated with cytotoxic drugs. However, it can cause spindle disorganization of microtubules, putting the zygote at risk for aneuploidy. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim of this study was to investigate the suitable concentration of paclitaxel for adding to the vitrification media to improve the developmental potential of post-thawed mature oocytes to blastocyst formation in mice. A total of 300 MII oocytes were retrieved from superovulated mice, and were divided into three groups of control, Experimental I, and Experimental II. Oocytes in Experimental I and Experimental II were cryopreserved in the presence of 0.5μM or 1μM of paclitaxel in vitrification media, respectively. After thawing, all oocytes were incubated in G-IVF medium for 1 hour. From each group,12 oocytes were selected for viability evaluation by Hoechst/propidium iodide nuclear staining. Standard in vitro fertilization was performed on the rest of the oocytes and embryo development was followed to the blastocyst stage. Fertilization rate was not significantly different between the three groups. However, the cleavage rate (55%) in Experimental II group was significantly lower compared to Experimental I (88%) and control groups (83%). There was a detectable difference between the three groups at the blastocyst rate (Experimental I and control groups, p = 0.004; Experimental II vs. control and Experimental I, p < 0.001). The highest rates of parthenogenesis and arrest were in Experimental II (16% and 21%, respectively) compared with control (6% and 5%, respectively) and Experimental I (5% and 3%, respectively). There was also a significant decrease in viability rate of oocytes in Experimental II compared to the other groups. A high concentration of paclitaxel, an anticancer drug, interrupted the mouse oocyte competency when supplemented to

  18. PTK2b function during fertilization of the mouse oocyte.

    PubMed

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

    2014-08-01

    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. Published by Elsevier Inc.

  19. Size-specific follicle selection improves mouse oocyte reproductive outcomes

    PubMed Central

    Xiao, Shuo; Duncan, Francesca E.; Bai, Lu; Nguyen, Catherine T.; Shea, Lonnie D.; Woodruff, Teresa K.

    2015-01-01

    Encapsulated in vitro follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, follicles are cultured for a defined period of time and analyzed as a cohort. However, follicle growth is not synchronous, and culturing follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of follicles based on size, rather than absolute culture time, better predict follicle maturity and oocyte quality. Multilayer secondary mouse follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when follicles were cultured for specific days. However, if follicles were cultured to a terminal diameter of 300–350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from follicles with diameters of 300–350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed 2 pronuclei after in vitro fertilization (IVF), 81% developed into the 2-cell embryo stage, and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other follicle size groups. Our study demonstrates that size-specific follicle selection can be used as a non-invasive marker to identify high quality oocytes and improve reproductive outcomes during eIVFG. PMID:26116002

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

    PubMed

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

    2016-03-01

    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. 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. The survival rate of vitrified-warmed Atg7(f/f) ;Zp3-Cre (Atg7(d/d) ) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7(f/f) ) oocytes. Fertilization and development in the Atg7(d/d) oocytes were significantly lower than the Atg7(f/f) oocytes, comparable to the Atg5(d/d) oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7(d/d) MII oocytes when compared to fresh Atg7(d/d) oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. We confirmed that the LC3-positive signal is nearly absent in Atg7(d/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.

  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. Extra- and intracellular ice formation in mouse oocytes.

    PubMed

    Mazur, Peter; Seki, Shinsuke; Pinn, Irina L; Kleinhans, F W; Edashige, Keisuke

    2005-08-01

    The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best

  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. Sensitivity of mouse oocytes to nicotine-induced perturbations during oocyte meiotic maturation and aneuploidy in vivo and in vitro.

    PubMed

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

    2000-03-01

    Oocyte meiosis is sensitive to endogenous and exogenous perturbations that upset the temporal sequence of biochemical reactions during oocyte maturation (OM) and predispose oocytes to aneuploidy. Nicotine is an alkaloid that has been reported to disrupt the rate of OM, reduce ovulation and fertilization rates, and increase diploidy. The objective of this study was to test the hypothesis that nicotine perturbs the rate of OM and induces aneuploidy in mouse oocytes in vivo and in vitro. Female mice were given 7.5 IU pregnant mare's serum and either 0, 5.0, 7.5, or 10 mg/kg nicotine in vivo at -3, 0, and +3 h relative to a 5 IU injection of HCG. Oocytes were also cultured in vitro in the presence of 0, 1.0, 5.0, or 10.0 mmol/l nicotine. In vivo, significant (P < 0.05) differences in the proportions of oocytes with premature centromere separation and premature anaphase were found at 10.0 mg/kg nicotine suggesting that the rate of OM was advanced. Also, at this dose the proportion of ovulated oocytes was reduced by approximately 50% relative to controls. In vitro, only non-significant differences were found among the parameters measured. Although nicotine reduced the ovulation rate and perturbed the rate of OM in vivo, these data show that the rate of aneuploidy was not significantly elevated.

  5. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation

    PubMed Central

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-01-01

    ABSTRACT Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1–PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  6. 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.

  7. 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

  8. Mouse Oocytes Acquire Mechanisms that Permit Independent Cell Volume Regulation at the End of Oogenesis.

    PubMed

    Richard, Samantha; Tartia, Alina P; Boison, Detlev; Baltz, Jay M

    2016-09-02

    Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [(3) H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. This article is protected by copyright. All rights reserved.

  9. 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

  10. 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

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

    PubMed

    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.

  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.

  13. 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. Copyright © 2016, American Association for the Advancement of Science.

  14. Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

    PubMed

    Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

    2015-01-01

    Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

  15. Epsin2 promotes polarity establishment and meiotic division through activating Cdc42 in mouse oocyte

    PubMed Central

    Zhang, Jiaqi; Liu, Xiaohui; Ma, Rujun; Hou, Xiaojing; Ge, Juan; Wang, Qiang

    2016-01-01

    Epsins are a conserved family of endocytic adaptors essential for diverse biological events. However, its role in oocytes remains completely unknown. Here, we report that specific depletion of Epsin2 in mouse oocytes significantly disrupts meiotic progression. Confocal microscopy reveals that Epsin2 knockdown results in the failure of actin cap formation and polar body extrusion during meiosis, indicative of the importance of Epsin2 in polarity establishment and cytokinesis. In addition, spindle defects and chromosome misalignment are readily observed in oocytes depleted of Epsin2. Moreover, we find that Epsin2 knockdown markedly decreases the activity of Cdc42 in oocytes and importantly, that the dominant-positive mutant of Cdc42 (Cdc42Q61L) is capable of partially rescuing the deficient phenotypes of Epsin2-knockdown oocytes. Together, our data identify Epsin2 as a novel player in regulating oocyte maturation, and demonstrate that Epsin2 promotes polarity establishment and meiotic division via activating Cdc42. PMID:27463009

  16. Amino Acid Correction of Regulatory Volume Decrease Evoked by Hypotonic Stress in Mouse Oocytes In Vitro.

    PubMed

    Pogorelova, M A; Golichenkov, V A; Pogorelova, V N; Panait, A I; Smirnov, A A; Pogorelov, A G

    2015-05-01

    Regulatory volume decrease in response to hypotonic stress is typical of the oocytes and early mouse embryos. Changes in the kinetics of osmotic reaction can be used as a marker of the modulating effect of the incubation medium on transmembrane transport in embryonic cells. Quantitative laser scanning microtomography (QLSM) was used to measure oocyte volume. In this paper, it is shown that addition of 5 μM glycine, taurine, or GABA, as well as ATP to Dulbecco's medium abolished the regulatory volume decrease in mature mouse oocytes.

  17. Analysis of the Phospholipid Profile of Metaphase II Mouse Oocytes Undergoing Vitrification

    PubMed Central

    Bang, Soyoung; Mok, Hyuck Jun; Suh, Chang Suk; Kim, Kwang Pyo; Lim, Hyunjung Jade

    2014-01-01

    Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation. PMID:25033391

  18. A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus-oocyte complexes.

    PubMed

    Greco, F; Perrin, J; Auffan, M; Tassistro, V; Orsière, T; Courbiere, B

    2015-07-01

    Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original-CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus-oocyte complexes (COC-CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC-CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC-CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC-CA is less time-consuming and simplifies the protocol of the original-CA, because COC-CA is easier to perform without the washing-out procedure.

  19. Effects of melatonin on oocyte maturation in PCOS mouse model.

    PubMed

    Nikmard, Fatemeh; Hosseini, Elham; Bakhtiyari, Mehrdad; Ashrafi, Mahnaz; Amidi, Fardin; Aflatoonian, Reza

    2017-04-01

    The purpose of oocyte in vitro maturation is generation of mature oocytes that could support future development. Efforts have been made to enhance oocyte developmental competence by developing optimal culture conditions. The present study is conducted to determine melatonin effects on quality of polycystic ovarian syndrome (PCOS) oocytes when it has been added during in vitro maturation, and immature oocytes were cultured in defined conditioned medium with and without different melatonin concentrations. Melatonin could significantly improve nuclear maturation of PCOS oocytes (81.1% vs. 56.3%, P < 0.05 were achieved with 10(-6) mol/L concentration). Cleavage rate was significantly higher in 10(-5) mol/L concentration compared to untreated oocytes in PCOS (54% vs. 35%, respectively) and it was significantly higher with 10(-6) mol/L concentration in the control group, 55% versus 38%, compared to untreated oocytes. This study showed that melatonin has the potential to induce oocyte nuclear maturation and guarantee fertilization potential. © 2016 Japanese Society of Animal Science.

  20. Cyclic AMP in oocytes controls meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary.

    PubMed

    Wang, Yijing; Teng, Zhen; Li, Ge; Mu, Xinyi; Wang, Zhengpin; Feng, Lizhao; Niu, Wanbao; Huang, Kun; Xiang, Xi; Wang, Chao; Zhang, Hua; Xia, Guoliang

    2015-01-15

    In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary. © 2015. Published by The Company of Biologists Ltd.

  1. G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes.

    PubMed

    Gill, Arvind; Hammes, Stephen R

    2007-02-01

    In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.

  2. JMY is required for asymmetric division and cytokinesis in mouse oocytes.

    PubMed

    Sun, Shao-Chen; Sun, Qing-Yuan; Kim, Nam-Hyung

    2011-05-01

    JMY is a transcriptional co-factor of p53. Latest work has revealed that JMY is also an actin nucleation factor that regulates new filament assembly and activates Arp2/3 complex in somatic cells; however, roles of JMY in mouse oocyte are unknown. Here we showed the expression and functions of JMY during mouse oocyte meiotic maturation. JMY mRNA is expressed largely from germinal vesicle to metaphase I stage, and gradually decreased during anaphase I, telophase I (TI) and metaphase II (MII) stages. Immunostaining results showed that JMY localized at the spindle and cytoplasm of oocytes. Depletion of JMY by RNAi resulted in symmetric division, failure of spindle migration and cytokinesis during oocyte meiotic maturation, showing a 2-cell-like MII oocyte and TI stage arrest. Actin cap and cortical granules-free domain formation were also disrupted after JMY RNAi, indicating the failure of spindle migration. JMY antibody injection results were consistent with those of JMY RNAi, further confirming the involvement of JMY in oocyte polarity. Our data indicate that JMY is required for spindle migration, asymmetric division and cytokinesis during mouse oocyte maturation.

  3. Follicle-stimulating hormone accelerates mouse oocyte development in vivo.

    PubMed

    Demeestere, Isabelle; Streiff, Agathe K; Suzuki, João; Al-Khabouri, Shaima; Mahrous, Enas; Tan, Seang Lin; Clarke, Hugh J

    2012-07-01

    During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.

  4. Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes

    PubMed Central

    2014-01-01

    Background Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. Results In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell–like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each “blastomere” of the 2-cell–like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each “blastomere” and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Conclusion Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI. PMID:24953160

  5. Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes.

    PubMed

    Zhou, Dongjie; Shen, Xinghui; Gu, Yanli; Zhang, Na; Li, Tong; Wu, Xi; Lei, Lei

    2014-06-21

    Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.

  6. Effect of Warming Rate on the Survival of Vitrified Mouse Oocytes and on the Recrystallization of Intracellular Ice1

    PubMed Central

    Seki, Shinsuke; Mazur, Peter

    2008-01-01

    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°C. They were then cooled rapidly to −70°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°C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140°C/min to 3300°C/min. Survivals after warming at 140°C/min and 250°C/min were low (<30%). Survivals after warming at ≥2200°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

  7. 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.

  8. Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.

    PubMed

    Rossi, Gianna; Palmerini, Maria Grazia; Macchiarelli, Guido; Buccione, Roberto; Cecconi, Sandra

    2006-07-01

    In this study the effects of mancozeb, a widely used ethylenebisdithiocarbamate fungicide, on mouse oocyte meiotic maturation and fertilization were analyzed. Oocyte cumulus cell-complexes were matured in vitro with or without increasing concentrations of the fungicide (from 0.001 to 1 microg/ml) that, due to its different stability in organic solvents and in water, was resuspended either in dimethyl sulfoxide or in culture medium. Although, about 95% of oocytes reached the metaphase II stage; mancozeb-exposed oocytes showed a dose-dependent increase of alterations in spindle morphology, and this negative effect was more evident when the fungicide was resuspended in culture medium. Under the latter culture condition, oocytes matured in the presence of 0.1 and 1 microg/ml mancozeb showed a significant reduction also in the formation of male and female pronuclei. These results indicate that mancozeb can adversely affect mammalian reproductive performance, likely by perturbing microtubular organization during meiotic maturation.

  9. Inhibition of denuded mouse oocyte meiotic maturation by forskolin, an activator of adenylate cyclase.

    PubMed

    Urner, F; Herrmann, W L; Baulieu, E E; Schorderet-Slatkine, S

    1983-09-01

    Forskolin, a diterpene that activates rapidly adenylate cyclase activity in several somatic cell systems, prevents spontaneous meiotic maturation of denuded mouse oocytes (ED50 of inhibition approximately 2.5 microM), unlike cholera toxin. The oocyte is sensitive to the action of forskolin during the period preceding germinal vesicle breakdown (GVBD). Washing of the cells abolishes the effect. The diterpene potentiates the inhibitory effect of iso-butyl-methyl-xanthine (IBMX), a phosphodiesterase inhibitor, and it increases cAMP concentration in the oocytes. These findings not only confirm the antagonistic effect of cAMP on the first step of meiosis reinitiation (GVBD) in mammalian oocytes, but also provide the first demonstration of a functional adenylate cyclase system in mammalian oocytes upon which regulatory signals may act.

  10. The Role of RING Box Protein 1 in Mouse Oocyte Meiotic Maturation

    PubMed Central

    Zhou, Lin; Yang, Ye; Zhang, Juanjuan; Guo, Xuejiang; Bi, Ye; Li, Xin; Zhang, Ping; Zhang, Junqiang; Lin, Min; Zhou, Zuomin; Shen, Rong; Guo, Xirong; Huo, Ran; Ling, Xiufeng; Sha, Jiahao

    2013-01-01

    RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCFβTrCP-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation. PMID:23874827

  11. 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. © 2016. Published by The Company of Biologists Ltd.

  12. 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

  13. Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization.

    PubMed

    Monti, Manuela; Redi, Carlo Alberto

    2016-01-07

    This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis. Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

  14. Resveratrol protects mouse oocytes from methylglyoxal-induced oxidative damage.

    PubMed

    Liu, Yu; He, Xiao-Qin; Huang, Xin; Ding, Lu; Xu, Lin; Shen, Yu-Ting; Zhang, Fei; Zhu, Mao-Bi; Xu, Bai-Hui; Qi, Zhong-Quan; Wang, Hai-Long

    2013-01-01

    Methylglyoxal, a reactive dicarbonyl compound, is mainly formed from glycolysis. Methylglyoxal can lead to the dysfunction of mitochondria, the depletion of cellular anti-oxidation enzymes and the formation of advanced glycation ends. Previous studies showed that the accumulation of methylglyoxal and advanced glycation ends can impair the oocyte maturation and reduce the oocyte quality in aged and diabetic females. In this study, we showed that resveratrol, a kind of phytoalexin found in the skin of grapes, red wine and other botanical extracts, can alleviate the adverse effects caused by methylglyoxal, such as inhibition of oocyte maturation and disruption of spindle assembly. Besides, methylglyoxal-treated oocytes displayed more DNA double strands breaks and this can also be decreased by treatment of resveratrol. Further investigation of these processes revealed that methylglyoxal may affect the oocyte quality by resulting in excessive reactive oxygen species production, aberrant mitochondrial distribution and high level lipid peroxidation, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can protect the oocytes from methylglyoxal-induced cytotoxicity and this was mainly through the correction of the abnormity of cellular reactive oxygen species metabolism.

  15. 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.

  16. 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

  17. Lin28a is dormant, functional, and dispensable during mouse oocyte-to-embryo transition.

    PubMed

    Flemr, Matyas; Moravec, Martin; Libova, Veronika; Sedlacek, Radislav; Svoboda, Petr

    2014-06-01

    The oocyte-to-embryo transition (OET) denotes transformation of a highly differentiated oocyte into totipotent blastomeres of the early mammalian embryo. OET depends exclusively on maternal RNAs and proteins accumulated during oocyte growth, which implies importance of post-transcriptional control of gene expression. OET includes replacement of abundant maternal microRNAs (miRNAs), enriched also in differentiated cells and exemplified by the Let-7 family, with embryonic miRNAs common in pluripotent stem cells (the miR-290 family in the mouse). Lin28a and its homolog Lin28b encode RNA-binding proteins, which interfere with Let-7 maturation and facilitate reprogramming of induced pluripotent stem cells. Both Lin28a and Lin28b transcripts are abundant in mouse oocytes. To test the role of maternal expression of Lin28a and Lin28b during oocyte-to-zygote reprogramming, we generated mice with oocyte-specific knockdown of both genes by using transgenic RNA interference. Lin28a and Lin28b are dispensable during oocyte growth because their knockdown has no effect on Let-7a levels in fully grown germinal vesicle (GV)-intact oocytes. Furthermore, transgenic females were fertile and produced healthy offspring, and their overall breeding performance was comparable to that of wild-type mice. At the same time, 2-cell embryos derived from transgenic females showed up-regulation of mature Let-7, suggesting that maternally provided LIN28A and LIN28B function during zygotic genome activation. Consistent with this conclusion is increased translation of Lin28a transcripts upon resumption of meiosis. Our data imply dual repression of Let-7 during OET in the mouse model, the selective suppression of Let-7 biogenesis by Lin28 homologs superimposed on previously reported global suppression of miRNA activity. © 2014 by the Society for the Study of Reproduction, Inc.

  18. Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

    PubMed

    Valsangkar, Deepa S; Downs, Stephen M

    2015-09-01

    In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

  19. Kif4 Is Essential for Mouse Oocyte Meiosis.

    PubMed

    Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

  20. Kif4 Is Essential for Mouse Oocyte Meiosis

    PubMed Central

    Camlin, Nicole J.; McLaughlin, Eileen A.; Holt, Janet E.

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age. PMID:28125646

  1. Regulation of peripheral spindle movement and spindle rotation during mouse oocyte meiosis: new perspectives.

    PubMed

    Ai, Jun-Shu; Wang, Qiang; Yin, Shen; Shi, Li-Hong; Xiong, Bo; OuYang, Ying-Chun; Hou, Yi; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2008-08-01

    Spindle movement, including spindle migration during first meiosis and spindle rotation during second meiosis, is essential for asymmetric divisions in mouse oocytes. Previous studies by others and us have shown that microfilaments are required for both spindle migration and rotation. In the present study, we aimed to further investigate the mechanism controlling spindle movement during mouse oocyte meiosis. By employing drug treatment and immunofluorescence microscopy, we showed that dynamic microtubule assembly was involved in both spindle migration and rotation. Furthermore, we found that the calcium/CaM/CaMKII pathway was important for regulating spindle rotation.

  2. The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis.

    PubMed

    Huang, Chun-Jie; Yuan, Yi-Feng; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiao-Fei; Huo, Li-Jun

    2017-03-01

    Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.

  3. Cytoskeleton-associated protein 5 and clathrin heavy chain binding regulates spindle assembly in mouse oocytes

    PubMed Central

    Wang, Dong-Hui; Han, Zhe; Kong, Xiang-Wei; Ma, Yu-Zhen; Yun, Zhi-Zhong; Liang, Cheng-Guang

    2017-01-01

    Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation. PMID:28177917

  4. Development of fertile mouse oocytes from mitotic germ cells in vitro.

    PubMed

    Morohaku, Kanako; Hirao, Yuji; Obata, Yayoi

    2017-09-01

    Mammalian fetal ovaries contain numerous primordial germ cells (PGCs), although few mature oocytes are obtained from females, owing to apoptosis and follicle atresia. The regulatory mechanisms underlying oogenesis/folliculogenesis remain unknown. Development of methods for obtaining mature oocytes from PGCs in fetal ovaries in vitro could contribute to clarifying these mechanisms. The failure of follicle assembly has been found to be the most challenging aspect in conventional culture conditions. Recently, we established novel culture conditions that enable successful follicle assembly, sustaining interactions between the oocyte and somatic cells, and, in turn, promoting oocyte growth and maturation. Mature oocytes were differentiated from PGCs after a 1-month culture period. A hundred mouse offspring were obtained from approximately a thousand mature oocytes, indicating that oocytes that were differentiated from PGCs in vitro acquired totipotency after fertilization. Here we provide a detailed protocol for using this in vitro system. This in vitro system will potentially provide a novel platform for studying oogenesis and preservation of female germ cells.

  5. Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides.

    PubMed

    McWilliams, R B; Gibbons, W E; Leibo, S P

    1995-05-01

    To survive cryopreservation, oocytes, zygotes and embryos must tolerate a sequence of volumetric contractions and expansions. These result as an egg or an embryo is exposed to a permeating cryoprotective additive, then to an increase followed by a decrease in the osmolality of its extracellular milieu as water freezes during cooling and then melts during warming, and finally to the dilution of the cryoprotective additive solution. Measurements of the extent to which mouse zygotes and human oocytes undergo osmotic contraction have been made by exposing them to solutions of monosaccharides (fructose, galactose, glucose) or disaccharides (maltose, sucrose, trehalose), ranging in concentration from 0.25 to 1.50 M. Mouse zygotes and human oocytes exhibit very similar responses to these solutions. Their volumes contract linearly as a function of 1/(osmolality) of the solutions, yielding estimates of non-osmotic volumes of 13-23%. Mouse zygotes exposed to 1.5 M concentrations of these solutions for 10 min lose 85% of their cell water. Yet > 75% of treated zygotes develop into hatching blastocysts. Human oocytes also appear to survive such extreme dehydration, based on a vital dye assay. These results suggest that solutions of various non-permeating saccharides can serve as osmotic buffers for the recovery of cryopreserved oocytes, zygotes and embryos.

  6. Hazardous Apoptotic Effects of 2-Bromopropane on Maturation of Mouse Oocytes, Fertilization, and Fetal Development

    PubMed Central

    Chan, Wen-Hsiung

    2010-01-01

    2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. Here, we further investigate the effects of 2-BP on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, 2-BP induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 2-BP during in vitro maturation (IVM) resulted in increased resorption of postimplantation embryos and decreased fetal weights. Experiments with a mouse model disclosed that consumption of drinking water containing 20 μM 2-BP led to decreased oocyte maturation in vivo and fertilization in vitro, as well as impairment of early embryonic development. Interestingly, pretreatment with a caspase-3-specific inhibitor effectively prevented 2-BP-triggered hazardous effects, suggesting that embryonic impairment by 2-BP occurs via a caspase-dependent apoptotic process. A study using embryonic stem cells as the assay model conclusively demonstrated that 2-BP induces cell death processes through apoptosis and not necrosis, and inhibits early embryo development in mouse embryonic stem cells. These results collectively confirm the hazardous effects of 2-BP on embryos derived from pretreated oocytes. PMID:21151443

  7. Smc1β is required for activation of SAC during mouse oocyte meiosis.

    PubMed

    Miao, Yilong; Zhou, Changyin; Cui, Zhaokang; Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Xiong, Bo

    2017-03-19

    Smc1β is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1β-deficient mice in both sexes are sterile. Ablation of Smc1β during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1β during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1β maintains the correct meiotic progression in mouse oocytes have not been clearly defined. Here, we find that GFP-fused Smc1β is expressed and localized to the chromosomes from GV to MII stages during mouse oocyte meiotic maturation. Knockdown of Smc1β by microinjection of gene-specific morpholino causes the impaired spindle apparatus and chromosome alignment which are highly correlated with the defective kinetochore-microtubule attachments, consequently resulting in a prominently higher incidence of aneuploid eggs. In addition, the premature extrusion of polar bodies and escape of metaphase I arrest induced by low dose of nocodazole treatment in Smc1β-depleted oocytes indicates that Smc1β is essential for activation of spindle assembly checkpoint (SAC) activity. Collectively, we identify a novel function of Smc1β as a SAC participant beyond its role in chromosome cohesion during mouse oocyte meiosis.

  8. Induced melanin reduces mutations and cell killing in mouse melanoma.

    PubMed

    Li, W; Hill, H Z

    1997-03-01

    When melanin absorbs light energy, it can produce potentially damaging active oxygen species. There is little doubt that constitutive pigment in dark-skinned individuals is photoprotective against skin cancer, but induced pigment-as in tanning-may not be. The first step in cancer induction is mutation in DNA. The most suitable systems for evaluating the role of melanin are those in which pigment can be varied and mutations can be measured. Several cell lines from Cloudman S91 mouse melanoma can be induced to form large quantities of melanin pigment after treatment with a number of different agents enabling comparison of mutant yields in the same cells differing principally in pigment concentration. In these studies, melanin was induced with synthetic alpha-melanocyte-stimulating hormone and with isobutyl methyl xanthine in the cell line S91/mel. The former inducer produced about 50% more pigment than the latter. Survival and mutation induction at the Na+/K(+)-ATPase locus were studied using ethyl methane sulfonate (EMS), a standard mutagen and five UV lamps emitting near monochromatic and polychromatic UV light in the three wave-length ranges of UV. There was greater protection against killing and mutation induction in the more heavily pigmented cells after exposure to EMS and after irradiation with monochromatic UVC and UVB. There was significant protection against killing by polychromatic UVB + UVA (FS20), but the small degree of protection against mutation was not significant. No significant change in killing and mutation using the same protocol was seen in S91/amel, a related cell line that does not respond to these inducers. No mutants were produced by either monochromatic or polychromatic UVA at doses that killed 50% of the cells. Our results show that induced pigment-shown earlier to be eumelanin (K. A. Cieszka et al., Exp. Dermatol. 4, 192-198, 1995)-is photo- and chemoprotective, but it is less effective in protection against mutagenesis by polychromatic

  9. MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism.

    PubMed

    LaRosa, Cean; Downs, Stephen M

    2005-02-01

    The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.

  10. RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

    PubMed

    Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

    2016-10-01

    Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

  11. Uranium in drinking water: effects on mouse oocyte quality.

    PubMed

    Kundt, Miriam S; Martinez-Taibo, Carolina; Muhlmann, Maria C; Furnari, Juan C

    2009-05-01

    The aim of this work was to evaluate the reproductive toxicological effects of uranium (U) at 2.5, 5, and 10 mgU/kg/d chronically administered in drinking water for 40 d. Swiss female control mice (n = 28) and mice chronically contaminated with uranyl nitrate in drinking water (n = 36) were tested. The number and quality of ovulated oocytes, chromatin organization, and nuclear integrity were evaluated. No significant differences were obtained in the numbers of ovulated oocytes between the different groups. Nevertheless, in 1,520 of the oocytes examined, dysmorphism increased from 11.99% in the control group to 27.99%, 27.19%, and 27.43% in each of the contaminated groups, respectively, in a dose-independent manner. On the other hand, morphological chromatin organization from 880 oocytes examined showed an increase in metaphase plate abnormalities from 37.20% (+/-7.21) in the control group to 55.13% (+/-21.36), 58.29% (+/-21.72), and 64.10% (+/-12.62) in each of the contaminated groups, respectively. Cumulus cell (CC) micronucleation, a parameter of nuclear integrity, increased from 0.21% (+/-0.31) in the control group to 1.92 (+/-0.95), 2.98 (+/-0.97), and 3.2 (+/-0.98), respectively. Both metaphase plate abnormalities and CC micronucleation showed an increase in a dose-dependent manner (r = 0.9; p < 0.001). The oocyte and its microenvironment showed high sensitivity to uranium contamination by drinking water. The lowest observed adverse effect level for this system is estimated at a level below 2.5 mgU/kg/d for female mice.

  12. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary

    PubMed Central

    Xu, Na; Zhu, Rui-Lou; Wang, Xiu-Xing; Chen, Zhong; Tao, Wei-Wei; Yao, Bing; Sun, Hai-Xiang; Huang, Xing-Xu; Xue, Bin; Li, Chao-Jun

    2017-01-01

    Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP), a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps) levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function. PMID:28072828

  13. 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.

  14. Water transport and estimated transmembrane potential during freezing of mouse oocytes.

    PubMed

    Toner, M; Cravalho, E G; Armant, D R

    1990-05-01

    The kinetics of water transport and the changes in transmembrane potential during freezing of mouse oocytes in isotonic phosphate buffered saline (PBS) were simulated using thermodynamic models. The permeability to water at 0 degree C, Lpg, and the activation energy, ELp, of metaphase II mouse oocytes from B6D2F1 mice were determined to be 0.044 +/- 0.008 micron/min-atm and 13.3 +/- 2.5 kcal/mol during freezing at 2 degrees C/min. The inactive cell volume was determined to be 0.214 with a correlation coefficient of 0.995, indicating that the oocytes closely follow the ideal Boyle-van't Hoff relation. The mean value of the oocyte diameter was 79.41 +/- 4.62 microns. These results were used to predict the behavior of mouse oocytes under various freezing conditions. The effect of the cooling rate on the cell volume and cytoplasm undercooling was investigated. The changes in transmembrane potential were also investigated during freezing of mouse oocytes. The computer simulations showed that at the beginning of the freezing process (-1 degrees C), the fast growth of ice in the extracellular solution causes a sharp increase of the membrane potential. It is predicted that the change in membrane potential is substantial for almost all cooling rates. Estimations show that values as high as -90 mV may be reached during freezing. The hyperpolarization of the membrane may cause orientation of the dipoles within the membrane. For membrane proteins with 300 debye dipole moment, the theoretical prediction suggests that the percentage of dipoles aligned with the membrane potential increases from 16% at 0 degrees C prior to freezing to 58% at -8 degrees C after seeding of the external ice followed with a cooling at 120 degrees C/min.

  15. 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

  16. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

    PubMed

    Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

    2014-02-01

    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

  17. Mitochondrial dynamics controlled by mitofusins define organelle positioning and movement during mouse oocyte maturation.

    PubMed

    Wakai, Takuya; Harada, Yuichirou; Miyado, Kenji; Kono, Tomohiro

    2014-11-01

    Mitochondria are abundant in fully grown mammalian oocytes with a unique spherical morphology, but the mechanisms controlling mitochondria behavior are not well understood. Here we describe for the first time the control of mitochondrial behavior in mouse oocytes by a fusion/fission mechanism. Mitofusins (Mfn1 and Mfn2) and OPA1 proteins are required for outer and inner mitochondrial membrane fusion, respectively, whereas Drp1 is the key regulator of mitochondrial fission. We show that mouse oocytes express the Mfn1, Mfn2, Opa1 and Drp1 proteins, both in immature and mature oocytes at similar levels. Overexpression of Mfn1 or Mfn2 causes marked mitochondrial aggregation, particularly in the perinuclear region during meiotic progression. Tracking of mitochondria with chromosomes or endoplasmic reticulum (ER) throughout oocyte maturation demonstrates that Mfn1 and Mfn2-promoted mitochondrial aggregation disturbs the spatiotemporal dynamic of the chromosomes and ER, respectively. Our findings suggest that organelle dynamics are co-ordinately controlled during meiotic division, and an imbalance of mitochondrial fusion/fission leads to disorganization of the organelle compartments. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes.

    PubMed

    Shen, Yu-Ting; Song, Yue-Qiang; He, Xiao-Qin; Zhang, Fei; Huang, Xin; Liu, Yu; Ding, Lu; Xu, Lin; Zhu, Mao-Bi; Hu, Wen-Feng; Qi, Zhong-Quan; Wang, Hai-Long; Yang, Xiang-Jun

    2014-10-01

    Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10μgmL(-1) TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P<0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10mgkg(-1) TPTCL (P<0.05). GVBD decreased in a non-significant, dose-dependent manner (P>0.05). PBE was inhibited with 10mgkg(-1) TPTCL (P<0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10mgkg(-1) TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

  19. Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations

    PubMed Central

    Lee, Ho-Joon; Elmoazzen, Heidi; Wright, Diane; Biggers, John; Rueda, Bo R; Heo, Yun Seok; Toner, Mehmet; Toth, Thomas L

    2010-01-01

    The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000°C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l propane-1,2-diol and 0.5 mol/l trehalose and achieve survival rates of 92.5%. Fertilization and blastocyst formation rates of vitrified–warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified–warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification. PMID:20113958

  20. 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

  1. 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 (H(2)O(2)), 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 H(2)O(2) and (·)OH insult at lower concentrations, but this protection was lost at higher concentrations (>50 μmol/L). At higher H(2)O(2) 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.

  2. Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion.

    PubMed

    Balboula, Ahmed Z; Blengini, Cecilia S; Gentilello, Amanda S; Takahashi, Masashi; Schindler, Karen

    2017-06-01

    During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. LIM kinase activity is required for microtubule organising centre positioning in mouse oocyte meiosis.

    PubMed

    Li, Xin; Zhu, Yubo; Cao, Yan; Wang, Qian; Du, Juan; Tian, Jianhui; Liang, Yuanjing; Ma, Wei

    2016-01-06

    LIM kinase 1 (LIMK1) activity is essential for cell migration and cell cycle progression. Little is known about LIMK1 expression and function in mammalian oocytes. In the present study we assessed LIMK1 protein expression, subcellular distribution and function during mouse oocyte meiosis. Western blot analysis revealed high and stable expression of LIMK1 from the germinal vesicle (GV) to MII stage. In contrast, activated LIMK1 (i.e. LIMK1 phosphorylated at threonine 508 (pLIMK1Thr508)) was only detected after GV breakdown, with levels increasing gradually to peak at MI and MII. Immunofluorescence showed pLIMK1Thr508 was colocalised with the microtubule organising centre (MTOC) components pericentrin and γ-tubulin at the spindle poles. A direct interaction between γ-tubulin and pLIMK1Thr508 was confirmed by co-immunoprecipitation. LIMK inhibition with 1 μM BMS3 damaged MTOC protein localisation to spindle poles, undermined the formation and positioning of functional MTOC and thus disrupted spindle formation and chromosome alignment. These effects were phenocopied by microinjection of LIMK1 antibody into mouse oocytes. In summary, the data demonstrate that LIMK activity is essential for MTOC organisation and distribution and so bipolar spindle formation and maintenance in mouse oocytes.

  4. Inhibition of calcineurin by FK506 stimulates germinal vesicle breakdown of mouse oocytes in hypoxanthine-supplemented medium

    PubMed Central

    Wang, Li; Zhen, Yan-Hong; Liu, Xiao-Ming; Cao, Jing; Wang, Yan-Ling

    2017-01-01

    Calcineurin (CN) is a serine/threonine phosphatase which plays important roles in meiosis maturation in invertebrate oocytes; however, the role of CN in mouse oocytes is relatively unexplored. In this study, we examined the expression, localization and functional roles of CN in mouse oocytes and granulosa cells. The RT-PCR results showed that the β isoform of calcineurin A subunit (Cn A) expressed significantly higher than α and γ isoforms, and the expression of Cn Aβ mRNA obviously decreased in oocytes in which germinal vesicle breakdown (GVBD) occurred, while only B1 of calcineurin B subunit (Cn B) was detected in oocytes and stably expressed during oocytes maturation. The following fluorescence experiment showed that Cn A was mainly located in the nucleus of germinal vesicle (GV) stage oocytes and gruanlosa cells, and subsequently dispersed into the entire cytoplasm after GVBD. The decline of Cn A in oocytes suggested that it may play an important role in GVBD. To further clarify the role of calcineurin during meiotic maturation, FK506 (a calcineurin inhibitor) was used in the culture medium contained hypoxanthine (HX) which could keep mouse oocytes staying at GV stage. As expected, FK506 could induce a significant elevation of GVBD rate and increase the MPF level of denuded oocytes (DOs). Furthermore, FK506 could also play an induction role of GVBD of oocytes in COCs and follicles, and the process could be counteracted by MAPK kinase inhibitor (U0126). Above all, the results implied that calcineurin might play a crucial role in development of mouse oocytes and MPF and MAPK pathways are involved in this process. PMID:28243539

  5. Mancozeb exposure in vivo impairs mouse oocyte fertilizability.

    PubMed

    Rossi, Gianna; Buccione, Roberto; Baldassarre, Massimiliano; Macchiarelli, Guido; Palmerini, Maria Grazia; Cecconi, Sandra

    2006-02-01

    Mancozeb is known to alter reproductive performance in exposed animals, but its specific mechanism of action is still unclear. We investigated whether in female mice of the F1 generation, mancozeb could affect oocyte ability to undergo complete meiotic maturation and fertilization. Female mice were treated with 50 and 500 mg/kg of mancozeb (or vehicle in the controls) from gestational day 2 to postnatal day 20. Results demonstrated that only at the highest dose, mancozeb induced a significant decrease in the number of ovulated eggs. Moreover, at this dose mancozeb caused a significant decrease of fertilizability related to a reduction of the formation of male and female pronuclei.

  6. 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.

  7. Quick freezing of unfertilized mouse oocytes using ethylene glycol with sucrose or trehalose.

    PubMed

    Rayos, A A; Takahashi, Y; Hishinuma, M; Kanagawa, H

    1994-01-01

    Unfertilized mouse oocytes were frozen by directly plunging them into liquid nitrogen vapour after equilibration in a freezing medium containing 3 mol ethylene glycol l-1 with 0.25 mol sucrose or trehalose l-1 for 5-40 min. After thawing and dilution of the cryoprotectant, oocytes of normal morphology were inseminated in vitro and the effect of equilibration period on the rates of fertilization and development in vitro was examined. Regardless of the equilibration in the freezing medium, no significant difference was observed on the fertilization rate of frozen-thawed oocytes. However, higher fertilization and higher normal fertilization rates were obtained with equilibration in 3 mol ethylene glycol l-1 with either 0.25 mol sucrose l-1 or trehalose for 20 and 40 min than with 5 and 10 min equilibration. Development rates to two-cell embryos and expanded blastocysts of in vitro fertilized frozen-thawed oocytes that were equilibrated in the freezing medium for 20 and 40 min were significantly higher (P < 0.05 or P < 0.01) than with 5 min equilibration. Development in vivo was assessed by transferring blastocysts derived from unfertilized oocytes frozen by the optimum treatment (20 min equilibration in the freezing medium before freezing) into the uterine horns of day 3 pseudopregnant female recipients. The development rate of frozen-thawed oocytes to the blastocyst stage after insemination in vitro was significantly lower than that of the non-frozen control (P < 0.001). However, transfer of the blastocysts derived from frozen-thawed oocytes to the uterine horns of the recipients in fetal development and implantation rates similar to those of the control.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.

    PubMed

    Stachecki, J J; Cohen, J; Willadsen, S M

    1998-12-01

    Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro. Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2 plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30 degreesC water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.

  9. The beneficial effect of repaglinide on in vitro maturation and development ability of immature mouse oocytes.

    PubMed

    Kalehoei, Eshrat; Azadbakht, Mehri

    2017-08-01

    Repaglinide is a hypoglycemic drug, causing depolarization of the cell membrane, opening the voltage-gated calcium channels, and then increasing intracellular calcium in the pancreatic B cells by inhibition of the K-ATP-sensitive channels. Oocyte in vitro maturation (IVM) is influenced by different factors such as calcium signaling. In this study, we examined the effects of repaglinide on in vitro maturation and fertilization ability of mouse oocyte. Immature oocytes were isolated from female Naval Medical Research Institute mice which are 6-8 wk old mechanically and then cultured in 30 μl droplets of T6 medium with different concentrations of repaglinide. The control group did not receive repaglinide (R0). Treatment groups received different concentrations (5, 10, and 100 nM and 1 and 10 μM) of repaglinide (R1, R2, R3, R4, and R5, respectively). Oocyte in vitro maturation rate was assessed after 24 h. In vitro fertilization was performed using metaphase II oocytes obtained from R0 and R4 treatments. Embryo cleavage rate was calculated at 48 h post-IVF. Chi-square test was used for evaluating difference between control and treatment groups (p < 0.05). Oocyte maturation rate after 24 h in treatment groups R2, R3, R4, and R5 was significantly higher than that in the control (p < 0.05). Supplementation of medium with 1 μM of repaglinide (R4) during IVM significantly improved outcome of embryo cleavage rate than control at 48 h post-IVF (p < 0.05). In conclusion, repaglinide can be considered as an effective agent for in vitro oocyte maturation and embryo cleavage.

  10. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte.

    PubMed

    Dalton, Caroline M; Carroll, John

    2013-07-01

    A fundamental rule of cell division is that daughter cells inherit half the DNA complement and an appropriate proportion of cellular organelles. The highly asymmetric cell divisions of female meiosis present a different challenge because one of the daughters, the polar body, is destined to degenerate, putting at risk essential maternally inherited organelles such as mitochondria. We have therefore investigated mitochondrial inheritance during the meiotic divisions of the mouse oocyte. We find that mitochondria are aggregated around the spindle by a dynein-mediated mechanism during meiosis I, and migrate together with the spindle towards the oocyte cortex. However, at cell division they are not equally segregated and move instead towards the oocyte-directed spindle pole and are excluded from the polar body. We show that this asymmetrical inheritance in favour of the oocyte is not caused by bias in the spindle itself but is dependent on an intact actin cytoskeleton, spindle-cortex proximity, and cell cycle progression. Thus, oocyte-biased inheritance of mitochondria is a variation on rules that normally govern organelle segregation at cell division, and ensures that essential maternally inherited mitochondria are retained to provide ATP for early mammalian development.

  11. 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.

  12. 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.

  13. Stag3 regulates microtubule stability to maintain euploidy during mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Mianqun; Dai, Xiaoxin; Sun, Yalu; Lu, Yajuan; Zhou, Changyin; Miao, Yilong; Wang, Ying; Xiong, Bo

    2017-01-01

    Stag3, a meiosis-specific subunit of cohesin complex, has been demonstrated to function in both male and female reproductive systems in mammals. However, its roles during oocyte meiotic maturation have not been fully defined. In the present study, we report that Stag3 uniquely accumulates on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Depletion of Stag3 by gene-targeting morpholino disrupts normal spindle assembly and chromosome alignment in oocytes. We also find that depletion of Stag3 reduces the acetylated level of tubulin and microtubule resistance to microtubule depolymerizing drug, suggesting that Stag3 is required for microtubule stability. Consistent with these observations, kinetochore-microtubule attachment, an important mechanism controlling chromosome alignment, is severely impaired in Stag3-depleted oocytes, resultantly causing the significantly increased incidence of aneuploid eggs. Collectively, our data reveal that Stag3 is a novel regulator of microtubule dynamics to ensure euploidy during moue oocyte meiotic maturation. PMID:27906670

  14. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte

    PubMed Central

    Dalton, Caroline M.; Carroll, John

    2013-01-01

    Summary A fundamental rule of cell division is that daughter cells inherit half the DNA complement and an appropriate proportion of cellular organelles. The highly asymmetric cell divisions of female meiosis present a different challenge because one of the daughters, the polar body, is destined to degenerate, putting at risk essential maternally inherited organelles such as mitochondria. We have therefore investigated mitochondrial inheritance during the meiotic divisions of the mouse oocyte. We find that mitochondria are aggregated around the spindle by a dynein-mediated mechanism during meiosis I, and migrate together with the spindle towards the oocyte cortex. However, at cell division they are not equally segregated and move instead towards the oocyte-directed spindle pole and are excluded from the polar body. We show that this asymmetrical inheritance in favour of the oocyte is not caused by bias in the spindle itself but is dependent on an intact actin cytoskeleton, spindle–cortex proximity, and cell cycle progression. Thus, oocyte-biased inheritance of mitochondria is a variation on rules that normally govern organelle segregation at cell division, and ensures that essential maternally inherited mitochondria are retained to provide ATP for early mammalian development. PMID:23659999

  15. Subcellular Distribution of Mitochondrial Ribosomal RNA in the Mouse Oocyte and Zygote

    PubMed Central

    Ninomiya, Youichirou; Ichinose, Shizuko

    2007-01-01

    Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote. PMID:18043748

  16. Glycine increases preimplantation development of mouse oocytes following vitrification at the germinal vesicle stage

    PubMed Central

    Cao, Xin-Yan; Rose, Jack; Wang, Shi-Yong; Liu, Yong; Zhao, Meng; Xing, Ming-Jie; Chang, Tong; Xu, Baozeng

    2016-01-01

    Ice-free cryopreservation, referred to as vitrification, is receiving increased attention in the human and animal assisted reproduction. However, it introduces the detrimental osmotic stress by adding and removing high contents of cryoprotectants. In this study, we evaluated the effects of normalizing cell volume regulation by adding glycine, an organic osmolyte, during vitrification of mouse germinal vesicle stage oocyte and/or subsequent maturation on its development. The data showed that glycine supplementation in either vitrification/thawing or maturation medium significantly improved the cytoplasmic maturation of MII oocytes manifested by spindle assembly, chromosomal alignment, mitochondrial distribution, euploidy rate, and blastocyst development following fertilization in vitro, compared to the control without glycine treatment. Furthermore, glycine addition during both vitrification/thawing and maturation further enhanced the oocyte quality demonstrated by various markers, including ATP contents and embryo development. Lastly, the effect of anti-apoptosis was also observed when glycine was added during vitrification. Our result suggests that reducing osmotic stress induced by vitrification could improve the development of vitrified mouse oocyte. PMID:27845423

  17. Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

    PubMed Central

    Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

    2012-01-01

    Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20–40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process. PMID:23203041

  18. Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes.

    PubMed

    Dietzel, E; Floehr, J; Van de Leur, E; Weiskirchen, R; Jahnen-Dechent, W

    2017-01-01

    Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. The astacin

  19. Combined inhibitory effects of low temperature and N-acetyl-l-cysteine on the postovulatory aging of mouse oocytes.

    PubMed

    Li, Qian; Cui, Long-Bo

    2016-04-01

    The postovulatory aging of oocytes eventually affects the development of oocytes and embryos. Oxidative stress is known to accelerate the onset of apoptosis in oocytes and influence their capacity for fertilisation. This study aimed to reveal the roles of temperature and the antioxidant N-acetyl-l-cysteine in preventing the aging of postovulatory mouse oocytes. First, newly ovulated mouse oocytes were cultured at various temperature and time combinations in HCZB medium with varying concentrations of N-acetyl-l-cysteine to assess signs of aging and developmental potential. When cultured in HCZB with 300 μM N-acetyl-l-cysteine at different temperature and incubation time combinations (namely 25°C for 12 h, 15°C for 24 h and 5°C for 12 h), the increase in the susceptibility of oocytes to activating stimuli was efficiently prevented, and the developmental potential was maintained following Sr2+ activation or in vitro fertilisation. After incubation at either 15°C for 36 h or 5°C for 24 h, oocytes that had decreased blastocyst rates displayed unrecoverable abnormal cortical granule distribution together with decreased BCL2 levels, total glutathione concentrations and glutathione/glutathione disulphide (GSH/GSSG) ratios. In conclusion, postovulatory oocyte aging could be effectively inhibited by appropriate N-acetyl-l-cysteine addition at low temperatures. In addition, a simple method for the temporary culture of mature oocytes was established.

  20. Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation.

    PubMed

    Yoon, Jeong; Juhn, Kyoung-Mi; Yoon, San-Hyun; Ko, Yong; Lim, Jin-Ho

    2017-03-01

    The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca(2)(+) chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca(2)(+) chelator to investigate the effect of Ca(2)(+) oscillations on their maturation. As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca(2)(+) chelator-treated group. The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca(2)(+) chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca(2)(+) oscillations driven by fertilization.

  1. De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes.

    PubMed

    Chiba, Hatsune; Hirasawa, Ryutaro; Kaneda, Masahiro; Amakawa, Yuko; Li, En; Sado, Takashi; Sasaki, Hiroyuki

    2008-12-01

    Mouse blastocyst stage embryo stained for histone H3 lysine-27 trimethylation (red) and DNA (blue). H3K27me3 marks the inactive X chromosome. The study by Chiba et al. in this issue suggests that de novo DNA methyltransferases are dispensable for setting the imprint on the maternally-derived X chromsome in growing oocytes. See Chiba et al. in this issue.

  2. Melatonin prevents postovulatory oocyte aging in the mouse and extends the window for optimal fertilization in vitro.

    PubMed

    Lord, Tessa; Nixon, Brett; Jones, Keith T; Aitken, R John

    2013-03-01

    The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence, and increased likelihood of embryonic resorption. Because oxidative stress accelerates the onset of apoptosis in oocytes and influences their capacity for fertilization, this study aimed to characterize the significance of such stress in the postovulatory aging of mouse oocytes in vitro. We investigated the ability of the potent antioxidant melatonin to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence. Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the time-dependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 and 16 h in vitro (P < 0.05) and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro.

  3. 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.

  4. 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.

  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. In Vivo acrylamide exposure may cause severe toxicity to mouse oocytes through its metabolite glycidamide

    PubMed Central

    Aras, Duru; Cakar, Zeynep; Ozkavukcu, Sinan; Can, Alp; Cinar, Ozgur

    2017-01-01

    High acrylamide (ACR) content in heat-processed carbohydrate-rich foods, as well as roasted products such as coffee, almonds etc., has been found to be as a risk factor for carcinogenicity and genotoxicity by The World Health Organization. Glycidamide (GLY), the epoxide metabolite of ACR, is processed by the cytochrome P-450 enzyme system and has also been found to be a genotoxic agent. The aim of this study was to determine whether ACR and/or GLY have any detrimental effect on the meiotic cell division of oocytes. For this purpose, germinal vesicle-stage mouse oocytes were treated with 0, 100, 500, or 1000 μM ACR or 0, 25, or 250 μM GLY in vitro. In vivo experiments were performed after an intraperitoneal injection of 25 mg/kg/day ACR of female BALB/c mice for 7 days. The majority of in vitro ACR-treated oocytes reached the metaphase-II stage following 18 hours of incubation, which was not significantly different from the control group. Maturation of the oocytes derived from in vivo ACR-treated mice was impaired significantly. Oocytes, reaching the M-II stage in the in vivo ACR-treated group, were characterized by a decrease in meiotic spindle mass and an increase in chromosomal disruption. In vitro GLY treatment resulted in the degeneration of all oocytes, indicating that ACR toxicity on female germ cells may occur through its metabolite, GLY. Thus, ACR exposure must be considered, together with its metabolite GLY, when female fertility is concerned. PMID:28182799

  7. Extracellular stimulation by serum proteins required for maximal intracellular killing of microorganisms by mouse peritoneal macrophages.

    PubMed Central

    Leijh, P C; van Zwet, T L; van Furth, R

    1984-01-01

    Intracellular killing of catalase-positive Staphylococcus aureus by resident mouse peritoneal macrophages was very low in the absence of serum but maximal in the presence of fresh normal serum. A large proportion of catalase-negative Streptococcus pyogenes were killed in the absence of extracellular serum, and maximal killing was reached only when serum was present extracellularly. Further investigations revealed that stimulation of intracellular killing by extracellular serum is dependent on the interaction of immunoglobulin G and Fc receptors and of complement component C3b with C3b receptors in the macrophage membrane. PMID:6238911

  8. Inhibitory effects of pre- and posttest drugs on mouse-killing by rats.

    PubMed

    Gay, P E; Leaf, R C; Arble, F B

    1975-01-01

    Mouse-killing in rats was gradually inhibited by repeated posttest injections of d-amphetamine (1.5 mg/kg), l-amphetamine (1.5 mg/kg) or pilocarpine (7.5 mg/kg), but not by control substances. Of these drugs, only d-amphetamine inhibited killing when given prior to a mouse-killing test. Further experiments suggested that anorexia per se did not contribute to drug-induced inhibitory effects, but that changes in internal state were important to the development of inhibition. Pretest injections appear to inhibit predatory killing by a direct pharmacological action on some target site or sites, while posttest injections produce a learned aversion to predatory killing.

  9. Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

    PubMed

    Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

    2012-01-01

    Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

  10. Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth.

    PubMed

    Thomas, Fiona H; Vanderhyden, Barbara C

    2006-04-12

    Ovarian folliculogenesis is regulated by both endocrine and intraovarian mechanisms that coordinate the processes of oocyte growth and somatic cell proliferation and differentiation. Within the follicle, paracrine interactions between the oocyte and surrounding granulosa cells are critical for normal cell development and function. This review focuses on the role of paracrine interactions during early oocyte and follicular development that ensure proper coordination of oocyte and somatic cell function. Particular emphasis is given to granulosa cell-derived Kit Ligand (KitL), whose functional importance for oocyte growth has been demonstrated by a wide range of in vivo and in vitro studies. Reported interactions between KitL and oocyte-derived growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) suggest the molecular basis of oocyte-granulosa cell interactions, but also hint at the complexity of these communications. These paracrine interactions and the structure of the oocyte-granulosa cell interface are follicle stage-specific and regulated by FSH. Elucidation of the molecular mechanisms that promote the development of healthy oocytes with good developmental competence has potential applications for improving fertility and for in vitro growth systems for oocytes from domestic animals and humans.

  11. Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment.

    PubMed

    Niikura, Yuichi; Niikura, Teruko; Tilly, Jonathan L

    2009-12-12

    Of all the major organ systems in the body, the ovaries of females are the first to exhibit impaired function with advancing age. Until recently, traditional thinking was that female mammals are provided with a non-renewable pool of oocyte-containing follicles at birth that are depleted during postnatal life to exhaustion, driving ovarian failure. However, a growing body of evidence, including the isolation of germline stem cells (GSC) from adult mouse ovaries that produce developmentally-competent oocytes, has challenged this belief. In addition, rare germline stem-like cells capable of generating oocytes in vitro that undergo parthenogenesis to form blastocyst-like structures have recently been identified in postmenopausal human ovaries. Here we show that the germline-specific meiosis-commitment genes,Stimulated by retinoic acid gene 8 (Stra8) and Deleted in azoospermia-like (Dazl), are highly expressed in aged mouse ovaries. However, histological and marker analyses fail to demonstrate the presence of oocytes, supporting that Stra8 and Dazl are expressed in premeiotic germ cells that do not undergo further differentiation. Through the use of aged germline-specific GFP-expressing transgenic mice, we further show that these germ cells can generate GFP-positive oocytes that co-express the primordial oocyte marker NOBOX and form follicles when grafted into young adult wild-type female hosts. Thus, aged mouse ovaries possess a rare population of premeiotic germ cells that retain the capacity to form oocytes if exposed to a young host environment.

  12. DYNLT3 is required for chromosome alignment during mouse oocyte meiotic maturation.

    PubMed

    Huang, Xin; Wang, Hai-Long; Qi, Shu-Tao; Wang, Zhen-Bo; Tong, Jing-Shan; Zhang, Qing-Hua; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Qi, Zhong-Quan; Sun, Qing-Yuan

    2011-10-01

    Dynein light chain, Tctex-type 3 (DYNLT3), is a member of the cytoplasmic dynein DYNLT light chain family and has been reported to have a potential role in chromosome congression in human mitosis. However, its role in mammalian meiosis is unclear. In this study, we examined its localization, expression, and functions in mouse oocyte meiosis. Immunofluorescent staining showed that DYNLT3 was restricted to the germinal vesicle and associated with kinetochores at the germinal vesicle breakdown stage, metaphase I and metaphase II. The expression level of DYNLT3 was similar at all meiotic stages. Depletion of DYNLT3 by antibody injection resulted in chromosome misalignment and decrease of the polar body extrusion rate. We further found that DYNLT3-depleted oocytes displayed kinetochore-microtubule detachments. Chromosome-spread experiments showed that depletion of DYNLT3 inhibited the metaphase-anaphase transition by preventing homologous chromosome segregation in meiosis I. Our data suggest that DYNLT3 is required for chromosome alignment and homologous chromosome segregation during mouse oocyte meiosis.

  13. Abce1 orchestrates M-phase entry and cytoskeleton architecture in mouse oocyte

    PubMed Central

    Jiao, Xiao-Fei; Huang, Chun-Jie; Wu, Di; Zhang, Jia-Yu; Long, Yu-Ting; Chen, Fan; Li, Xiang; Huo, Li-Jun

    2017-01-01

    ATP-binding cassette E1 (ABCE1) is a member of the ATP-binding cassette transporters and essential for diverse biological events regulating abroad range of biological functions including viral infection, cell proliferation, anti-apoptosis, translation initiation and ribosome biogenesis. Here, we discovered that Abce1 also plays indispensable roles in mouse oocyte meiotic progression. In the present study, we examined the expression, localization, and function of Abce1 during mouse oocyte meiotic maturation. Immunostaining and confocal microscopy identified that Abce1 localized as small dots in nucleus in germinal vesicle stage. After germinal vesicle breakdown, it dispersedly localized around the whole spindle apparatus. During the anaphase and telophase stages, Abce1 was just like a cap to localize around the two pole region of spindle but not the midbody and chromosome. Knockdown of Abce1 by specific siRNA injection delayed the resumption of meiosis (GVBD) and affected the extrusion of first polar body. Moreover, the process of spindle assembly and chromosome alignment were severely impaired. Abce1-RNAi led to the dissociation of γ-tubulin and p-MAPK from spindle poles, thus caused mounts of spindle morphology abnormities and chromosome alignment defects, leading to high incidence of aneuploidy. Our findings refresh the knowledge of Abce1 function by exploring its role in oocyte meiotic resumption, spindle assembly and chromosome alignment. PMID:28380459

  14. Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

    PubMed Central

    Salisbury, Jesse; Hutchison, Keith W.; Wigglesworth, Karen; Eppig, John J.; Graber, Joel H.

    2009-01-01

    Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries. PMID:19834616

  15. Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.

    PubMed

    Bui, Thi Thu Hien; Belli, Martina; Fassina, Lorenzo; Vigone, Giulia; Merico, Valeria; Garagna, Silvia; Zuccotti, Maurizio

    2017-02-24

    Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species.

  16. Successful Cryopreservation of Mouse Oocytes by Using Low Concentrations of Trehalose and Dimethylsulfoxide1

    PubMed Central

    Eroglu, Ali; Bailey, Sarah E.; Toner, Mehmet; Toth, Thomas L.

    2008-01-01

    Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., −15°C), which diminishes with further cooling of the oocytes to −30°C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to −196°C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes. PMID:18815355

  17. Successful cryopreservation of mouse oocytes by using low concentrations of trehalose and dimethylsulfoxide.

    PubMed

    Eroglu, Ali; Bailey, Sarah E; Toner, Mehmet; Toth, Thomas L

    2009-01-01

    Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., -15 degrees C), which diminishes with further cooling of the oocytes to -30 degrees C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to -196 degrees C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes.

  18. 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.

  19. In mouse oocytes the mitochondrion-originated germinal body-like structures accumulate mouse Vasa homologue (MVH) protein.

    PubMed

    Reunov, Arkadiy A; Reunova, Yulia A

    2015-08-01

    Mouse Vasa homologue (MVH) antibodies were applied to mouse Graafian oocytes to clarify if mitochondrion-originated germinal body-like structures, described previously by conventional electron microscopy, were associated with the germ plasm. It was found that both the mitochondrion-like structures with cristae and the germinal body-like structures that lacked any signs of cristae were labelled specifically by the anti-MVH antibody. Moreover, some granules were MVH-positive ultrastructural hybrids of the mitochondria and germinal body-like structures, the presence of which clearly supported the idea of a mitochondrial origin for the germinal body-like structures. This finding is the first evidence that mitochondrion-originated germinal body-like granules represent mouse germ plasm.

  20. Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

    PubMed Central

    2010-01-01

    Background RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. Results Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. Conclusions We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte. PMID:20939886

  1. Four-dimensional visualization and quantitative analysis of meiotic spindle movements in live mouse oocytes.

    PubMed

    Tian, N; Zhang, L; Liu, B; Wang, P; Li, Y; Ma, W

    2012-09-01

    This paper made a different attempt of real-time observation of the meiotic spindle movements in living mouse oocyte using a convenient method. This method was based on an experimental phenomenon discovered in our work. In living mouse oocytes, a high concentration of calcium ions (Ca(2+)) was observed throughout the region occupied by the initial meiotic spindle. After Ca(2+) labelling with Fura-2, a weakly fluorescent area (WFA) appeared on each side of the chromosomes. The activities of the WFAs changed during spindle development. By real-time tracking of WFAs, we were able to indirectly observe the meiotic spindle movements. Occasionally, it was observed that the first meiotic spindle rotated from an orientation parallel to the cortex to become perpendicular, instead of migrating from the oocyte centre to the cortex along its axis. Moreover, we analysed this uncommon rotation of the first meiotic spindle and found that the whole rotation process can be divided into two phases: the early slow-speed rotation and the subsequent rapid-speed rotation. We further characterized this rotation with respect to rotational speed and acceleration at all the stages of development. By using a two-photon laser-scanning microscope in combination with Fura-2 dye that is nondamaging to oocytes, we provide a convenient method for indirect visualization and quantitative analysis of spindle movements by real-time tracking of WFAs. This method is easy to operate and master, and economical with time and effort. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

  2. Melatonin prevents hypochlorous acid-induced alterations in microtubule and chromosomal structure in metaphase-II mouse oocytes.

    PubMed

    Banerjee, Jashoman; Maitra, Dhiman; Diamond, Michael P; Abu-Soud, Husam M

    2012-09-01

    Hypochlorous acid (HOCl) is generated by myeloperoxidase, using chloride and hydrogen peroxide as substrates. Here we demonstrate that HOCl alters metaphase-II mouse oocyte microtubules and chromosomal (CH) alignment which can be prevented by melatonin. Metaphase-II mouse oocytes, obtained commercially, were grouped as: control, melatonin (150, 200nmol/mL), HOCl (10, 20, 50, and 100nmol/mL), and HOCl (50nmol/mL) pretreated with 150 and 200 nmol/mL of melatonin. Microtubule and CH alignment was studied utilizing an indirect immunofluorescence technique and scored by two observers. Pearson chi-square test and Fisher's exact test were used to compare outcomes between controls and treated groups and also among each group. Poor scores for the spindle and chromosomes increased significantly at 50nmol/mL of HOCl (P<0.001). Oocytes treated with melatonin only at 150 and 200 nmol/mL showed no changes; significant differences (P<0.001) were observed when oocytes exposed to 50nmol/mL of HOCl were compared to oocytes pretreated with 200 nmol/mL melatonin. Fifty percent of the oocytes demonstrated good scores, both in microtubule and CH alterations, when pretreated with melatonin at 150 nmol/mL compared to 0% in the HOCl-only group. HOCl alters the metaphase-II mouse oocyte spindle and CH alignment in a dose-dependant manner, which might be a potential cause of poor oocyte quality (e.g., in patients with endometriosis). Melatonin prevented the HOCl-mediated spindle and CH damage, and therefore, may be an attractive therapeutic option to prevent oocyte damage in endometriosis or inflammatory diseases where HOCl levels are known to be elevated. © 2012 John Wiley & Sons A/S.

  3. 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

  4. 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.

  5. Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca2+ oscillations required for mammalian oocyte activation

    PubMed Central

    Nomikos, Michail; Theodoridou, Maria; Elgmati, Khalil; Parthimos, Dimitris; Calver, Brian L.; Buntwal, Luke; Nounesis, George; Swann, Karl; Lai, F. Anthony

    2014-01-01

    A sperm-specific phospholipase C-zeta (PLCζ) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLCζ has been shown to trigger a prolonged series of repetitive Ca2+ transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca2+ signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLCζ from disparate mammalian species to trigger Ca2+ oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLCζ species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLCζ relative to mouse PLCζ in generating Ca2+ oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLCζ chimeras to enable study of the potential role of discrete PLCζ domains in conferring the enhanced Ca2+ signalling potency of human PLCζ. Functional analysis of these human/mouse PLCζ domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLCζ activity. Our empirical observations are compatible with a basic mathematical model for the Ca2+ dependence of generating cytoplasmic Ca2+ oscillations in mammalian oocytes by sperm PLCζ. PMID:24478462

  6. Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols.

    PubMed

    Berthelot-Ricou, Anais; Perrin, Jeanne; di Giorgio, Carole; de Meo, Michel; Botta, Alain; Courbiere, Blandine

    2013-09-01

    To assess the genotoxicity of three oocyte vitrification protocols. Murine assay. Biogenotoxicology research laboratory. CD1 female mice. Three mouse oocyte groups were exposed to three commercialized human oocyte vitrification protocols. Protocols 1 and 2 contained dimethyl sulfoxide and ethylene glycol (EG), and protocol 3 contained EG and 1,2-propanediol (PrOH). DNA damage was first evaluated by comet assay after oocyte exposure to the three different equilibration and vitrification solutions. Comet assay was also performed after full vitrification and warming procedure and compared with a negative control group (oocytes stored in medium culture only) and a positive control group (oocytes exposed to hydrogen peroxide just before comet assay). DNA damage was quantified as Olive tail moment (OTM). Statistical analysis consisted of a Shapiro-Wilk test. Then, median protocol OTM was compared with the negative control group with the Mann-Whitney U test. The difference was considered to be statistically significant if the P value was <.05. In both parts of our study, protocols 1 and 2 did not induce significant DNA damage, whereas protocol 3 induced statistically higher DNA damage compared with the negative control group. Vitrification protocols containing PrOH induced significant DNA damage on mouse oocytes, both before cooling and after warming. Therefore, for the moment, we prefer vitrification techniques without PrOH while we await more studies on PrOH toxicity and long-term evaluation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  7. Assessment of Mouse Germinal Vesicle Stage Oocyte Quality by Evaluating the Cumulus Layer, Zona Pellucida, and Perivitelline Space

    PubMed Central

    Liu, Ying-Lei; Chen, Ying; Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Liang, Cheng-Guang

    2014-01-01

    To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications. PMID:25144310

  8. Cross species fertilization and development investigated by cat sperm injection into mouse oocytes.

    PubMed

    Xu, Yong-Nan; Cui, Xiang-Shun; Sun, Shao-Chen; Jin, Yong-Xun; Kim, Nam-Hyung

    2011-07-01

    The use of intracytoplasmic sperm injection (ICSI) in model animals is a powerful approach for the study of species-specific fertilization processes and multiploidy embryogenesis. In this study, we examined the fertilization process in mouse oocytes following injection of a single mouse or cat sperm, two mouse spermatozoa or mouse and cat spermatozoa. These treatments did not affect histone H3K9 acetylation or methylation, although the pattern of DNA methylation differed following the injection of cat sperm. Immunocytochemical staining revealed that sperm chromatin was normally incorporated with female mouse chromatin following any of the four injection scenarios. Furthermore, metaphase was successfully entered to reach a normal two-cell stage, and cell division could even persist to produce blastocyst stage embryos. In addition, both mouse and cat Pou5l and Nanog mRNA were expressed in the hybrid embryos. These results suggest that, although epigenetic modification of DNA is affected by the sperm injection treatment, fertilization and cleavage occur in a non-species-specific manner. In addition, despite abnormal division of the chromosomes, intra- and inter-species ICSI produced embryos that could develop into blastocysts.

  9. 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

  10. Multiple conductance classes of mouse nicotinic acetylcholine receptors expressed in Xenopus oocytes.

    PubMed Central

    Kullberg, R; Owens, J L; Camacho, P; Mandel, G; Brehm, P

    1990-01-01

    Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were injected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of alpha-, beta-, gamma-, and delta-subunit mRNAs produced two predominant channel classes with conductances of approximately 50 and approximately 12 pS, while infrequent openings of approximately 25-pS channels were also observed. Injection of alpha-, beta-, and gamma-subunit mRNAs produced a single class of approximately 12-pS AcChoR channels, which resembled the smallest conductance channels present in alpha beta gamma omega-injected oocytes. Assembly of delta-less channels may thus explain the lowest conductance AcChoR channels in alpha beta gamma delta-injected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle. Images PMID:2315303

  11. Ultrastructure of the oocytes of the Egyptian sping mouse (Acomys cahirinus).

    PubMed

    Kang, Y H; Anderson, W A

    1975-06-01

    The oocytes of types 2, 3, 4 and 5 follicles from the normal spiny mouse were examined withe the electron microsome. Multiple juxtanuclear Golgi bodies, mitochondria associated with flattened granular endoplasmic reticulum, and large nucleus are the main features of the type 2 follicle oocyte. The numbers of mitochondria and Golgi apparati increase significantly at later stages. Small mitochondrial aggregates lacking "intermitochondrial cement" are seen in the ooplasm of types 3, 4 and 5 follicles. "Lamellar complexes" comprising two to six elongate flattened rough ER cisternae and intercisternal filaments begin to appear in the occyte of the type 3 follicle. The intercisternal filaments may be observed as punctate-, dashed-, and solid-lines in cross sections. In tangential sections the filaments display a paracrystalline structure. In the type 4 follicle oocytes, the "lamellar complex" becomes more extensive; polysomes and ribosomal fibrils are juxtaposed to the "lamellar complexes." Bundles of ribosomal fibrils are abundant in the ooplasm of the type 5 follicle. The origin of ribosomal filaments and the functional significance of "lamellar complexes" are discussed.

  12. 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

  13. Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

    PubMed

    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

  14. Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation

    PubMed Central

    Yoon, Jeong; Juhn, Kyoung-Mi; Yoon, San-Hyun; Lim, Jin-Ho

    2017-01-01

    Objective The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca2+ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). Methods MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca2+ chelator to investigate the effect of Ca2+ oscillations on their maturation. Results As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca2+ chelator-treated group. Conclusion The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca2+ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca2+ oscillations driven by fertilization. PMID:28428939

  15. Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis.

    PubMed

    Li, Mo; Li, Sen; Yuan, Ju; Wang, Zhen-Bo; Sun, Shao-Chen; Schatten, Heide; Sun, Qing-Yuan

    2009-11-02

    In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1-3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.

  16. Ionic currents activated via purinergic receptors in the cumulus cell-enclosed mouse oocyte.

    PubMed

    Arellano, Rogelio O; Martínez-Torres, Ataulfo; Garay, Edith

    2002-09-01

    Several chemical signals synthesized in the ovary, including neurotransmitters, have been proposed to serve as regulators of folliculogenesis, however, their mechanisms of action have not been completely elucidated. Here, electrophysiological and molecular biology techniques were used to study responses generated via purinergic stimulation in cultured mouse cumulus cell-enclosed oocytes (CEOs). Application of extracellular ATP elicited depolarizing responses in CEOs. Using the voltage clamp technique by impaling oocytes with two microelectrodes, we determined that these responses were mainly due to activation of two distinct ionic currents. The first corresponded to the opening of Ca2+-dependent Cl- channels (I(Cl(Ca))) and the second to the opening of Ca2+-independent channels that are permeable to Na+ (I(c+)). The potency order for different nucleotides (50 micro M) was UTP > ATP > 2meS-ATP > ADP, and alpha,betame-ATP and adenosine were found to be inactive. Suramin (100 micro M) blocked the response elicited by ATP or UTP. In addition, voltage dependent K+ currents activated by depolarization of CEOs were characterized. All CEO ionic currents recorded from the oocyte were completely inhibited by octanol (1 mM), a gap junction blocker. Thus, purinergic responses and K+ currents originate mainly in the membrane of cumulus cells. Transcripts of the purinergic receptor P2Y2 subtype were amplified by polymerase chain reaction from the cDNA of granulosa cells or cumulus cells. This study shows that P2Y2 receptors are expressed in CEOs, and that their stimulation opens at least two different types of ion channels. Both the ion channels and the receptors seemed to be located in the cumulus cells, which transmit their corresponding electrical signals to the oocyte via gap junction channels.

  17. Endoplasmic reticulum-derived multilamellar bodies in oocytes of mouse follicle cultures under oxidized low-density lipoprotein treatment.

    PubMed

    Spanel-Borowski, Katharina; Nowicki, Marcin; Borlak, Juergen; Trapphoff, Tom; Eichenlaub-Ritter, Ursula

    2013-01-01

    Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL). Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12-48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy. Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress. Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy. Copyright © 2012 S. Karger AG, Basel.

  18. 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

  19. 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.

  20. Effects of Trichostatin A on Cumulus Expansion during Mouse Oocyte Maturation.

    PubMed

    Du, Ming; Fu, Xiangwei; Zhou, Yanhua; Zhu, Shien

    2013-11-01

    This study was conducted to investigate the effects of Trichostatin A (TSA) on cumulus expansion during mouse oocyte maturation. TSA treatment inhibited cumulus expansion and significantly reduced the cumulus expansion index (CEI) (p<0.05). To determine the underlying mechanism, the expression levels of several key factors that play crucial roles in cumulus expansion including components of extracellular matrix (ECM) (Has2, Ptgs2, Ptx3, and Tnfaip6) and Growth differentiation factor 9 (GDF9) were measured in control and TSA treated samples by real-time PCR. The effect of TSA on ERK phosphorylation (p-ERK1/2) in cumulus cells and GDF9 protein level in fully grown oocytes (FGOs) were detected by Western blotting. The expression levels of the ECM genes were significantly decreased (p<0.05) by TSA treatment while GDF9 expression did not response to TSA (p>0.05). TSA treatment blocked the activation of ERK1/2 (p<0.05) and had no significant effect on GDF9 protein expression (p>0.05). Collectively, these results suggested that TSA treatment altered ECM gene expression and blocked ERK1/2 activation to inhibit cumulus expansion in the mouse.

  1. APC(FZR1) prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing.

    PubMed

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

    2012-10-01

    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 APC(CDC20) 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 APC(CDC20) activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction.

  2. 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

  3. Regulation of fatty acid oxidation in mouse cumulus-oocyte complexes during maturation and modulation by PPAR agonists.

    PubMed

    Dunning, Kylie R; Anastasi, Marie R; Zhang, Voueleng J; Russell, Darryl L; Robker, Rebecca L

    2014-01-01

    Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of ³H₂O from [³H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid

  4. 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

  5. Chronic Unpredictable Stress Decreases Expression of Brain-Derived Neurotrophic Factor (BDNF) in Mouse Ovaries: Relationship to Oocytes Developmental Potential

    PubMed Central

    Tong, Xian-Hong; Han, Hui; Shen, Ni; Jin, Ren-Tao; Wang, Wei; Zhou, Gui-Xiang; He, Guo-Ping; Liu, Yu-Sheng

    2012-01-01

    Background Brain-derived neurotropic factor (BDNF) was originally described in the nervous system but has been shown to be expressed in ovary tissues recently, acting as a paracrine/autocrine regulator required for developments of follicles and oocytes. Although it is generally accepted that chronic stress impairs female reproduction and decreases the expression of BDNF in limbic structures of central nervous system, which contributes to mood disorder. However, it is not known whether chronic stress affects oocytes developments, nor whether it affects expression of BDNF in ovary. Methods Mice were randomly assigned into control group, stressed group, BDNF-treated group and BDNF-treated stressed group. The chronic unpredictable mild stress model was used to produce psychosocial stress in mice, and the model was verified by open field test and hypothalamic-pituitary-adrenal (HPA) axis activity. The methods of immunohistochemistry and western blotting were used to detect BDNF protein level and distribution. The number of retrieved oocytes, oocyte maturation, embryo cleavage and the rates of blastocyst formation after parthenogenetic activation were evaluated. Results Chronic unpredictable stress decreased the BDNF expression in antral follicles, but didn’t affect the BDNF expression in primordial, primary and secondary follicles. Chronic unpredictable stress also decreased the number of retrieved oocytes and the rate of blastocyst formation, which was rescued by exogenous BDNF treatment. Conclusion BDNF in mouse ovaries may be related to the decreased number of retrieved oocytes and impaired oocytes developmental potential induced by chronic unpredictable stress. PMID:23284991

  6. Mouse cumulus-denuded oocytes restore developmental capacity completely when matured with optimal supplementation of cysteamine, cystine, and cumulus cells.

    PubMed

    Zhou, Ping; Wu, Yan-Guang; Wei, De-Li; Li, Qing; Wang, Gang; Zhang, Jie; Luo, Ming-Jiu; Tan, Jing-He

    2010-04-01

    Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

  7. 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

  8. Injury effects of ginkgolide B on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo.

    PubMed

    Shiao, Nion-Heng; Chan, Wen-Hsiung

    2009-07-10

    Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1-6 microM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3-6 microM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.

  9. 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

  10. KIF2A regulates the spindle assembly and the metaphase I-anaphase I transition in mouse oocyte

    PubMed Central

    Chen, Ming-Huang; Liu, Yu; Wang, Ya-Long; Liu, Rui; Xu, Bai-Hui; Zhang, Fei; Li, Fei-Ping; Xu, Lin; Lin, Yan-Hong; He, Shu-Wen; Liao, Bao-Qiong; Fu, Xian-Pei; Wang, Xiao-Xue; Yang, Xiang-Jun; Wang, Hai-Long

    2016-01-01

    KIF2A, a member of the kinesin-13 family, has been reported to play a role in spindle assembly in mitosis. However, its function in mammalian meiosis remains unknown. In this research, we examined the expression, localization and function of KIF2A during mouse oocyte meiosis. KIF2A was expressed in some key stages in mouse oocyte meiosis. Immunofluorescent staining showed that KIF2A distributed in the germinal vesicle at the germinal vesicle stage and as the spindle assembling after meiosis resumption, KIF2A gradually accumulated to the entire spindle. The treatment of oocytes with taxol and nocodazole demonstrated that KIF2A was co-localized with α-tubulin. Depletion of KIF2A by specific short interfering (si) RNA injection resulted in abnormal spindle assembly, failure of spindle migration, misaligned chromosomes and asymmetric cell division. Meanwhile, SKA1 expression level was decreased and the TACC3 localization was disrupted. Moreover, depletion of KIF2A disrupted the actin cap formation, arrested oocytes at metaphase I with spindle assembly checkpoint protein BubR1 activated and finally reduced the rate of the first polar body extrusion. Our data indicate that KIF2A regulates the spindle assembly, asymmetric cytokinesis and the metaphase I-anaphase I transition in mouse oocyte. PMID:27991556

  11. Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment

    PubMed Central

    Niikura, Yuichi; Niikura, Teruko; Tilly, Jonathan L.

    2009-01-01

    Of all the major organ systems in the body, the ovaries of females are the first to exhibit impaired function with advancing age. Until recently, traditional thinking was that female mammals are provided with a non-renewable pool of oocyte-containing follicles at birth that are depleted during postnatal life to exhaustion, driving ovarian failure. However, a growing body of evidence, including the isolation of germline stem cells (GSC) from adult mouse ovaries that produce developmentally-competent oocytes, has challenged this belief. In addition, rare germline stem-like cells capable of generating oocytes in vitro that undergo parthenogenesis to form blastocyst-like structures have recently been identified in postmenopausal human ovaries. Here we show that the germline-specific meiosis-commitment genes,Stimulated by retinoic acid gene 8 (Stra8) and Deleted in azoospermia-like (Dazl), are highly expressed in aged mouse ovaries. However, histological and marker analyses fail to demonstrate the presence of oocytes, supporting that Stra8 and Dazl are expressed in premeiotic germ cells that do not undergo further differentiation. Through the use of aged germline-specific GFP-expressing transgenic mice, we further show that these germ cells can generate GFP-positive oocytes that co-express the primordial oocyte marker NOBOX and form follicles when grafted into young adult wild-type female hosts. Thus, aged mouse ovaries possess a rare population of premeiotic germ cells that retain the capacity to form oocytes if exposed to a young host environment. PMID:20157580

  12. 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.

  13. Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes.

    PubMed

    Watanabe, H; Tateno, H; Kusakabe, H; Matsuoka, T; Kamiguchi, Y; Fujise, Y; Ishikawa, H; Ohsumi, S; Fukui, Y

    2007-02-01

    Prior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3-97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2-93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.

  14. Effects of various combinations of cryoprotectants and cooling speed on the survival and further development of mouse oocytes after vitrification.

    PubMed

    Cha, Soo Kyung; Kim, Bo Yeun; Kim, Mi Kyung; Kim, You Shin; Lee, Woo Sik; Yoon, Tae Ki; Lee, Dong Ryul

    2011-03-01

    The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen (SN(2)). Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into SN(2) or liquid nitrogen (LN(2)). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using SN(2) were increased in both the EG only and EG+DMSO groups. A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, SN(2) may improve the efficiency of vitrification by reducing cryoinjury.

  15. Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes.

    PubMed

    Lee, Hyun-Seo; Kim, Kyeoung-Hwa; Kim, Eun-Young; Lee, Su-Yeon; Ko, Jung-Jae; Lee, Kyung-Ah

    2016-04-01

    Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.

  16. Oocyte activation ability correlates with head flatness and presence of perinuclear theca substance in human and mouse sperm.

    PubMed

    Ito, Chizuru; Akutsu, Hidenori; Yao, Ryoji; Kyono, Koichi; Suzuki-Toyota, Fumie; Toyama, Yoshiro; Maekawa, Mamiko; Noda, Tetsuo; Toshimori, Kiyotaka

    2009-10-01

    Recent studies indicate that round-headed sperm cannot activate oocytes and lack the postacrosomal sheath (PAS) or perinuclear theca (PT), although normal flat-headed sperm can activate oocytes and do have PAS (PT). In this study, we investigated how oocyte activation ability correlates with sperm head morphology (round and flat) and the presence of PT, by studying MN13, a representative molecule of the PT. We analyzed sperm with flat and round heads from infertile patients with globozoospermia (n = 1) and teratozoospermia (n = 1), and also from GOPC(-/-) mice, an animal model of human globozoospermia. Differential interference contrast image analysis, immunocytochemistry with MN13 antibody, transmission electron microscopy and an oocyte activation assay (assessing pronucleus formation) with ICSI were used. Flat-headed (control) sperm from both a healthy fertile volunteer man and wild-type mice had MN13 and PAS (PT). Flat-headed sperm (<5% of the population) from GOPC(-/-) mice also had both MN13 and PAS (PT), and they showed high oocyte activation ability. In contrast, round-headed sperm from a globozoospermia patient (100%) and GOPC(-/-) mice (>95% of the population) had neither MN13, nor PAS (PT), nor oocyte activation ability. Oocyte activation was higher in flat- versus round-headed sperm from GOPC(-/-) mice (P < 0.05). Oocyte activation ability may be related to sperm head flatness and presence of MN13 and PAS (PT) in human and mouse sperm. This information is a first step towards the possibility of selecting good-quality sperm with high oocyte activation ability for ICSI.

  17. 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.

  18. Activin A accelerates the progression of fetal oocytes throughout meiosis and early oogenesis in the mouse.

    PubMed

    Liang, Gui-Jin; Zhang, Xi-Feng; Wang, Jun-Jie; Sun, Yuan-Chao; Sun, Xiao-Feng; Cheng, Shun-Feng; Li, Lan; De Felici, Massimo; Shen, Wei

    2015-10-15

    Activins can exert several roles in ovary development. However, little is known about their involvement in early mammalian oogenesis. In this study, we reported that activin receptors (including ActRIA, ActRIB, ActRIIA, and ActRIIB) are expressed throughout the development of the mouse ovaries from 12.5 days postcoitum (dpc) to 21 days postparturition (dpp). Moreover, we found that in vitro, the addition of activin A (ActA) to the culture medium of 12.5 dpc ovarian tissues accelerated the progression of oocytes throughout meiotic prophase I stages. This result was reproduced in vivo following administration of ActA to pregnant mice. The in vitro effect of ActA was associated with increased expression of premeiotic and meiotic genes (including Dazl, Spo11, Stra8, Scp3, and Rec8) in the ovarian tissues. Mechanistically, ActA-dependent SMAD3 signaling modulated the expression of members of the retinoic acid (RA) system, including the RA degradation CYP26B1 enzyme and the RA receptors. Finally, ActA promoted the survival and growth of fetal and early postnatal oocytes and primordial follicle assembly both in vitro and in vivo. In conclusion, the present study identifies new roles of ActA in early oogenesis and suggested that ActA and RA might cooperate in promoting meiosis in female germ cells.

  19. Pronuclear epigenetic modification of protamine deficient human sperm following injection into mouse oocytes.

    PubMed

    Rajabi, Hoda; Mohseni-Kouchesfehani, Homa; Mohammadi-Sangcheshmeh, Abdollah; Farifteh-Nobijari, Fattaneh; Salehi, Mohammad

    2016-01-01

    Epigenetic abnormalities and abnormal chromatin structure in sperm may lead to male infertility. Protamine deficiency is among the disorders of chromatin structure in sperm. The study of epigenetic changes in male pronuclei is necessary since abnormal sperm is sometimes used to create embryos using assisted reproductive techniques. The present study was carried out to compare epigenetic global marks in male pronuclei derived from normal and protamine deficient sperm cells. To do so, interspecies fertilization was used to obtain the male pronucleus. Normal and protamine deficient sperm cells, which were identified by chromomycin A3 staining, were injected into mouse oocytes. Oocytes were cultured until pronuclear formation and were then labeled with different antibodies (anti 5-methylcytosine, anti 5-hydroxymethylcytosine, and anti acetyl H4K12). Based on the fluorescence intensity, the level of each of these epigenetic factors was determined and they revealed a significant relationship between the level of sperm protamine deficiency and sperm epigenetic factors. Protamine deficiency was found to be associated with an increased methylation (p=0) and decreased hydroxymethylation rate (p=0.015) of the male pronucleus chromatin. However, no association was found between protamine deficiency and the level of H4K12 acetylation (p=0.548). Also, the efficiency of fertilization in protamine deficient sperm cells was less than normal. These results suggest that protamine deficient sperm cells lead to the formation of epigenetically altered pronuclei.

  20. Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes

    PubMed Central

    1985-01-01

    A human autoantiserum (5051) directed against pericentriolar material (PCM) was used to study the distribution of microtubule-organizing centers (MTOCs) in the oocyte and during the first cell cycle of mouse development. In oocytes, the PCM was found not only at the poles of the barrel-shaped metaphase II spindle but also at many discrete loci around the cytoplasm near the cell cortex. The spindle poles were also composed of several PCM foci. In metaphase-arrested eggs only the PCM foci located near the chromosomes acted as MTOCs. However, after reduction of the critical concentration for tubulin polymerization by taxol, the cytoplasmic PCM foci were also found to be associated with nucleation of microtubules. After fertilization the cortical PCM foci remained in a peripheral position until the end of the S phase, when they appeared to migrate centrally towards the pronuclei. At prometaphase of the first mitotic division, numerous MTOCs were found around the two sets of chromosomes; these MTOCs then aligned to form two bands on either side of the metaphase plate of the first mitosis. PMID:2865266

  1. Maternal diet-induced obesity alters mitochondrial activity and redox status in mouse oocytes and zygotes.

    PubMed

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

    2010-04-09

    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.

  2. Recovery of cell nuclei from 15,000 years old mammoth tissues and its injection into mouse enucleated matured oocytes

    PubMed Central

    Kato, Hiromi; Anzai, Masayuki; Mitani, Tasuku; Morita, Masahiro; Nishiyama, Yui; Nakao, Akemi; Kondo, Kenji; Lazarev, Petr A.; Ohtani, Tsuyoshi; Shibata, Yasuyuki; Iritani, Akira

    2009-01-01

    Here, we report the recovery of cell nuclei from 14,000–15,000 years old mammoth tissues and the injection of those nuclei into mouse enucleated matured oocytes by somatic cell nuclear transfer (SCNT). From both skin and muscle tissues, cell nucleus-like structures were successfully recovered. Those nuclei were then injected into enucleated oocytes and more than half of the oocytes were able to survive. Injected nuclei were not taken apart and remained its nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation (PCC) at 1 hour after injection and did not form pronuclear-like structures at 7 hours after injection. As half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures, it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pre-treatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues. PMID:19644224

  3. Recovery of cell nuclei from 15,000 years old mammoth tissues and its injection into mouse enucleated matured oocytes.

    PubMed

    Kato, Hiromi; Anzai, Masayuki; Mitani, Tasuku; Morita, Masahiro; Nishiyama, Yui; Nakao, Akemi; Kondo, Kenji; Lazarev, Petr A; Ohtani, Tsuyoshi; Shibata, Yasuyuki; Iritani, Akira

    2009-01-01

    Here, we report the recovery of cell nuclei from 14,000-15,000 years old mammoth tissues and the injection of those nuclei into mouse enucleated matured oocytes by somatic cell nuclear transfer (SCNT). From both skin and muscle tissues, cell nucleus-like structures were successfully recovered. Those nuclei were then injected into enucleated oocytes and more than half of the oocytes were able to survive. Injected nuclei were not taken apart and remained its nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation (PCC) at 1 hour after injection and did not form pronuclear-like structures at 7 hours after injection. As half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures, it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pre-treatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues.

  4. Cytotoxic effects of CdSe quantum dots on maturation of mouse oocytes, fertilization, and fetal development.

    PubMed

    Hsieh, Ming-Shu; Shiao, Nion-Heng; Chan, Wen-Hsiung

    2009-05-14

    Quantum dots (QDs) are useful novel luminescent markers, but their embryonic toxicity is yet to be fully established, particularly in oocyte maturation and sperm fertilization. Earlier experiments by our group show that CdSe-core QDs have cytotoxic effects on mouse blastocysts and are associated with defects in subsequent development. Here, we further investigate the influence of CdSe-core QDs on oocyte maturation, fertilization, and subsequent pre- and postimplantation development. CdSe-core QDs induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryo development, but not ZnS-coated CdSe QDs. Treatment of oocytes with 500 nM CdSe-core QDs during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. To our knowledge, this is the first study to report the negative impact of CdSe-core QDs on mouse oocyte development. Moreover, surface modification of CdSe-core QDs with ZnS effectively prevented this cytotoxicity.

  5. 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.

  6. Method of euthanasia influences the oocyte fertilization rate with fresh mouse sperm.

    PubMed

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

    2014-11-01

    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.

  7. Expression of Variant Ribosomal RNA Genes in Mouse Oocytes and Preimplantation Embryos1

    PubMed Central

    Ihara, Motomasa; Tseng, Hung; Schultz, Richard M.

    2011-01-01

    Ribosomal DNA (rDNA) is not composed of multiple copies of identical transcription units, as commonly believed, but rather of at least seven rDNA variant subtypes that are expressed in somatic cells. This finding raises the possibility that ribosome function may be modulated as proposed by the ribosome filter hypothesis. We report here that mouse oocytes and preimplantation embryos express all the rDNA variants except variant V and that there is no marked developmental change in the qualitative pattern of variant expression. The maternal and embryonic ribosome pools are therefore quite similar, minimizing the likelihood that developmental changes in composition of the ribosome population are critical for preimplantation development. PMID:21209414

  8. 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

  9. Expression of Nox genes in rat organs, mouse oocytes, and sea urchin eggs.

    PubMed

    Maru, Yoshiro; Nishino, Takeshi; Kakinuma, Katsuko

    2005-04-01

    Degenerate primers were designed to isolate new homologs of Nox family genes in rat organs and sea urchin eggs. The primers were capable of amplifying Nox1, Nox2, Nox3, Nox4, Duox1 and Duox2 but not Nox5, and failed to isolate novel homologs in rat. However, a novel homolog (named as Nox-U1) was identified in sea urchin eggs. In the most conserved region (amino acid 336--417 in human Nox2) Nox-U1 has the highest identity with Nox2, which appears to be abundant in mouse oocytes. However, phylogenetic analysis of the entire sequence has revealed that Nox-U1 is closer to Nox4 or Nox5 than Nox2 or Nox3. Histidine residues assumed to be responsible for heme ligation, motifs for FAD- and NADPH-binding, and two asparagine-linked glycosylation sites are conserved.

  10. Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent, Carboxylated ε-poly-L-lysine

    PubMed Central

    Watanabe, Hitomi; Kohaya, Natsuki; Kamoshita, Maki; Fujiwara, Katsuyoshi; Matsumura, Kazuaki; Hyon, Suong-Hyu; Ito, Junya; Kashiwazaki, Naomi

    2013-01-01

    In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), called as carboxylated ε-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes. PMID:24376724

  11. Overexpression of SETβ, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocytes.

    PubMed

    Qi, Shu-Tao; Wang, Zhen-Bo; Ouyang, Ying-Chun; Zhang, Qing-Hua; Hu, Meng-Wen; Huang, Xin; Ge, Zhaojia; Guo, Lei; Wang, Ya-Peng; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2013-04-01

    Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETβ is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETβ might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETβ during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETβ was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETβ accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETβ still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETβ partly colocalized with PP2A at the inner centromere area. Overexpression of SETβ in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETβ by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETβ, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

  12. Identification, characterization and metagenome analysis of oocyte-specific genes organized in clusters in the mouse genome

    PubMed Central

    Paillisson, Amélie; Dadé, Sébastien; Callebaut, Isabelle; Bontoux, Martine; Dalbiès-Tran, Rozenn; Vaiman, Daniel; Monget, Philippe

    2005-01-01

    Background Genes specifically expressed in the oocyte play key roles in oogenesis, ovarian folliculogenesis, fertilization and/or early embryonic development. In an attempt to identify novel oocyte-specific genes in the mouse, we have used an in silico subtraction methodology, and we have focused our attention on genes that are organized in genomic clusters. Results In the present work, five clusters have been studied: a cluster of thirteen genes characterized by an F-box domain localized on chromosome 9, a cluster of six genes related to T-cell leukaemia/lymphoma protein 1 (Tcl1) on chromosome 12, a cluster composed of a SPErm-associated glutamate (E)-Rich (Speer) protein expressed in the oocyte in the vicinity of four unknown genes specifically expressed in the testis on chromosome 14, a cluster composed of the oocyte secreted protein-1 (Oosp-1) gene and two Oosp-related genes on chromosome 19, all three being characterized by a partial N-terminal zona pellucida-like domain, and another small cluster of two genes on chromosome 19 as well, composed of a TWIK-Related spinal cord K+ channel encoding-gene, and an unknown gene predicted in silico to be testis-specific. The specificity of expression was confirmed by RT-PCR and in situ hybridization for eight and five of them, respectively. Finally, we showed by comparing all of the isolated and clustered oocyte-specific genes identified so far in the mouse genome, that the oocyte-specific clusters are significantly closer to telomeres than isolated oocyte-specific genes are. Conclusion We have studied five clusters of genes specifically expressed in female, some of them being also expressed in male germ-cells. Moreover, contrarily to non-clustered oocyte-specific genes, those that are organized in clusters tend to map near chromosome ends, suggesting that this specific near-telomere position of oocyte-clusters in rodents could constitute an evolutionary advantage. Understanding the biological benefits of such an

  13. Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs.

    PubMed

    Coonrod, S A; Calvert, M E; Reddi, P P; Kasper, E N; Digilio, L C; Herr, J C

    2004-01-01

    In order to gain a deeper understanding of the molecular underpinnings of sperm-egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode's solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning

  14. 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.

  15. 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

  16. 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

  17. L-carnitine supplementation during vitrification of mouse oocytes at the germinal vesicle stage improves preimplantation development following maturation and fertilization in vitro.

    PubMed

    Moawad, Adel R; Tan, Seang Lin; Xu, Baozeng; Chen, Hai Ying; Taketo, Teruko

    2013-04-01

    Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

  18. Mitoguardin-1 and -2 promote maturation and the developmental potential of mouse oocytes by maintaining mitochondrial dynamics and functions

    PubMed Central

    Liu, Xiao-Man; Zhang, Yong-Ping; Ji, Shu-Yan; Li, Bo-Tai; Tian, Xuejun; Li, Dali; Tong, Chao; Fan, Heng-Yu

    2016-01-01

    Mitochondrial dynamics change mitochondrial morphological features and numbers as a part of adaptive cellular metabolism, which is vital for most eukaryotic cells and organisms. A disease or even death of an animal can occur if these dynamics are disrupted. Using large-scale genetic screening in fruit flies, we previously found the gene mitoguardin (Miga), which encodes a mitochondrial outer-membrane protein and promotes mitochondrial fusion. Knockout mouse strains were generated for the mammalian Miga homologs Miga1 and Miga2. Miga1/2−/− females show greatly reduced quality of oocytes and early embryos and are subfertile. Mitochondria became clustered in the cytoplasm of oocytes from the germinal-vesicle stage to meiosis II; production of reactive oxygen species increased in mitochondria and caused damage to mitochondrial ultrastructures. Additionally, reduced ATP production, a decreased mitochondrial-DNA copy number, and lower mitochondrial membrane potential were detected in Miga1/2−/− oocytes during meiotic maturation. These changes resulted in low rates of polar-body extrusion during oocyte maturation, reduced developmental potential of the resulting early embryos, and consequently female subfertility. We provide direct evidence that MIGA1/2-regulated mitochondrial dynamics is crucial for mitochondrial functions, ensure oocyte maturation, and maintain the developmental potential. PMID:26716412

  19. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

    PubMed Central

    Carvacho, Ingrid; Ardestani, Goli; Lee, Hoi Chang; McGarvey, Kaitlyn; Fissore, Rafael A.; Lykke-Hartmann, Karin

    2016-01-01

    The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. PMID:27681336

  20. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature.

    PubMed

    Mazur, Peter; Pinn, Irina L; Kleinhans, F W

    2007-04-01

    We have previously reported [Cryobiology 51 (2005) 29-53] that intracellular ice formation (IIF) in mouse oocytes suspended in various concentrations of glycerol and ethylene glycol (EG) occurs at temperatures where the percentage of unfrozen water is about 6% and 12%, respectively, even though the IIF temperatures varied from -14 to -41 degrees C. However, because of the way the solutions were prepared, the concentrations of salt and glycerol or EG in that unfrozen fraction at IIF were also rather tightly grouped. The experiments reported in the present paper were designed to separate the effects of the unfrozen fraction at IIF from that of the solute concentration in the unfrozen fraction. This separation makes use of two facts. One is that the concentration of solutes in the residual liquid at a given subzero temperature is fixed regardless of their concentration in the initial unfrozen solution. However, second, the fraction unfrozen at a given temperature is dependent on the initial solute concentration. Experimentally, oocytes were suspended in solutions of glycerol/buffered saline and EG/buffered saline of varying total solute concentration with the restriction that the mass ratios of glycerol and EG to salts are held constant. The oocytes were then cooled rapidly enough (20 degrees C/min) to avoid significant osmotic shrinkage, and the temperature at which IIF occurred was noted. When this is done, we find, as previously that the fraction of water remaining unfrozen at the temperature of IIF remains nearly constant at 5-8% for both glycerol and EG even though the IIF temperatures vary from -14 to -50 degrees C. But unlike the previous results, the salt and CPA concentrations in the unfrozen fraction vary by a factor of three. The present procedure for preparing the solutions produces a potentially complicating factor; namely, the cell volumes vary substantially prior to freezing: substantially greater than isotonic in some solutions; substantially smaller

  1. Mouse oocytes fertilised by ICSI during in vitro maturation retain the ability to be activated after refertilisation in metaphase II and can generate Ca2+ oscillations

    PubMed Central

    Jędrusik, Agnieszka; Ajduk, Anna; Pomorski, Paweł; Maleszewski, Marek

    2007-01-01

    Background At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca2+]i oscillations through the generation of inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release by binding to IP3 receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation. Results Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca2+]i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP3 receptors. Conclusion Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca2+]i transients, and to be activated after completion of maturation. PMID:17584490

  2. 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

  3. Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures.

    PubMed

    Wang, Liang; Liu, Jun; Zhou, Guang-Bin; Hou, Yun-Peng; Li, Jun-Jie; Zhu, Shi-En

    2011-11-01

    Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (L(p)) and permeability (P(s)) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10(-3) M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.

  4. 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

  5. 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

  6. Effects of Mono-(2-Ethylhexyl) Phthalate and Di-(2-Ethylhexyl) Phthalate Administrations on Oocyte Meiotic Maturation, Apoptosis and Gene Quantification in Mouse Model

    PubMed Central

    Absalan, Forouzan; Saremy, Sadegh; Mansori, Esrafil; Taheri Moghadam, Mahin; Eftekhari Moghadam, Ali Reza; Ghanavati, Razie

    2017-01-01

    Objective Phthalates, which are commonly used to render plastics into soft and flexible materials, have also been determined as developmental and reproductive toxicants in human and animals. The purpose of this study was to evaluate the effect of mono-(2- ethylhexyl) phthalate (MEHP) and di-(2-ethylhexyl) phthalate (DEHP) oral administrations on maturation of mouse oocytes, apoptosis and gene transcription levels. Materials and Methods In this experimental study, immature oocytes recovered from Naval Medical Research Institute (NMRI) mouse strain (6-8 weeks), were divided into seven different experimental and control groups. Control group oocytes were retrieved from mice that received only normal saline. The experimental groups I, II or III oocytes were retrieved from mice treated with 50, 100 or 200 µl DEHP (2.56 µM) solution, respectively. The experimental groups IV, V or VI oocytes were retrieved from mouse exposed to 50, 100 or 200 µl MEHP (2.56 µM) solution, respectively. Fertilization and embryonic development were carried out in OMM and T6 medium. Apoptosis was assessed by annexin V-FITC/Dead Cell Apoptosis Kit, with PI staining. In addition, the mRNA levels of Pou5f1, Ccna1 and Asah1 were examined in oocytes. Finally, mouse embryo at early blastocyst stage was stained with acridine-orange (AO) and ethidium-bromide (EB), in order to access their viability. Results The proportion of oocytes that progressed up to metaphase II (MII) and 2-cells embryo formation stage was significantly decreased by exposure to MEHP or DEHP, in a dose-dependent manner. Annexin V and PI positive oocytes showed greater quantity in the treated mice than control. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) revealed that expression levels of Pou5f1, Asah1 and Ccna1 were significantly lower in the treated mouse oocytes than control. The total cell count for blastocyst developed from the treated mouse oocytes was lower than the controls. Conclusion These

  7. Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes.

    PubMed

    Adhikari, Deepak; Zheng, Wenjing; Shen, Yan; Gorre, Nagaraju; Ning, Yao; Halet, Guillaume; Kaldis, Philipp; Liu, Kui

    2012-06-01

    Mammalian oocytes are arrested at the prophase of meiosis I during fetal or postnatal development, and the meiosis is resumed by the preovulatory surge of luteinizing hormone. The in vivo functional roles of cyclin-dependent kinases (Cdks) during the resumption of meiosis in mammalian oocytes are largely unknown. Previous studies have shown that deletions of Cdk3, Cdk4 or Cdk6 in mice result in viable animals with normal oocyte maturation, indicating that these Cdks are not essential for the meiotic maturation of oocytes. In addition, conventional knockout of Cdk1 and Cdk2 leads to embryonic lethality and postnatal follicular depletion, respectively, making it impossible to study the functions of Cdk1 and Cdk2 in oocyte meiosis. In this study, we generated conditional knockout mice with oocyte-specific deletions of Cdk1 and Cdk2. We showed that the lack of Cdk1, but not of Cdk2, leads to female infertility due to a failure of the resumption of meiosis in the oocyte. Re-introduction of Cdk1 mRNA into Cdk1-null oocytes largely resumed meiosis. Thus, Cdk1 is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. We also found that Cdk1 maintains the phosphorylation status of protein phosphatase 1 and lamin A/C in oocytes in order for meiosis resumption to occur.

  8. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    PubMed

    Wang, Lu; Lu, Angeleem; Zhou, Hong-Xia; Sun, Ran; Zhao, Jie; Zhou, Cheng-Jie; Shen, Jiang-Peng; Wu, Sha-Na; Liang, Cheng-Guang

    2013-01-01

    Casein kinase I alpha (CK1α) is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1) extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP), an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  9. Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole.

    PubMed

    Generoso, W M; Katoh, M; Cain, K T; Hughes, L A; Foxworth, L B; Mitchell, T J; Bishop, J B

    1989-02-01

    The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.

  10. TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: the effect of microorganisms within human follicular fluid collected during IVF cycles.

    PubMed

    Pelzer, Elise S; Harris, Jessica E; Allan, John A; Waterhouse, Mary A; Ross, Tara; Beagley, Kenneth W; Knox, Christine L

    2013-09-01

    Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours' incubation within human follicular fluids (n=5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (<29%) were associated with extensive fragmentation in mouse oocytes (80-100%). Bacteria colonizing human follicular fluid in vivo may cause DNA fragmentation in mouse oocytes following 12h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Fyn Kinase Activity Is Required for Normal Organization and Functional Polarity of the Mouse Oocyte Cortex

    PubMed Central

    Luo, Jinping; Mcginnis, Lynda K.; Kinsey, William H.

    2014-01-01

    Summary The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm–egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II, however, pronuclei eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally. PMID:19363790

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

    PubMed

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

    1998-08-01

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

  13. Oocyte maturation and expression pattern of follicular genes during in-vitro culture of vitrified mouse pre-antral follicles.

    PubMed

    Jamalzaei, Parisa; Valojerdi, Mojtaba Rezazadeh; Ebrahimi, Bita; Farrokhi, Ali

    2016-01-01

    Our aim was to evaluate the oocyte maturation rate and follicular genes expression pattern during in-vitro culture of vitrified mouse pre-antral follicles. Middle sized pre-antral follicles were isolated mechanically from the ovaries of pre-pubertal mice and distributed in vitrification and control groups. In the vitrification group, follicles were washed in equilibration and vitrification solutions and then were immersed in liquid nitrogen after loading on cryotop tips. After warming in descending concentrations of sucrose solutions, fresh and vitrified-warmed follicles were cultured for 13 days. Follicles survival rate and follicular genes expression were assessed during in vitro culture. Finally, at the end of the culture period oocytes maturation rate were compared in both groups. In the vitrification group, follicles survival rate was lower significantly comparing to the control group (P < 0.05), whereas oocytes maturation rate were similar. Although at the beginning of the culture period, expression of some genes such as Gdf9, Bmp15, Tgfβ1 and BmprII were higher in the vitrification group (P < 0.05), during the rest of the culture period expression pattern of all follicular genes were similar in both groups. In conclusion, survival rate of cryotop vitrified pre-antral follicles reduced during culture period while oocytes maturation and follicular genes expression did not show any noticeable alteration.

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

    PubMed Central

    Franciosi, Federica; Manandhar, Shila

    2016-01-01

    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 Ptenfl/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

  15. 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.

  16. Nicotine-induced Disturbances of Meiotic Maturation in Cultured Mouse Oocytes: Alterations of Spindle Integrity and Chromosome Alignment.

    PubMed

    Zenzes, Maria Teresa; Bielecki, Ryszard

    2004-09-15

    We investigated whether nicotine exposure in vitro of mouse oocytes affects spindle and chromosome function during meiotic maturation (M-I and M-II). Oocytes in germinal vesicle (GV) stage were cultured in nicotine for 8 h or for 16 h, to assess effects in M-I and in metaphase II (M-II). The latter culture setting used the three protocols: 8 h nicotine then 8 h medium (8N + 8M); 16 h nicotine (16N); 8 h medium then 8 h nicotine (8M + 8N). Non-toxic concentrations of nicotine at 1.0, 2.5, 5.0 and 10.0 mmol/L were used. Spindle-chromosome configurations were analyzed with wide-field optical sectioning microscopy. In 8 h cultures, nicotine exposure resulted in dose-related increased proportions of M-I oocytes with defective spindle-chromosome configurations. A dose-related delayed entry into anaphase I was also detected. In 16 h cultures, nicotine exposure for the first 8 h (8N + 8M), or for 16 h (16N), resulted in dose- and time-related increased proportions of oocytes arrested in M-I (10 mmol/L; 8 h: 53.2%, controls 9.6%; 16 h: 87.6%, controls 8.5%). Defects in M-I spindles and chromosomes caused M-I arrest leading to dose-related decreased proportions of oocytes that reached metaphase-II (10 mmol/L 8 h: 46.8%, controls 90.4%;16 h: 12.4%, controls 91.5%). A delayed anaphase-I affected the normal timing of M-II, leading to abnormal oocytes with dispersed chromosomes, or with double spindles and no polar body. Nicotine exposure during the second 8 h (8M + 8N) resulted in dose-related, increased proportions of M-II oocytes with defective spindles and chromosomes (10 mmol/L: 42.9%, controls 2.0%). Nicotine has no adverse effects on GV break down, but induces spindle and chromosome defects compromising oocyte meiotic maturation and development.

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

    PubMed

    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.

  18. 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

  19. Deformation of a single mouse oocyte in a constricted microfluidic channel

    PubMed Central

    Luo, ZhengYuan; Guven, Sinan; Gozen, Irep; Chen, Pu; Tasoglu, Savas; Anchan, Raymond M; Bai, BoFeng; Demirci, Utkan

    2015-01-01

    Single oocyte manipulation in microfluidic channels via precisely controlled flow is critical in microfluidic-based in vitro fertilization. Such systems can potentially minimize the number of transfer steps among containers for rinsing as often performed during conventional in vitro fertilization and can standardize protocols by minimizing manual handling steps. To study shape deformation of oocytes under shear flow and its subsequent impact on their spindle structure is essential for designing microfluidics for in vitro fertilization. Here, we developed a simple yet powerful approach to (i) trap a single oocyte and induce its deformation through a constricted microfluidic channel, (ii) quantify oocyte deformation in real-time using a conventional microscope, and (iii) retrieve the oocyte from the microfluidic device to evaluate changes in their spindle structures. We found that oocytes can be significantly deformed under high flow rates, e.g., 10 μl/min in a constricted channel with a width and height of 50 and 150 μm, respectively. Oocyte spindles can be severely damaged, as shown here by immunocytochemistry staining of the microtubules and chromosomes. The present approach can be useful to investigate underlying mechanisms of oocyte deformation exposed to well-controlled shear stresses in microfluidic channels, which enables a broad range of applications for reproductive medicine. PMID:26696793

  20. 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.

  1. Deformation of a single mouse oocyte in a constricted microfluidic channel.

    PubMed

    Luo, ZhengYuan; Guven, Sinan; Gozen, Irep; Chen, Pu; Tasoglu, Savas; Anchan, Raymond M; Bai, BoFeng; Demirci, Utkan

    2015-10-01

    Single oocyte manipulation in microfluidic channels via precisely controlled flow is critical in microfluidic-based in vitro fertilization. Such systems can potentially minimize the number of transfer steps among containers for rinsing as often performed during conventional in vitro fertilization and can standardize protocols by minimizing manual handling steps. To study shape deformation of oocytes under shear flow and its subsequent impact on their spindle structure is essential for designing microfluidics for in vitro fertilization. Here, we developed a simple yet powerful approach to (i) trap a single oocyte and induce its deformation through a constricted microfluidic channel, (ii) quantify oocyte deformation in real-time using a conventional microscope, and (iii) retrieve the oocyte from the microfluidic device to evaluate changes in their spindle structures. We found that oocytes can be significantly deformed under high flow rates, e.g., 10 μl/min in a constricted channel with a width and height of 50 and 150 μm, respectively. Oocyte spindles can be severely damaged, as shown here by immunocytochemistry staining of the microtubules and chromosomes. The present approach can be useful to investigate underlying mechanisms of oocyte deformation exposed to well-controlled shear stresses in microfluidic channels, which enables a broad range of applications for reproductive medicine.

  2. The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes

    PubMed Central

    Kim, Boram; Zhang, Xuesen; Kan, Rui; Cohen, Roy; Mukai, Chinatsu; Travis, Alexander J.; Coonrod, Scott A.

    2014-01-01

    Ca2+ oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca2+ homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Matertm/tm (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Matertm/tm oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca2+ oscillations was altered in Matertm/tm oocytes after fertilization in vitro. Intriguingly, Ca2+ oscillations in Matertm/tm oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca2+ oscillation defect in Matertm/tm oocytes was likely caused by a reduced

  3. Post-ovulatory aging of mouse oocytes in vivo and in vitro: Effects of caffeine on exocytosis and translocation of cortical granules.

    PubMed

    Zheng, Jie; Yin, Xun-Qiang; Ge, Wei; He, Gui-Fang; Qian, Wei-Ping; Ma, Jun-Yu; Shen, Wei; Yin, Shen; Sun, Qing-Yuan

    2016-11-01

    The developmental potential of post-ovulatory oocytes decreases with aging in vivo and in vitro. In this study, we aimed to investigate the effects of a potent antioxidant caffeine on cortical granules (CGs) distribution in mouse oocytes aging in vivo and in vitro. We found that in vivo administration of 150 mg/kg caffeine caused ovulation of some morphologically abnormal oocytes showing premature exocytosis or congregation of CGs, but significantly decreased abnormal distribution of CGs in oocytes aging for 6 h, 12 h and 18 h in vivo compared to those without caffeine treatment. Unexpectedly, supplementation of oocyte culture medium with 10 mmol/L caffeine accelerated CGs release of oocytes and the normal CG distribution rate dramatically decreased from 6 h in oocytes aging in vitro. It appeared that oocytes showed a high degree of abnormal CG distribution by aging for 18 h, and caffeine might delay oocyte CG exocytosis in vivo, but accelerates CG exocytosis in vitro. Our findings may have implications for improving assisted reproduction technologies.

  4. 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

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

    PubMed Central

    Sriraman, Kalpana; Anand, Sandhya; Bhutda, Smita

    2015-01-01

    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

  6. High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

    PubMed Central

    Purcell, Scott H.; Chi, Maggie; Jimenez, Patricia T.; Grindler, Natalia; Schedl, Tim; Moley, Kelle H.

    2012-01-01

    Background Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment. Methodology/Principal Findings We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage. Conclusions/Significance Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes. PMID:23152876

  7. Prophase I arrest of mouse oocytes mediated by natriuretic peptide precursor C requires GJA1 (connexin-43) and GJA4 (connexin-37) gap junctions in the antral follicle and cumulus-oocyte complex.

    PubMed

    Richard, Samantha; Baltz, Jay M

    2014-06-01

    Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMPs) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface. © 2014 by the Society for the Study of Reproduction, Inc.

  8. EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

    PubMed Central

    Qu, Yi; Lu, Danyu; Jiang, Hao; Chi, Xiaochun; Zhang, Hongquan

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes. Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes. However, the role of EZH2 in oocyte meiotic maturation was unknown. Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes. We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion. EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes. Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation. However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly. Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy. Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation. Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation. PMID:27226494

  9. 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.

  10. A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes.

    PubMed

    Downs, S M

    2001-02-01

    This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a

  11. Defying DNA Double-Strand Break-Induced Death during Prophase I Meiosis by Temporal TAp63α Phosphorylation Regulation in Developing Mouse Oocytes

    PubMed Central

    Kim, Dal-Ah

    2014-01-01

    The dichotomy in DNA damage sensitivity of developing mouse oocytes during female germ line development is striking. Embryonic oocytes withstand hundreds of programmed DNA double-strand breaks (DSBs) required for meiotic recombination. Postnatal immature oocytes fail to tolerate even a few DSBs induced by gamma radiation treatment. TAp63α, a p53 family member, undergoes phosphorylation and mediates postnatal immature oocyte death following gamma radiation treatment, which is thought important for germ line quality maintenance. Whether prenatal meiotic oocytes tolerate DNA DSBs simply because they lack TAp63α expression is not clear. We found a significant number of oocytes in newborn mice initiate TAp63α expression and simultaneously carry meiotic DNA DSBs. However, the risk of premature death appears unlikely, because newborn oocytes strongly abate TAp63α phosphorylation induction and resist normally lethal doses of ionizing radiation damage. A calyculin A-sensitive Ser/Thr phosphatase activity downregulates TAp63α phosphorylation and ATM kinase mediates phosphorylation. Possible alterations in the relative balance of these counteracting activities during development may first temper TAp63α phosphorylation and death induction during meiotic DNA DSB repair and recombination, and afterward, implement germ line quality control in later stages. Insights into inherent DNA DSB resistance mechanisms in newborn oocytes may help prevent infertility in women in need of radiation or chemotherapy. PMID:24515437

  12. Maternal chromatin remodeling during maturation and after fertilization in mouse oocytes.

    PubMed

    Spinaci, Marcella; Seren, Eraldo; Mattioli, Mauro

    2004-10-01

    Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation.

  13. Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest

    PubMed Central

    Touati, Sandra A.; Buffin, Eulalie; Cladière, Damien; Hached, Khaled; Rachez, Christophe; van Deursen, Jan M.; Wassmann, Katja

    2015-01-01

    Mammalian female meiosis is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that a gradual decline of BubR1 contributes to age-related aneuploidization. Here we employ a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I and the establishment of robust kinetochore-microtubule attachments in a meiosis-specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multifaceted role in chromosome segregation during the first meiotic division and suggest that age-related decline of BubR1 is a key determinant of the formation of aneuploid oocytes as women approach menopause. PMID:25897860

  14. WNT4-like protein is a cortical granule component in mouse oocytes and functions in regulating preimplantation embryogenesis.

    PubMed

    Liu, Min; Yang, Huei-Ting

    2016-01-01

    Mammalian cortical granules (CG) are membrane-bound organelles located in the cortex of the unfertilized oocytes. Upon fertilization, CG undergo exocytosis to function in blocking polyspermy. While cortical granules are important in fertilization, their exact biochemical composition and reproductive function have not been fully defined. In the present study, a 66 kDa wingless-type MMTV integration site family, member 4 (WNT4)-like protein, with mouse CG origin was identified. Oocytes that were double labeled with lectin Lens culinaris agglutinin (LCA) and WNT4 antibody showed colocalization of the WNT4 molecules and cortical granules. The disappearance of WNT4 molecules in the artificially activated oocytes that were devoid of cortical granules confirmed their granule origin. Following fertilization, WNT4 remained associated with zygotes and blastomeres of 2-cell and 8-cell embryos; however the amount of protein present was reduced more than 2-fold as embryos developed. Prior to implantation, WNT4 appeared to be detectable only in the trophoblast cells. Our functional study revealed that WNT4 molecules were involved in regulating zygotic cleavage and early embryogenesis. To our knowledge, this is the first study demonstrating mammalian cortical granules contain signaling molecules that are involved in the regulation of the first phase of embryonic development.

  15. Melatonin protects oocyte quality from Bisphenol A-induced deterioration in the mouse.

    PubMed

    Zhang, Mianqun; Dai, Xiaoxin; Lu, Yajuan; Miao, Yilong; Zhou, Changyin; Cui, Zhaokang; Liu, Honglin; Xiong, Bo

    2017-02-08

    Bisphenol A (BPA) has been reported to adversely affect the mammalian reproductive system in both sexes. However, the underlying mechanisms regarding how BPA disrupts the mammalian oocyte quality and how to prevent it have not been fully defined. Here, we document that BPA weakens oocyte quality by impairing both oocyte meiotic maturation and fertilization ability. We find that oral administration of BPA (100 μg/kg body weight per day for 7 days) compromises the first polar body extrusion (78.0% vs 57.0%, P<.05) by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment. This defect could be remarkably ameliorated (76.7%, P<.05) by concurrent oral administration of melatonin (30 mg/kg body weight per day for 7 days). In addition, BPA administration significantly decreases the fertilization rate of oocytes (87.2% vs 41.1%, P<.05) by reducing the number of sperm binding to the zona pellucida, which is consistent with the premature cleavage of ZP2 as well as the mis-localization and decreased protein level of ovastacin. Also, the localization and protein level of Juno, the sperm receptor on the egg membrane, are strikingly impaired in BPA-administered oocytes. Finally, we show that melatonin administration substantially elevates the in vitro fertilization rate (63.0%, P<.05) by restoring above defects of fertilization proteins and events, which might be mediated by the improvement of oocyte quality via reduction of ROS levels and inhibition of apoptosis. Collectively, our data reveal that melatonin has a protective action against BPA-induced deterioration of oocyte quality in mice.

  16. Stability of Mouse Oocytes at −80 °C: The Role of the Recrystallization of Intracellular Ice

    PubMed Central

    Seki, Shinsuke; Mazur, Peter

    2013-01-01

    The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities world-wide. 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 hour to three months, and warmed to 25 °C at one of three rates. With the highest warming rate (2,950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next two 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 2,950 °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-months storage at −80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes world-wide in Dry Ice. PMID:21239524

  17. 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.

  18. Activation of Mouse Cumulus-Oocyte Complex Maturation In Vitro Through EGF-Like Activity of Versican.

    PubMed

    Dunning, Kylie R; Watson, Laura N; Zhang, Voueleng J; Brown, Hannah M; Kaczmarek, Adrian K; Robker, Rebecca L; Russell, Darryl L

    2015-05-01

    In vitro maturation of oocytes is suboptimal to in vivo maturation with altered gene expression and compromised oocyte quality. The large proteoglycan versican is abundant in mouse cumulus-oocyte complexes (COCs) matured in vivo but is absent in cultured COCs. Versican is also positively correlated with human oocyte quality. Versican contains an epidermal growth factor (EGF) motif, and based on EGF-like activities in other systems we hypothesized that versican acts as an EGF-like signaling factor during COC maturation. Here, we purified recombinant versican and compared its function with that of EGF during in vitro maturation (IVM). Versican significantly increased cumulus expansion and induced cumulus-specific genes Ptgs2, Tnfaip6, and Has2, which was blocked by EGF receptor antagonist. Microarray analysis revealed that versican has overlapping function with EGF; however, a subset of genes was uniquely altered following 6 h of IVM with either treatment. Following 6 h of IVM, both Areg and Ereg were significantly increased by both treatments, whereas Egln3, Nr4a1, Nr4a2, Nr4a3, and Adamts5 were significantly higher following versican treatment compared with EGF. In contrast, Sprr1a and Aqp3 were increased after 6 h of EGF but not versican treatment. To determine whether there were temporal differences, COCs were cultured with EGF or versican for 0-12 h. Versican-induced expression occurred later but remained elevated for longer compared with EGF for Ptgs2, Ereg, and Nr4a3. The unique expression profiles of Aqp3 and Nr4a3 during IVM were similarly regulated in vivo. These data indicate that versican has EGF-like effects on COC gene expression, but with distinct temporal characteristics.

  19. Di (2-ethylhexyl) phthalate exposure impairs meiotic progression and DNA damage repair in fetal mouse oocytes in vitro

    PubMed Central

    Liu, Jing-Cai; Lai, Fang-Nong; Li, Ling; Sun, Xiao-Feng; Cheng, Shun-Feng; Ge, Wei; Wang, Yu-Feng; Li, Lan; Zhang, Xi-Feng; De Felici, Massimo; Dyce, Paul W; Shen, Wei

    2017-01-01

    Di (2-ethylhexyl) phthalate (DEHP), is the most common member of the class of phthalates that are used as plasticizers and have become common environmental contaminants. A number of studies have shown that DEHP exposure impacts reproductive health in both male and female mammals by acting as an estrogen analog. Here, we investigated the effects of DEHP on meiotic progression of fetal mouse oocytes by using an in vitro model of ovarian tissue culture. The results showed that 10 or 100 μM DEHP exposure inhibited the progression of oocytes throughout meiotic prophase I, specifically from the pachytene to diplotene stages. DEHP possibly impairs the ability to repair DNA double-strand breaks induced by meiotic recombination and as a consequence activates a pachytene check point. At later stages, such defects led to an increased number of oocytes showing apoptotic markers (TUNEL staining, expression of pro-apoptotic genes), resulting in reduced oocyte survival, gap junctions, and follicle assembly in the ovarian tissues. Microarray analysis of ovarian tissues exposed to DEHP showed altered expression of several genes including some involved in apoptosis and gonad development. The expression changes of some genes clustered in cell-cell communication and signal transduction, along with plasma membrane, extracellular matrix and ion channel function classes, were dependent on the DEHP concentration. Together, these results bring new support to the notion that exposure to DEHP during gestation might exert deleterious effects on ovary development, perturbing germ cell meiosis and the expression of genes involved in a wide range of biological processes including ovary development. PMID:28771232

  20. A Single Bivalent Efficiently Inhibits Cyclin B1 Degradation and Polar Body Extrusion in Mouse Oocytes Indicating Robust SAC during Female Meiosis I

    PubMed Central

    Hoffmann, Steffen; Maro, Bernard; Kubiak, Jacek Z.; Polanski, Zbigniew

    2011-01-01

    The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size. PMID:22125605

  1. A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I.

    PubMed

    Hoffmann, Steffen; Maro, Bernard; Kubiak, Jacek Z; Polanski, Zbigniew

    2011-01-01

    The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size.

  2. 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

    2015-06-25

    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.

  3. 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

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

    PubMed Central

    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

  5. DNA methylation, histone modifications and behaviour of AKAP95 during mouse oocyte growth and upon nuclear transfer of foreign chromatin into fully grown prophase oocytes.

    PubMed

    Hoffmann, Steffen; Tomasik, Grzegorz; Polanski, Zbigniew

    2012-01-01

    The poor efficiency of mammalian cloning is due to inappropriate/incomplete epigenetic reprogramming of the donor chromatin. As the success in reprogramming of the donor nucleus may require activity of similar mechanisms which reprogram the chromatin in the course of gametogenesis, we decided to follow the status of some epigenetic markers in the late phase of oogenesis in mice, i.e. in prophase oocytes during their growth and after completion of the growth phase. Our analysis reveals an increase in the level of global DNA methylation starting in oocytes with diameters around 60 microm which was further elevated until completion of oocyte growth. A similar increase was observed in respect to the acetylation of histone H4. On the other hand, the methylation of histone H4 Arg3 was constantly high until the end of oocyte growth, although it differed between fully grown oocytes depending on the type of spatial chromatin organization. We have also studied the AKAP95 protein which was abundant at earlier stages but decreased in fully grown oocytes according to changes in their chromatin organization. The nuclear transfer of different types of donor nuclei with hypomethylated DNA into fully grown prophase oocytes did not increase the global level of methylation of transferred foreign chromatin, regardless if the recipient oocyte was devoid of its own nucleus or its nucleus was left intact. This suggests a major problem in the ability of recipient oocytes to modify donor DNA methylation.

  6. 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

  7. The influence of reduced glutathione in fertilization medium on the fertility of in vitro-matured C57BL/6 mouse oocytes.

    PubMed

    Ishizuka, Y; Nishimura, M; Matsumoto, K; Miyashita, M; Takeo, T; Nakagata, N; Hosoi, Y; Anzai, M

    2013-09-15

    It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo-matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.

  8. 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. © 2016. Published by The Company of Biologists Ltd.

  9. Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape.

    PubMed

    Veselovska, Lenka; Smallwood, Sebastien A; Saadeh, Heba; Stewart, Kathleen R; Krueger, Felix; Maupetit-Méhouas, Stéphanie; Arnaud, Philippe; Tomizawa, Shin-Ichi; Andrews, Simon; Kelsey, Gavin

    2015-09-25

    Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated. Yet, the mechanisms by which transcription regulates DNA methylation in oocytes remain unclear. Here, we systematically test the link between transcription and the methylome. We perform deep RNA-Seq and de novo transcriptome assembly at different stages of mouse oogenesis. This reveals thousands of novel non-annotated genes, as well as alternative promoters, for approximately 10 % of reference genes expressed in oocytes. In addition, a large fraction of novel promoters coincide with MaLR and ERVK transposable elements. Integration with our transcriptome assembly reveals that transcription correlates accurately with DNA methylation and accounts for approximately 85-90 % of the methylome. We generate a mouse model in which transcription across the Zac1/Plagl1 locus is abrogated in oocytes, resulting in failure of DNA methylation establishment at all CpGs of this locus. ChIP analysis in oocytes reveals H3K4me2 enrichment at the Zac1 imprinted control region when transcription is ablated, establishing a connection between transcription and chromatin remodeling at CpG islands by histone demethylases. By precisely defining the mouse oocyte transcriptome, this work not only highlights transcription as a cornerstone of DNA methylation establishment in female germ cells, but also provides an important resource for developmental biology research.

  10. Knockdown of UCHL5IP causes abnormalities in γ-tubulin localisation, spindle organisation and chromosome alignment in mouse oocyte meiotic maturation.

    PubMed

    Wang, Ya-Peng; Qi, Shu-Tao; Wei, Yanchang; Ge, Zhao-Jia; Chen, Lei; Hou, Yi; Ouyang, Ying-Chun; Schatten, Heide; Zhao, Jian-Guo; Sun, Qing-Yuan

    2013-01-01

    UCHL5IP is one of the subunits of the haus complex, which is important for microtubule generation, spindle bipolarity and accurate chromosome segregation in Drosophila and human mitotic cells. In this study, the expression and localisation of UCHL5IP were explored, as well as its functions in mouse oocyte meiotic maturation. The results showed that the UCHL5IP protein level was consistent during oocyte maturation and it was localised to the meiotic spindle in MI and MII stages. Knockdown of UCHL5IP led to spindle defects, chromosome misalignment and disruption of γ-tubulin localisation in the spindle poles. These results suggest that UCHL5IP plays critical roles in spindle formation during mouse oocyte meiotic maturation.

  11. A Gs-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-Gs signaling

    PubMed Central

    Norris, Rachael P.; Freudzon, Leon; Freudzon, Marina; Hand, Arthur R.; Mehlmann, Lisa M.; Jaffe, Laurinda A.

    2008-01-01

    The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a Gs G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, Gs is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-Gs signaling, we examined the effect of LH on the localization of Gαs. Gs activation in response to stimulation of an exogenously expressed β2-adrenergic receptor causes Gαs to move from the oocyte plasma membrane into the cytoplasm, whereas Gs inactivation in response to inhibition of the β2-adrenergic receptor causes Gαs to move back to the plasma membrane. However, LH does not cause a change in Gαs localization, indicating that LH does not act by terminating receptor-Gs signaling. PMID:17850783

  12. 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.

  13. Chromosomal aberrations in in-vitro matured oocytes influence implantation and ongoing pregnancy rates in a mouse model undergoing intracytoplasmic sperm injection.

    PubMed

    Li, Min; Zhao, Hong-Cui; Li, Rong; Yu, Yang; Qiao, Jie

    2014-01-01

    Implantation failure and early pregnancy loss have been reported to be closely related to the quality of mammalian oocytes; however, the pregnant outcome of embryos from in-vitro matured (IVM) oocytes remains unknown. In this study we examined spindle assembly and chromosome segregation during differentiation, and the duration of IVM of mouse oocytes. The resulting implantation and pregnancy outcomes were analyzed to clarify the relationship between the spindle and chromosomes of IVM oocytes and implantation and early pregnancy. Cumulus-enclosed germinal vesicle oocytes were collected and randomly cultured in IVM medium with different IVM durations. One part of IVM oocytes were analyzed the spindle and chromosome morphology by immunofluorescence method, and the other part of them were fertilized by intracytoplasmic sperm injection. The resulting embryos were transferred into pseudo-pregnant female mice, and the post-implantation and full term development was observed. The chromosome aberrations and incorrect spindle assembly seems not affect the early development and blastocyst cell number derived from IVM oocytes, however the development potential of the resulting embryos after implantation were significant decreased with the ratio increasing of chromosome aberrations and incorrect spindle assembly. Accordingly, the full-term development was also decreased. In conclusion, the present study showed the spindle assembly of in vitro-matured oocytes was one of the most important factors that affected the implantation and ongoing pregnancy rates of IVM oocytes, and the improvement by an appropriate duration of maturation in vitro will enhance the post-implantation development potential of the resulting embryos, and decrease implantation failure and early pregnancy loss.

  14. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes.

    PubMed

    Cheeseman, Liam P; Boulanger, Jérôme; Bond, Lisa M; Schuh, Melina

    2016-12-19

    An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

  15. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes

    PubMed Central

    Cheeseman, Liam P.; Boulanger, Jérôme; Bond, Lisa M.; Schuh, Melina

    2016-01-01

    An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy. PMID:27991490

  16. In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages.

    PubMed Central

    Silva, M T; Appelberg, R; Silva, M N; Macedo, P M

    1987-01-01

    We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli. Images PMID:3623691

  17. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse

    PubMed Central

    Rossi, Valerio; Lispi, Monica; Longobardi, Salvatore; Mattei, Maurizio; Rella, Francesca Di; Salustri, Antonietta; De Felici, Massimo; Klinger, Francesca G

    2017-01-01

    Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy. PMID:27689876

  19. ZP3 is Required for Germinal Vesicle Breakdown in Mouse Oocyte Meiosis

    PubMed Central

    Gao, Lei-Lei; Zhou, Chun-Xiang; Zhang, Xiao-Lan; Liu, Peng; Jin, Zhen; Zhu, Gang-Yi; Ma, Yang; Li, Jing; Yang, Zhi-Xia; Zhang, Dong

    2017-01-01

    ZP3 is a principal component of the zona pellucida (ZP) of mammalian oocytes and is essential for normal fertility, and knockout of ZP3 causes complete infertility. ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction; however, additional cellular roles for ZP3 in mammalian oocytes have not been yet reported. In the current study, we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP. Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown (GVBD) (marking the beginning of meiosis), significantly reducing the percentage of MII oocytes. To investigate the ZP3-mediated mechanisms governing GVBD, we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry. We identified Protein tyrosine phosphatase, receptor type K (Ptprk), Aryl hydrocarbon receptor-interacting protein-like 1 (Aipl1), and Diaphanous related formin 2 (Diaph2) as potential candidates, and established a working model to explain how ZP3 affects GVBD. Finally, we provided preliminary evidence that ZP3 regulates Akt phosphorylation, lamin binding to the nuclear membrane via Aipl1, and organization of the actin cytoskeleton via Diaph2. These findings contribute to our understanding of a novel role played by ZP3 in GVBD. PMID:28145526

  20. 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

  1. 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

    2016-08-09

    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.

  2. Small molecule inhibitor of formin homology 2 domains (SMIFH2) reveals the roles of the formin family of proteins in spindle assembly and asymmetric division in mouse oocytes.

    PubMed

    Kim, Hak-Cheol; Jo, Yu-Jin; Kim, Nam-Hyung; Namgoong, Suk

    2015-01-01

    Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.

  3. 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.

  4. Cyclin A2 is required for sister chromatid segregation, but not separase control, in mouse oocyte meiosis.

    PubMed

    Touati, Sandra A; Cladière, Damien; Lister, Lisa M; Leontiou, Ioanna; Chambon, Jean-Philippe; Rattani, Ahmed; Böttger, Franziska; Stemmann, Olaf; Nasmyth, Kim; Herbert, Mary; Wassmann, Katja

    2012-11-29

    In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.

  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.

  6. Lin28 regulates the expression of neuropeptide Y receptors and oocyte-specific homeobox genes in mouse embryonic stem cells.

    PubMed

    Park, Geon Tae; Seo, You-Mi; Lee, Su-Yeon; Lee, Kyung-Ah

    2012-06-01

    Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.

  7. Direct Microscale Measurement of Mouse Oocyte Membrane Permeability to Water and Ethylene Glycol at Subzero Temperatures Using Cryomicroscopy.

    PubMed

    Han, X

      BACKGROUND: Investigation of cell osmotic behavior at subzero temperatures is of critical importance to the optimization of cooling procedures for cryopreservation. Based on established thermodynamic models, plasma membrane permeability coefficients for water and cryoprotectant agent (CPA) (Lcpa, Pp) and their activation energies (Ea(Lp), Ea(Pcpa)) are essential to predict the change of cell volume and composition of intracellular solutions corresponding to different cooling procedures. However, currently available methods to measure Lp at subzero temperatures suffer from technical difficulties due to ice formation and there are no generalized methods to measure Pcpa at subzero temperatures. The present study aims to investigate cell osmotic behavior at subzero temperatures without ice formation. In the study cells were directly injected into super-cooled CPA solutions mounted on a cryomicroscope, and the corresponding osmotic properties were measured. Using ethylene glycol (EG), the value of PEG for mouse (CD-1) metaphase II oocytes at 0, -5, -10 degree C was determined to be 8.451.20, 7.430.91, 6.401.10, x10-6 cm/min, respectively, and Ea(PEG) was calculated to be 3.9 kCal/mol. Lp in the presence of EG (Lp(EG)) at 0, -5, -10 , -15 degree C was determined to be 7.0 1.15, 4.90 1.20, 2.44 0.31, 1.200.24, x 10(-2) µm/min/atm, respectively, and Ea(Lp) was calculated to be 15.5 kCal/mol. Comparing these values with those previously measured at superzero temperatures, we concluded that for mouse oocytes, the Arrhenius relationship for Lp(EG) is consistent at superzero and subzero temperatures, but the values of PEG at subzero temperatures are much lower than the extrapolated values from the Arrhenius relationship at superzero temperatures, possibly caused by membrane phase transition at low temperatures.

  8. 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

  9. Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.

    PubMed

    Ohtake, Jun; Sakurai, Masahiro; Hoshino, Yumi; Tanemura, Kentaro; Sato, Eimei

    2015-03-01

    We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.

  10. Effect of the expression of aquaporins 1 and 3 in mouse oocytes and morulae on the nucleation temperature for intracellular ice formation

    PubMed Central

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

    2013-01-01

    The occurrence of intracellular ice formation (IIF) is the most important factor determining whether or not 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 minutes, 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° to 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

  11. 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.

  12. Killing of Targets by CD8+ T Cells in the Mouse Spleen Follows the Law of Mass Action

    PubMed Central

    Ganusov, Vitaly V.; Barber, Daniel L.; De Boer, Rob J.

    2011-01-01

    It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV) in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number) of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response. PMID:21283669

  13. 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.

  14. 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

  15. Research on stress-induced apoptosis of natural killer cells and the alteration of their killing activity in mouse liver

    PubMed Central

    Ma, Zhen; Liu, Yang; Zhou, Xin; Yu, Hai-Long; Li, Ming-Qi; Tomiyama-Miyaji, Chikako; Abo, Toru; Bai, Xue-Feng

    2013-01-01

    AIM: To investigate the stress-induced apoptosis of natural killer (NK) cells and the changes in their killing activity in mouse livers. METHODS: A restraint stress model was established in mice. Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver. The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay. RESULTS: The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen, which decreased the cell number. The number and percentage of NK cells in the spleen decreased. However, the number of NK cells in the liver decreased, whereas the percentage of NK cells was significantly increased. The apoptosis of NK cells increased gradually with prolonged stress time, and the macrophage-1 (Mac-1)+ NK cells were more susceptible to apoptosis than Mac-1- NK cells. Large numbers of Mac-1- NK cells in the liver, which are more resistant to stress-induced apoptosis, were observed than the Mac-1- NK cells in the spleen. The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased, but the retention of numerous Mac-1- NK cells in the liver maintained the killing ability. CONCLUSION: Significant stress-induced apoptosis was observed among Mac-1+ NK cells, but not Mac-1- NK cells in the mouse liver. Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver. PMID:24115824

  16. Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium--a revisit.

    PubMed

    Mazur, Peter; Kleinhans, F W

    2008-02-01

    We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to -50 degrees C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006

  17. Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system.

    PubMed

    Tahaei, Leila Sadat; Eimani, Hussein; Yazdi, Poopak Eftekhari; Ebrahimi, Bita; Fathi, Rouhollah

    2011-06-01

    Evaluation of the all-trans retinoic acid (t-RA) effects on in vitro maturation (IVM) and in vitro fertilization (IVF) of immature mouse oocytes in the presence and absence of granulosa cell monolayer. Denuded oocytes isolated from mice ovaries and matured in IVM medium alone (Control I), IVM medium in the presence of granulosa cells (Control II), IVM medium with t-RA (Experimental I) and IVM medium simultaneously with t-RA and granulosa cells (Experimental II). After 24 h, matured oocytes were fertilized in T6 medium and their development was followed until the blastocyst stage. Metaphase II oocytes ploidy were evaluated by chromosome counting. The t-RA group compared to the control groups showed no obvious abnormalities. Additionally maturation and embryo development rates significantly increased in the t-RA treated granulosa cell co-culture system. In conclusion, association of t-RA with granulosa cell co-culture during in vitro maturation increases meiosis resumption, formation of metaphase II oocytes, as well as 2-cell and blastocyst stage embryos.

  18. Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes.

    PubMed

    Wang, Zhen-Bo; Jiang, Zong-Zhe; Zhang, Qing-Hua; Hu, Meng-Wen; Huang, Lin; Ou, Xiang-Hong; Guo, Lei; Ouyang, Ying-Chun; Hou, Yi; Brakebusch, Cord; Schatten, Heide; Sun, Qing-Yuan

    2013-12-01

    Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.

  19. SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

    PubMed

    Hwang, Grace; Sun, Fengyun; O'Brien, Marilyn; Eppig, John J; Handel, Mary Ann; Jordan, Philip W

    2017-05-01

    SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. © 2017. Published by The Company of Biologists Ltd.

  20. Transition Metal Chelator Induces Progesterone Production in Mouse Cumulus-Oocyte Complexes and Corpora Lutea.

    PubMed

    Tian, X; Anthony, K; Diaz, Francisco J

    2017-04-01

    Progesterone production is upregulated in granulosa cells (cumulus and mural) after the LH surge, but the intra-follicular mechanisms regulating this transition are not completely known. Recent findings show that the transition metal chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), impairs ovarian function. In this study, we provide evidence that chelating transition metals, including zinc, enhances progesterone production. The findings show that TPEN (transition metal chelator) increases abundance of Cyp11a1 and Star messenger RNA (mRNA) between 8- and 20-fold and progesterone production more than 3-fold in cultured cumulus-oocyte complexes (COC). Feeding a zinc-deficient diet for 10 days, but not 3 days, increased Star, Hsd3b, and prostaglandin F2 alpha receptor (Ptgfr) mRNA ~2.5-fold, suggesting that the effect of TPEN is through modulation of zinc availability. Progesterone from cumulus cells promotes oocyte developmental potential. Blocking progesterone production with epostane during maturation reduced subsequent blastocyst formation from 89 % in control to 18 % in epostane-treated complexes, but supplementation with progesterone restored blastocyst developmental potential to 94 %. Feeding a zinc-deficient diet for 5 days before ovulation did not affect the number of CL, STAR protein, or serum progesterone. However, incubating luteal tissue with TPEN increased abundance of Star, Hsd3b, and Ptgfr mRNA 2-3-fold and increased progesterone production 3-fold. TPEN is known to abolish SMAD2/3 signaling in cumulus cells. However, treatment of COC with the SMAD2/3 phosphorylation inhibitor, SB421542, did not by itself induce steroidogenic transcripts but did potentiate EGF-induced Star mRNA expression. Collectively, the results show that depletion of transition metals with TPEN acutely enhances progesterone biosynthesis in COC and luteal tissue.

  1. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    PubMed

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  2. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes

    PubMed Central

    Watanabe, Toshiaki; Takeda, Atsushi; Tsukiyama, Tomoyuki; Mise, Kazuyuki; Okuno, Tetsuro; Sasaki, Hiroyuki; Minami, Naojiro; Imai, Hiroshi

    2006-01-01

    Small RNAs ranging in size between 18 and 30 nucleotides (nt) are found in many organisms including yeasts, plants, and animals. Small RNAs are involved in the regulation of gene expression through translational repression, mRNA degradation, and chromatin modification. In mammals, microRNAs (miRNAs) are the only small RNAs that have been well characterized. Here, we have identified two novel classes of small RNAs in the mouse germline. One class consists of ∼20- to 24-nt small interfering RNAs (siRNAs) from mouse oocytes, which are derived from retroelements including LINE, SINE, and LTR retrotransposons. Addition of retrotransposon-derived sequences to the 3′ untranslated region (UTR) of a reporter mRNA destabilizes the mRNA significantly when injected into full-grown oocytes. These results suggest that retrotransposons are suppressed through the RNAi pathway in mouse oocytes. The other novel class of small RNAs is 26- to 30-nt germline small RNAs (gsRNAs) from testes. gsRNAs are expressed during spermatogenesis in a developmentally regulated manner, are mapped to the genome in clusters, and have strong strand bias. These features are reminiscent of Tetrahymena ∼23- to 24-nt small RNAs and Caenorhabditis elegans X-cluster small RNAs. A conserved novel small RNA pathway may be present in diverse animals. PMID:16766679

  3. Protective effects of resveratrol against mancozeb induced apoptosis damage in mouse oocytes

    PubMed Central

    Liu, Yu; Chen, Ming-Huang; Zhang, Zhen; Fu, Xian-Pei; Fu, Bin-Bin; Liao, Bao-Qiong; Lin, Yan-Hong; Qi, Zhong-Quan; Wang, Hai-Long

    2017-01-01

    Mancozeb, a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts, is one of the most widely used fungicides in agriculture. Mancozeb could lead to mitochondria dysfunction, cellular anti-oxidation enzymes depletion and apoptotic pathways activation. Previous studies indicated the exposure of mancozeb through mother would lead to irregular estrous cycles, decreased progesterone levels, reduced litter sizes, and more frequent delivery of dead fetuses. In this study, we investigated mancozeb inducing reproductive toxicity, especially focusing on its apoptotic effect and epigenetic modifications. We also showed that resveratrol, a kind of phytoalexin found in peanuts and grapes, can alleviate mancozeb's adverse effects, such as declined fertility, decreased ovary weight and primary follicles. Besides, mancozeb treated oocytes displayed suboptimal developmental competence and this can also be improved by treatment of resveratrol. More detailed investigation of these processes revealed that mancozeb increased reactive oxygen species, causing cell apoptosis and abnormal epigenetic modifications, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can alleviate mancozeb induced infertility and this was mainly through the correction of apoptotic tendency and the abnormity of cellular epigenetic modification. PMID:28031523

  4. Protective effects of resveratrol against mancozeb induced apoptosis damage in mouse oocytes.

    PubMed

    Liu, Yu; Wang, Ya-Long; He, Shu-Wen; Chen, Ming-Huang; Zhang, Zhen; Fu, Xian-Pei; Fu, Bin-Bin; Liao, Bao-Qiong; Lin, Yan-Hong; Qi, Zhong-Quan; Wang, Hai-Long

    2017-01-24

    Mancozeb, a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts, is one of the most widely used fungicides in agriculture. Mancozeb could lead to mitochondria dysfunction, cellular anti-oxidation enzymes depletion and apoptotic pathways activation. Previous studies indicated the exposure of mancozeb through mother would lead to irregular estrous cycles, decreased progesterone levels, reduced litter sizes, and more frequent delivery of dead fetuses. In this study, we investigated mancozeb inducing reproductive toxicity, especially focusing on its apoptotic effect and epigenetic modifications. We also showed that resveratrol, a kind of phytoalexin found in peanuts and grapes, can alleviate mancozeb's adverse effects, such as declined fertility, decreased ovary weight and primary follicles. Besides, mancozeb treated oocytes displayed suboptimal developmental competence and this can also be improved by treatment of resveratrol. More detailed investigation of these processes revealed that mancozeb increased reactive oxygen species, causing cell apoptosis and abnormal epigenetic modifications, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can alleviate mancozeb induced infertility and this was mainly through the correction of apoptotic tendency and the abnormity of cellular epigenetic modification.

  5. Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

    PubMed

    Matson, P L; Graefling, J; Junk, S M; Yovich, J L; Edirisinghe, W R

    1997-07-01

    Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

  6. Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases

    PubMed Central

    Shirane, Kenjiro; Toh, Hidehiro; Kobayashi, Hisato; Miura, Fumihito; Chiba, Hatsune; Ito, Takashi; Kono, Tomohiro; Sasaki, Hiroyuki

    2013-01-01

    DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes. PMID:23637617

  7. Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter.

    PubMed Central

    Perlmann, T; Wrange, O

    1991-01-01

    The mouse mammary tumor virus (MMTV) promoter is positively regulated by glucocorticoid hormone via binding of glucocorticoid receptor to a specific response element. Upon addition of hormone, a nucleosome containing the glucocorticoid response element is removed or structurally altered, suggesting that the nucleosome interferes with transcription. Accordingly, inhibition of chromatin assembly should relieve the repression and result in an increased constitutive activity. We have tested this hypothesis by injecting nonspecific competitor DNA into Xenopus laevis oocytes to titrate endogenous histones. The coinjection of competitor DNA altered chromatin structure: nucleosomal ladders produced by micrococcal nuclease were disrupted, and the unique helical setting of the MMTV promoter in a nucleosome was lost, as shown by in situ DNase I footprinting. Basal MMTV transcription was drastically increased by competitor DNA, whereas a coinjected, constitutively active adenovirus 2 major late promoter was not stimulated. These results show that the uninduced MMTV promoter is under negative control and provide direct support for the theory that the nucleosomal organization maintains the repression of this promoter in its uninduced state. Images PMID:1656227

  8. Endometriosis-induced alterations in mouse metaphase II oocyte microtubules and chromosomal alignment: a possible cause of infertility.

    PubMed

    Mansour, Gihan; Sharma, Rakesh K; Agarwal, Ashok; Falcone, Tommaso

    2010-10-01

    To examine the effect of peritoneal fluid (PF) of patients with endometriosis on the cytoskeleton of metaphase II oocytes and correlate the results with the stage of endometriosis and the duration of infertility. Prospective-controlled study. Center for reproductive medicine at a tertiary-care hospital. Women with endometriosis (n=23) and tubal ligation/reversal (n=15). Peritoneal fluid obtained from 38 women (23 with endometriosis and 15 tubal ligation/reversal) after laparoscopy. Four hundred metaphase II oocytes were used: 165 frozen metaphase II oocytes were incubated in the PF of patients with endometriosis, 135 oocytes incubated in the PF of nonendometriosis patients (control subjects) and 100 oocytes incubated in human tubal fluid (HTF) media. Spindle abnormalities (microtubule and chromosomal) were evaluated by confocal imaging. In the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment (score ≥3), indicating severe damage compared with the control groups. The proportions of abnormalities in microtubule and chromosome alterations in endometriosis (67.9% and 63.6%, respectively) were significantly higher than for oocytes incubated with PF of the nonendometriosis group (24.4% and 14.8%) as well as the HTF group (13% and 13%). Oocyte cytoskeleton damage positively correlated with the duration of infertility and the stage of endometriosis. Alteration of oocyte cytoskeleton might be one of the causes of poor oocyte quality in patients with endometriosis. Copyright © 2010. Published by Elsevier Inc.

  9. Cooperative effects of 17β-estradiol and oocyte-derived paracrine factors on the transcriptome of mouse cumulus cells.

    PubMed

    Emori, Chihiro; Wigglesworth, Karen; Fujii, Wataru; Naito, Kunihiko; Eppig, John J; Sugiura, Koji

    2013-12-01

    Oocyte-derived paracrine factors (ODPFs) and estrogens are both essential for the development and function of ovarian follicles in mammals. Cooperation of these two factors was assessed in vitro using intact cumulus-oocyte complexes, cumulus cells cultured after the removal of oocytes [oocytectomized (OOX) cumulus cells], and OOX cumulus cells cocultured with denuded oocytes, all in the presence or absence of 17β-estradiol (E2). Effects on the cumulus cell transcriptome were assessed by microarray analysis. There was no significant difference between the cumulus cell transcriptomes of either OOX cumulus cells cocultured with oocytes or intact cumulus-oocyte complexes. Therefore, oocyte-mediated regulation of the cumulus cell transcriptome is mediated primarily by ODPFs and not by gap junctional communication between oocytes and cumulus cells. Gene ontology analysis revealed that both ODPFs and E2 strongly affected the biological processes associated with cell proliferation in cumulus cells. E2 had limited effects on ODPF-regulated biological processes. However, in sharp contrast, ODPFs significantly affected biological processes regulated by E2 in cumulus cells. For example, only in the presence of ODPFs did E2 significantly promote the biological processes related to phosphorylation-mediated signal transduction in cumulus cells, such as the signaling pathways of epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor. Therefore, ODPFs and E2 cooperate to regulate the cumulus cell transcriptome and, in general, oocytes modulate the effects of estrogens on cumulus cell function.

  10. Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

    PubMed

    Kelly, Gemma L; Grabow, Stephanie; Glaser, Stefan P; Fitzsimmons, Leah; Aubrey, Brandon J; Okamoto, Toru; Valente, Liz J; Robati, Mikara; Tai, Lin; Fairlie, W Douglas; Lee, Erinna F; Lindstrom, Mikael S; Wiman, Klas G; Huang, David C S; Bouillet, Philippe; Rowe, Martin; Rickinson, Alan B; Herold, Marco J; Strasser, Andreas

    2014-01-01

    The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

  11. Role of PDE3A in regulation of cell cycle progression in mouse vascular smooth muscle cells and oocytes: implications in cardiovascular diseases and infertility.

    PubMed

    Begum, Najma; Shen, Weixing; Manganiello, Vincent

    2011-12-01

    Phosphodiesterase-3 (PDE3) is a major cAMP-hydrolyzing PDE in vascular smooth muscle cells (VSMCs) and oocytes. The exact role and contribution of the two PDE3 isoforms, PDE3A and PDE3B, in VSMC growth regulation and oocyte maturation was examined using PDE3A (3A) and PDE3B (3B) knockout (KO) mouse models. PDE3A-deficient VSMCs exhibit marked reduction in mitogen-induced cell growth due to cell cycle arrest at G₀-G₁ phase, which resulted from dysregulation of cAMP/protein kinase A (PKA)-activated and mitogen-activated protein kinase (MAPK)-signaling pathways, as well as from alterations in key cell cycle regulatory proteins. Similarly, PDE3A-deficient oocytes exhibit cell cycle arrest at G₂/M phase because increased cAMP/PKA signaling in KO oocytes most likely inhibits Cdc25B-catalyzed dephosphorylation/activation of Cdc2 (maturation promoting factor (MPF)), a key regulator of G₂/M transition. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Zinc supplementation of vitrification medium improves in vitro maturation and fertilization of oocytes derived from vitrified-warmed mouse ovaries.

    PubMed

    Geravandi, Shirin; Azadbakht, Mehri; Pourmoradi, Mahsa; Nowrouzi, Fatemeh

    2017-02-01

    Oocyte cryopreservation is an approach for fertility preservation for normal women and cancer patients facing chemo and radiotherapy. The present study evaluated the effect of adding zinc chloride to the vitrification medium used for whole mouse ovaries and then assessing the in vitro maturation and fertilization of oocytes when they were subsequently extracted from these vitrified ovarian tissues. Four vitrification solutions with 0, 100,150 and 200 μg/dl zinc (V0, V1, V2 and V3 respectively) were compared. The viability of oocytes isolated from ovaries vitrified-warmed in the highest concentration of zinc (V3) was significantly higher after 24 than in the control V0 group (72.99 vs 85.97). Progression to the MII stage, fertilization and cleavage by 48 h was also higher in the V3 than V0 control group (35.55 vs 44.73), (47.67 vs 63.74), (28.72 vs 43.03) (P < 0.05) respectively. These results indicate that supplementation of vitrification medium for intact ovaries with zinc can improve the oocyte viability and in vitro maturation-fertilization rate. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Attachment, ingestion and intracellular killing of Helicobacter pylori by human peripheral blood mononuclear leukocytes and mouse peritoneal inflammatory macrophages.

    PubMed

    Chmiela, M; Paziak-Domanska, B; Wadström, T

    1995-02-01

    The different steps of phagocytosis, attachment, ingestion and intracellular killing of cells of Helicobacter pylori strain 17874 (expressing sialic acid-specific haemagglutinin) and cells of H. pylori strain 17875 (expressing non-sialic acid-specific haemagglutinin) have been studied. More cells of sialopositive H. pylori strain 17874 have been found attached to human peripheral blood mononuclear leukocytes (PBM) and mouse peritoneal inflammatory macrophages (PIM) than cells of sialonegative H. pylori strain 17875. Binding of cells of H. pylori strain 17874 has been significantly inhibited by treatment of phagocytes with neuraminidase. Inhibition of adhesion of these bacteria preincubated with foetuin to normal phagocytic cells has also been found. Well adhering cells of H. pylori strain 17874 were more resistant to killing mechanisms of human PBM and mouse PIM than cells of strain 17875. Good, probably sialic acid-specific haemagglutinin dependent, adhesion of H. pylori bacteria to phagocytes can be considered as an important virulence factor which facilitates the pathogen to avoid the defence mechanisms.

  14. Taurine and beta-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA.

    PubMed

    Horikoshi, T; Asanuma, A; Yanagisawa, K; Anzai, K; Goto, S

    1988-09-01

    The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

  15. 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

    2015-03-19

    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.

  16. Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos.

    PubMed

    Yao, Li-Juan; Zhong, Zhi-Sheng; Zhang, Li-Sheng; Chen, Da-Yuan; Schatten, Heide; Sun, Qing-Yuan

    2004-05-01

    Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.

  17. RhoA-mediated FMNL1 regulates GM130 for actin assembly and phosphorylates MAPK for spindle formation in mouse oocyte meiosis.

    PubMed

    Wang, Fei; Zhang, Liang; Duan, Xing; Zhang, Guang-Li; Wang, Zhen-Bo; Wang, Qiang; Xiong, Bo; Sun, Shao-Chen

    2015-01-01

    Formin-like 1 (FMNL1) is a member of Formin family proteins which are the actin nucleators. Although FMNL1 activities have been shown to be essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis, the functional roles of mammalian FMNL1 during oocyte meiosis remain uncertain. In this study, we investigated the functions of FMNL1 in mouse oocytes using specific morpholino (MO) microinjection and live cell imaging. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD). FMNL1 knockdown caused the low rate of polar body extrusion and resulted in large polar bodies. Time-lapse microscopic and immunofluorescence intensity analysis indicated that this might be due to the aberrant actin expression levels. Cortical polarity was disrupted as shown by a loss of actin cap and cortical granule free domain (CGFD) formation, which was confirmed by a failure of meiotic spindle positioning. And this might be the reason for the large polar body formation. Spindle formation was also disrupted, which might be due to the abnormal localization of p-MAPK. These results indicated that FMNL1 affected both actin dynamics and spindle formation for the oocyte polar body extrusion. Moreover, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130. Finally, we found that the small GTPase RhoA might be the upstream regulator of FMNL1. Taken together, our data indicate that FMNL1 is required for spindle organization and actin assembly through a RhoA-FMNL1-GM130 pathway during mouse oocyte meiosis.

  18. Bone marrow transplantation generates immature oocytes and rescues long-term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure.

    PubMed

    Lee, Ho-Joon; Selesniemi, Kaisa; Niikura, Yuichi; Niikura, Teruko; Klein, Rachael; Dombkowski, David M; Tilly, Jonathan L

    2007-08-01

    Although early menopause frequently occurs in female cancer patients after chemotherapy (CTx), bone marrow (BM) transplantation (BMT) has been linked to an unexplained return of ovarian function and fertility in some survivors. Studies modeling this in mice have shown that BMT generates donor-derived oocytes in CTx-treated recipients. However, a subsequent report claimed that ovulated eggs are not derived from BM and that BM-derived oocytes reported previously are misidentified immune cells. This study was conducted to further clarify the impact of BMT on female reproductive function after CTx using a preclinical mouse model. Female mice were administered CTx followed by BMT using coat color-mismatched female donors. After housing with males, the number of pregnancies and offspring genotype were recorded. For cell tracking, BM from germline-specific green fluorescent protein-transgenic mice was transplanted into CTx-treated wild-type recipients. Immune cells were sorted from blood and analyzed for germline markers. BMT rescued long-term fertility in CTx-treated females, but all offspring were derived from the recipient germline. Cell tracking showed that donor-derived oocytes were generated in ovaries of recipients after BMT, and two lines of evidence dispelled the claim that these oocytes are misidentified immune cells. These data from a preclinical mouse model validate a testable clinical strategy for preserving or resurrecting ovarian function and fertility in female cancer patients after CTx, thus aligning with recommendations of the 2005 National Cancer Institute Breast Cancer Progress Review Group and President's Cancer Panel to prioritize research efforts aimed at improving the quality of life in cancer survivors.

  19. A Motor-Gradient and Clustering Model of the Centripetal Motility of MTOCs in Meiosis I of Mouse Oocytes

    PubMed Central

    2016-01-01

    Asters nucleated by Microtubule (MT) organizing centers (MTOCs) converge on chromosomes during spindle assembly in mouse oocytes undergoing meiosis I. Time-lapse imaging suggests that this centripetal motion is driven by a biased ‘search-and-capture’ mechanism. Here, we develop a model of a random walk in a drift field to test the nature of the bias and the spatio-temporal dynamics of the search process. The model is used to optimize the spatial field of drift in simulations, by comparison to experimental motility statistics. In a second step, this optimized gradient is used to determine the location of immobilized dynein motors and MT polymerization parameters, since these are hypothesized to generate the gradient of forces needed to move MTOCs. We compare these scenarios to self-organized mechanisms by which asters have been hypothesized to find the cell-center- MT pushing at the cell-boundary and clustering motor complexes. By minimizing the error between simulation outputs and experiments, we find a model of “pulling” by a gradient of dynein motors alone can drive the centripetal motility. Interestingly, models of passive MT based “pushing” at the cortex, clustering by cross-linking motors and MT-dynamic instability gradients alone, by themselves do not result in the observed motility. The model predicts the sensitivity of the results to motor density and stall force, but not MTs per aster. A hybrid model combining a chromatin-centered immobilized dynein gradient, diffusible minus-end directed clustering motors and pushing at the cell cortex, is required to comprehensively explain the available data. The model makes experimentally testable predictions of a spatial bias and self-organized mechanisms by which MT asters can find the center of a large cell. PMID:27706163

  20. HCO3−/Cl− Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

    PubMed Central

    Zhou, Chenxi; Tiberi, Mario; Liang, Binhui; Alper, Seth L.; Baltz, Jay M.

    2009-01-01

    Background Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO3−/Cl− exchange—a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO3−/Cl− exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO3−/Cl− exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established. Methodology/Principal Findings Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO3−/Cl− exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO3−/Cl− exchange in mature MII eggs. A GFP-tagged form of the HCO3−/Cl− exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation. Conclusions Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO3−/Cl− exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during

  1. cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte.

    PubMed

    Hwang, Heekyung; Cheon, Yong-Pil

    2016-12-01

    Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

  2. cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte

    PubMed Central

    Hwang, Heekyung; Cheon, Yong-Pil

    2016-01-01

    ABSTRACT Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest. PMID:28144640

  3. Review: Lamin A/C, caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.

    PubMed

    Arnault, Emilie; Doussau, Mireille; Pesty, Arlette; Lefèvre, Brigitte; Courtot, Anne-Marie

    2010-02-01

    After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.

  4. A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse.

    PubMed

    Chouinard, L A

    1975-05-01

    The ordered changes which occur in the structural organization of the mouse oocyte nucelus during the preparatory, the maturative and the preovulatory stages of antral follicle development, have been studied under both light and electron microscopy. All observations have been made on those antral follicles whose development is initiated on postnatal day 14 and completed by postnatal day 28 in prepubertal animals of the ICR albino mouse strain. The formed entities that can be recognized within the oocyte nucleus during that period are the condensing bivalents, the heterochromatic knobs, the nucleolus and the extranucleolar bodies. At the onset of antral follicle development, the highly unravelled dictyate bivalents are seen to take on a lampbrush-type configuration. Subsequent condensation of these lampbursh bivalents appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development. The shortening and thickening of the lampbrush bivalents are best interpreted as resulting from the withdrawal of their lateral loop-like projections into the chromosome axes and from the focal aggregation of these axes into compact chromatin masses. Electron-opaque granules, which appear within the oocyte nucleus during the preparatory and maturative follicle stages, are seen to be intimately associated with these condensing bivalents. A number of Feulgen-positive heterochromatic knobs make their appearance in contact with certain bivalents during the preparatory follicle stage. These knobs are not reincorporated as such into the condensing chromatin masses and undergo disintegration and dissolution during the preovulatory follicle stage. The size, shape and ultrastructural features of the nucleolus remain unchanged thoughout the period of antral follicle development. Breakdown and dissolution of the nucleolar mass is a swift process that takes place only in the fully mature preovulatory follicle and more or less concomitantly

  5. Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800

    PubMed Central

    1990-01-01

    Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo. PMID:2211822

  6. Oocyte aging-induced Neuronatin (NNAT) hypermethylation affects oocyte quality by impairing glucose transport in porcine

    PubMed Central

    Gao, Ying-Ying; Chen, Li; Wang, Tao; Nie, Zheng-Wen; Zhang, Xia; Miao, Yi-Liang

    2016-01-01

    DNA methylation plays important roles in regulating many physiological behaviors; however, few studies were focused on the changes of DNA methylation during oocyte aging. Early studies showed that some imprinted genes’ DNA methylation had been changed in aged mouse oocytes. In this study, we used porcine oocytes to test the hypothesis that oocyte aging would alter DNA methylation pattern of genes and disturb their expression in age oocytes, which affected the developmental potential of oocytes. We compared several different types of genes and found that the expression and DNA methylation of Neuronatin (NNAT) were disturbed in aged oocytes significantly. Additional experiments demonstrated that glucose transport was impaired in aged oocytes and injection of NNAT antibody into fresh oocytes led to the same effects on glucose transport. These results suggest that the expression of NNAT was declined by elevating DNA methylation, which affected oocyte quality by decreasing the ability of glucose transport in aged oocytes. PMID:27782163

  7. 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. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  8. Recent progress in reproduction of whale oocytes.

    PubMed

    Zheng, Yue-Liang

    2013-08-01

    Whale oocytes recovered from follicles can be matured in vitro. Whale sperm and mature oocytes can be used for in vitro fertilization (IVF), and IVF embryos have the ability to develop to morula stage. Whale sperm injected into bovine or mouse oocytes can activate the oocytes and form pronucleus. Interspecies somatic cell nuclear transfer embryos have been reconstructed with whale somatic cell nucleus and enucleated bovine or porcine oocytes, and interspecies cloned embryos can develop in vitro. This paper reviews recent progress in maturation, fertilization and development of whale oocytes.

  9. Ammonium accumulation and use of mineral oil overlay do not alter imprinting establishment at three key imprinted genes in mouse oocytes grown and matured in a long-term follicle culture.

    PubMed

    Anckaert, Ellen; Adriaenssens, Tom; Romero, Sergio; Smitz, Johan

    2009-10-01

    Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin-specific monoallelic expression. Follicle culture under oil overlay has been associated with altered imprinting establishment in mouse oocytes. We previously demonstrated normal imprinting establishment at four key imprinted genes in mouse oocytes grown and matured in a long-term in vitro follicle culture system without oil overlay. Ammonium (300 microM) has been linked to aberrant imprinting in in vitro preimplantation embryo culture. Compared to culture without oil, mineral oil overlay during follicle culture led to a dramatic increase in ammonia levels in culture medium: mean ammonia levels were, respectively, 39 and 290 microM at Day 4 of culture, 73 and 465 microM at Day 8, and 101 and 725 microM at Day 12 (P < 0.0001). Mineral oil overlay and high ammonia levels (comparable to the follicle culture system for which aberrant imprinting was previously described) during follicle culture did not affect follicle survival, metaphase II (MII) rate, or MII oocyte diameter. Bisulphite sequencing revealed that high levels of ammonia and mineral oil overlay during follicle culture did not alter the methylation status of differentially methylated regions of three key imprinted genes (Snrpn, Igf2r, and H19) in MII oocytes. In the current culture setup, ammonium accumulation and mineral oil overlay during follicle culture do not induce aberrant imprinting establishment at the studied regulatory sequences in mouse oocytes.

  10. Cytogenetic analysis and developmental assessment of mouse embryos derived from in vitro fertilization of oocytes reconstructed by meiosis-II chromosome transplantation.

    PubMed

    Mitsui, Akinori; Yoshizawa, Midori

    2007-04-01

    An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-II-t resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal.

  11. FOXO1, FOXO3, AND FOXO4 are differently expressed during mouse oocyte maturation and preimplantation embryo development.

    PubMed

    Kuscu, Nilay; Celik-Ozenci, Ciler

    2015-01-01

    Preimplantation embryo development is affected by its environment. FoxO transcription factors are regulated by PI3K/Akt signaling pathway that essentially supports growth and development. FoxO transcription factors are at the interface of crucial cellular processes, orchestrating programs of gene expression that regulate apoptosis, cell-cycle arrest, oxidative stress resistance, DNA repair, glucose metabolism, and differentiation. In the presence of growth factors, FoxO transcription factors are localized in the cytoplasm, whereas under stress conditions they move to the nucleus and trigger transcriptional activities of their target genes. The aim of the present study is to investigate whether FoxO transcription factors are present during in vivo oocyte maturation and preimplantation embryo development. Presence and localizations of FoxO1, FoxO3 and FoxO4 proteins have been determined with immunofluorescence staining. Our results have confirmed that FoxO1, FoxO3 and FoxO4 proteins are differentially expressed in prophase I, metaphase I, metaphase II oocytes, as well as in fertilized oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst. FoxOs translocate to nucleus in embryos with developmental delay. Our findings indicate that FoxO transcription factors are present during both oocyte and embryo in vivo maturation and provide fundamental knowledge that FoxOs may regulate in vitro embryo development under stress conditions.

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

    PubMed Central

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

    2016-01-01

    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

  13. Adverse Effects of High Concentrations of Fluoride on Characteristics of the Ovary and Mature Oocyte of Mouse.

    PubMed

    Yin, Songna; Song, Chao; Wu, Haibo; Chen, Xin; Zhang, Yong

    2015-01-01

    Reproductive toxicity has been an exciting topic of research in reproductive biology in recent years. Soluble fluoride salts are toxic at high concentrations; their reproductive toxicity was assessed in this study by administering different fluoride salt concentrations to mice. Continuous feeding for five weeks resulted in damage to the histological architecture of ovaries. The expression of genes, including Dazl, Stra8, Nobox, Sohlh1, and ZP3 gene, associated with oocyte formation were much lower in the experimental group as compared with the control group. The number of in vitro fertilization of mature oocytes were also much lower in the experimental group as compared with control. Moreover, the fertility of female mice, as assessed by mating with normal male mice, was also lower in experimental compared with control groups. The expression of the oocyte-specific genes: Bmp15, Gdf9, H1oo, and ZP2, which are involved in oocyte growth and the induction of the acrosome reaction, decreased with the fluoride administration. DNA methylation and histone acetylation (H3K18ac and H3K9ac) are indispensable for germline development and genomic imprinting in mammals, and fluoride administration resulted in reduced levels of H3K9ac and H3K18ac in the experimental group as compared with the control group, as detected by immunostaining. Our results indicate that the administration of high concentrations of fluoride to female mice significantly reduced the number of mature oocytes and hampered their development and fertilization. Thus, this study lays a foundation for future studies on fluoride-induced reproductive disorders in women.

  14. Data on morphology, large-scale chromatin configuration and the occurrence of proteins and rRNA in nucleolus-like bodies of fully-grown mouse oocytes in different fixatives.

    PubMed

    Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

    2016-06-01

    Here we provide data on accessibility of nucleolus-like bodies (NLBs) of fully-grown (GV) mouse oocytes to fluorescence in situ hybridization (FISH) probes and anti-nucleolar antibodies as well as on oocyte general morphology and large scale chromatin configuration, which relate to the research article "High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes" (Shishova et al., 2015 [1]). Experimental factors include: a cross-linking reagent formaldehyde and two denaturing fixatives, such as 70% ethanol and a mixture of absolute methanol and glacial acetic acid (3:1, v/v).

  15. A Small Molecule Inhibitor of Human RAD51 Potentiates Breast Cancer Cell Killing by Therapeutic Agents in Mouse Xenografts

    PubMed Central

    Huang, Fei; Mazin, Alexander V.

    2014-01-01

    The homologous recombination pathway is responsible for the repair of DNA double strand breaks. RAD51, a key homologous recombination protein, promotes the search for homology and DNA strand exchange between homologous DNA molecules. RAD51 is overexpressed in a variety of cancer cells. Downregulation of RAD51 by siRNA increases radio- or chemo-sensitivity of cancer cells. We recently developed a specific RAD51 small molecule inhibitor, B02, which inhibits DNA strand exchange activity of RAD51 in vitro. In this study, we used human breast cancer cells MDA-MB-231 to investigate the ability of B02 to inhibit RAD51 and to potentiate an anti-cancer effect of chemotherapeutic agents including doxorubicin, etoposide, topotecan, and cisplatin. We found that the combination of B02 with cisplatin has the strongest killing effect on the cancer cells. We then tested the effect of B02 and cisplatin on the MDA-MB-231 cell proliferation in mouse xenografts. Our results showed that B02 significantly enhances the therapeutic effect of cisplatin on tumor cells in vivo. Our current data demonstrate that use of RAD51-specific small molecule inhibitor represents a feasible strategy of a combination anti-cancer therapy. PMID:24971740

  16. AR-13, a Celecoxib Derivative, Directly Kills Francisella In Vitro and Aids Clearance and Mouse Survival In Vivo.

    PubMed

    Hoang, Ky V; Adcox, Haley E; Fitch, James R; Gordon, David M; Curry, Heather M; Schlesinger, Larry S; White, Peter; Gunn, John S

    2017-01-01

    Francisella tularensis (F. tularensis) is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the United States and treatment of tularemia is confined to few antibiotics. In this study, we demonstrate that AR-13, a derivative of the cyclooxygenase-2 inhibitor celecoxib, exhibits direct in vitro bactericidal killing activity against Francisella including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as toward the intracellular proliferation of LVS in macrophages, without causing significant host cell toxicity. Identification of an AR-13-resistant isolate indicates that this compound has an intracellular target(s) and that efflux pumps can mediate AR-13 resistance. In the mouse model of tularemia, AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4. Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death. Taken together, these data support the translational potential of AR-13 as a lead compound for the further development of new anti-Francisella agents.

  17. The transcriptome of human oocytes

    PubMed Central

    Kocabas, Arif Murat; Crosby, Javier; Ross, Pablo J.; Otu, Hasan H.; Beyhan, Zeki; Can, Handan; Tam, Wai-Leong; Rosa, Guilherme J. M.; Halgren, Robert G.; Lim, Bing; Fernandez, Emilio; Cibelli, Jose Bernardo

    2006-01-01

    The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. PMID:16968779

  18. Effect of sodium lithium and proton concentrations on the electrophysiological properties of the four mouse GABA transporters expressed in Xenopus oocytes.

    PubMed

    Grossman, Tamar R; Nelson, Nathan

    2003-01-01

    Mouse GABA transporters belong to the family of Na(+) and Cl(-) dependent neurotransmitter transporter. GABA transport, by these family members, was shown to be electrogenic and driven by sodium ions. It was demonstrated that, as in several other transporters, sodium binding and release by GAT1, GAT3 and BGT-1, the canine homolog of GAT2, resulted in the appearance of presteady-state currents. In this work we show that each of the four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to the transporters affinities to Na(+). At 100 mM GAT1 exhibited symmetric presteady-state currents at all imposed potentials, whereas GAT2 exhibited asymmetric presteady-state currents exclusively at negative imposed potentials, GAT3 or GAT4 exhibited presteady-state currents predominantly at positive imposed potentials. GABA uptake by GAT2 and GAT4 was much more sensitive to external pH than GAT1 and GAT3. Reducing the external Na(+) concentration rendered the GABA uptake activity by GAT1 and GAT3 to be sensitive to pH. Lowering the external pH reduced the Na(+) affinity of GAT1. Substitution of the external Na(+) to Li(+) resulted in the appearance of leak currents exclusively at negative potentials in Xenopus oocyte expressing GAT1 and GAT3. Low Na(+) concentrations inhibited the leak currents of GAT1 but Na(+) had little effect on the leak currents of GAT3. Washing of occluded Na(+) in GAT1 enhanced the leak currents. Similarly addition of GABA in the presence of 80 mM Li(+), that presumably accelerated the release of the bound Na(+), also induced the leak currents. Conversely, addition of GABA to GAT3 expressing oocytes, in the presence of 80 mM Li(+), inhibited the leak currents.

  19. Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

    PubMed Central

    Mouzaki, A; Weil, R; Muster, L; Rungger, D

    1991-01-01

    The Xenopus oocyte system was used to test functionally, putative trans-active elements involved in the transcriptional control of the mouse interleukin-2 (IL-2) gene in resting and mitogen-induced primary T-lymphocytes. The IL-2 gene injected into the oocyte is active over a wide range of DNA concentrations. This basal activity is silenced by the addition of protein extracts from G0-arrested spleen cells. Extracts from 8 h-stimulated spleen cells do not silence but moderately increase transcription over basal level. When IL-2 transcription is silenced first by an injection of extract from resting spleen cells, the addition of proteins from stimulated cells results in a strong increase in transcription (derepression). Use of proteins from purified splenic T-lymphocytes shows that both silencer(s) and activator(s) are contributed by these cells. Extracts from control tissues have neither a silencing nor stimulatory effect. None of the proteins tested affects the activities of co-injected control genes. Injections with IL-2 promoter mutants indicate that the main target sequence of the silencing and activating factors is a purine region (Pu-box) lying between positions -261 and -292 upstream of the IL-2 gene. Bandshift assays show differential binding of the Pu-box with proteins from resting or activated T-cells. Images PMID:2026141

  20. 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.

  1. 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.

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

    PubMed Central

    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. PMID:1577859

  3. 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.

  4. Role of Caspase-3 Cleaved IP3R1 on Ca2+ Homeostasis and Developmental Competence of Mouse Oocytes and Eggs

    PubMed Central

    Zhang, Nan; Fissore, Rafael. A.

    2014-01-01

    Apoptosis in most cell types is accompanied by altered Ca2+ homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) generates a 95-kDa C-terminal fragment (C-IP3R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca2+ homeostasis and express C-IP3R1, although whether or not C-IP3R1 expression contributes to Ca2+ misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding CIP3R1. We found that: 1) expression of C-IP3R1 in eggs lowered the Ca2+ content of the endoplasmic reticulum (ER), although, as C-IP3R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; 2) expression of CIP3R1 in eggs enhanced fragmentation associated with aging; 3) endogenous IP3R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP3R1 in eggs with downregulated IP3R1 partly restored fragmentation; 4) C-IP3R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca2+ pool that occurs during maturation, undermined the Ca2+ oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3R1 and C-IP3R1 in regulating Ca2+ homeostasis and the ER Ca2+ content during oocyte maturation. Nevertheless, the role of C-IP3R1 on Ca2+ homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3R1 remains intact and C-IP3R1 undergoes rapid turnover. PMID:24692207

  5. Mps1 kinase-dependent Sgo2 centromere localisation mediates cohesin protection in mouse oocyte meiosis I.

    PubMed

    El Yakoubi, Warif; Buffin, Eulalie; Cladière, Damien; Gryaznova, Yulia; Berenguer, Inés; Touati, Sandra A; Gómez, Rocío; Suja, José A; van Deursen, Jan M; Wassmann, Katja

    2017-09-25

    A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.In meiosis I centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage ensuring that sister chromatids are kept together until their separation in meiosis II. Here the authors demonstrate that Bub1 and Mps1 kinase activities are required for Sgo2 localisation to the centromere region.

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

    PubMed Central

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

    2010-01-01

    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

  7. 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.

  8. 2-Deoxy-D-glucose inhibits intracellular multiplication and promotes intracellular killing of Legionella pneumophila in A/J mouse macrophages.

    PubMed Central

    Ogawa, M; Yoshida, S; Mizuguchi, Y

    1994-01-01

    Legionella pneumophila can grow intracellularly in A/J mouse macrophages. 2-Deoxy-D-glucose (2dG) (0.1, 1, and 10 mM) inhibited intracellular multiplication and promoted intracellular killing of L. pneumophila dose dependently when it was added to the culture medium of macrophage monolayers, whereas it did not inhibit the bacterial growth in buffered yeast extract broth, which was used for an L. pneumophila culture. The effect of 2dG was reversible because the surviving bacteria resumed intracellular multiplication after the washing away of 2dG from the culture. The effect of 2dG was also competitively inhibited by high concentrations of glucose. The inhibitory effect of 2dG was not attributed to the inhibition of bacterial phagocytosis by macrophages. Furthermore, sodium fluoride (0.1 and 1 mM), cycloheximide (0.1 and 1 microgram/ml), and tunicamycin (1, 2, and 5 micrograms/ml) did not promote the killing of L. pneumophila in macrophages, implying that the inhibitory effect of 2dG cannot be attributed to the inhibition of glycolysis, protein synthesis, and protein glycosylation in macrophages. We suggest that 2dG promotes intracellular killing of L. pneumophila by activating some novel killing mechanism of macrophages. PMID:8262638

  9. Cryopreservation of unfertilized human oocytes.

    PubMed

    Stachecki, James J; Cohen, Jacques; Garrisi, John; Munné, Santiago; Burgess, Colleen; Willadsen, Steen M

    2006-08-01

    Previous investigations revealed that choline-based freezing media developed in our laboratory were superior to conventional sodium-based media for storing mouse oocytes. This paper examines the ability of the choline-based medium CJ2 and a modified form of this medium, CJ3, to cryopreserve unfertilized human oocytes. Oocytes that were consented for research and matured overnight, as well as freshly collected, donor, mature metaphase II (MII) oocytes, were cryopreserved using choline-based media and an optimized slow-cooling protocol. The results showed higher survival and fertilization rates when CJ3 supplemented with 0.2 mmol/l sucrose was used as compared with CJ2 supplemented with either 0.1 mmol/l or 0.2 mmol/l sucrose. Freshly collected oocytes were more difficult to cryopreserve than those matured in vitro. Modification of the base medium proved to be one of the key factors in obtaining survival rates over 90%. Fertilization rates, embryo development, and genetic analysis of embryos resulting from control and frozen-thawed oocytes are provided. There appears to be a high correlation between chromosomal anomalies and abnormal morphology in embryos from thawed oocytes.

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

    PubMed

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

    2011-08-28

    Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

  11. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

    PubMed Central

    Bonne-Année, Sandra; Kerepesi, Laura A.; Hess, Jessica A.; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B.; Nolan, Thomas J.; Abraham, David

    2014-01-01

    Neutrophils are multifaceted cells that are often the immune system’s first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. PMID:24642003

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

    SciTech Connect

    Tomita, T.; Blumenstock, E.; Kanegasaki, S.

    1981-06-01

    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.

  13. Measurement of the size of intracellular ice crystals in mouse oocytes using a melting point depression method and the influence of intracellular solute concentrations

    PubMed Central

    Han, Xu; Critser, John K.

    2009-01-01

    Characterization of intracellular ice formed during the cooling procedures of cells significantly benefits the development and optimization design of cryopreservation or cryosurgery techniques. In this study, we investigated the influence of the concentration of extracellular non-permeable and permeable solutes on the melting points of the intracellular ice in mouse oocytes using cryomicroscopy. The results showed that the melting points of the intracellular ice are always lower than the extracellular ice. Based on this observation and the Gibbs-Thomson relation, we established a physical model to calculate the size of intracellular ice crystals and described its relationship with the concentrations of intracellular permeating solutes and macromolecules. This model predicts that the increased concentration of macromolecules in cells, by increasing the extracellular non-permeating solute concentration, can significantly lower the required concentration of permeable solutes for intracellular vitrification. The prediction was tested through the cryomicroscopic observation of the co-existence of intracellular vitrification and extracellular crystallization during cooling at 100°C/min when the extracellular solutions contain 5 molal (m) ethylene glycol and 0.3 to 0.6 m NaCl. PMID:19729005

  14. Measurement of the size of intracellular ice crystals in mouse oocytes using a melting point depression method and the influence of intracellular solute concentrations.

    PubMed

    Han, Xu; Critser, John K

    2009-12-01

    Characterization of intracellular ice formed during the cooling procedures of cells significantly benefits the development and optimization design of cryopreservation or cryosurgery techniques. In this study, we investigated the influence of the concentration of extracellular non-permeable and permeable solutes on the melting points of the intracellular ice in mouse oocytes using cryomicroscopy. The results showed that the melting points of the intracellular ice are always lower than the extracellular ice. Based on this observation and the Gibbs-Thomson relation, we established a physical model to calculate the size of intracellular ice crystals and described its relationship with the concentrations of intracellular permeating solutes and macromolecules. This model predicts that the increased concentration of macromolecules in cells, by increasing the extracellular non-permeating solute concentration, can significantly lower the required concentration of permeable solutes for intracellular vitrification. The prediction was tested through the cryomicroscopic observation of the co-existence of intracellular vitrification and extracellular crystallization during cooling at 100 degrees C/min when the extracellular solutions contain 5 molal (m) ethylene glycol and 0.3 to 0.6m NaCl.

  15. 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

  16. Na+- and Cl−-coupled active transport of carnitine by the amino acid transporter ATB0,+ from mouse colon expressed in HRPE cells and Xenopus oocytes

    PubMed Central

    Nakanishi, Takeo; Hatanaka, Takahiro; Huang, Wei; Prasad, Puttur D; Leibach, Frederick H; Ganapathy, Malliga E; Ganapathy, Vadivel

    2001-01-01

    ATB0,+ is an amino acid transporter energized by transmembrane gradients of Na+ and Cl− and membrane potential. We cloned this transporter from mouse colon and expressed the clone functionally in mammalian (human retinal pigment epithelial, HRPE) cells and Xenopus laevis oocytes to investigate the interaction of carnitine and its acyl esters with the transporter. When expressed in mammalian cells, the cloned ATB0,+ was able to transport carnitine, propionylcarnitine and acetylcarnitine. The transport process was Na+ and Cl− dependent and inhibitable by the amino acid substrates of the transporter. The Michaelis constant for carnitine was 0.83 ± 0.08 mm and the Hill coefficient for Na+ activation was 1.6 ± 0.1. When expressed in Xenopus laevis oocytes, the cloned ATB0,+ was able to induce inward currents in the presence of carnitine and propionylcarnitine under voltage-clamped conditions. There was no detectable current in the presence of acetylcarnitine. Carnitine-induced currents were obligatorily dependent on the presence of Na+ and Cl−. The currents were saturable with carnitine and the Michaelis constant was 1.8 ± 0.4 mm. The analysis of Na+- and Cl−-activation kinetics revealed that 2 Na+ and 1 Cl− were involved in the transport of carnitine via the transporter. These studies describe the identification of a novel function for the amino acid transporter ATB0,+. Since this transporter is expressed in the intestinal tract, lung and mammary gland, it is likely to play a significant role in the handling of carnitine in these tissues. A Na+-dependent transport system for carnitine has already been described. This transporter, known as OCTN2 (novel organic cation transporter 2), is expressed in most tissues and transports carnitine with high affinity. It is energized, however, only by a Na+ gradient and membrane potential. In contrast, ATB0,+ is a low-affinity transporter for carnitine, but exhibits much higher concentrative capacity than OCTN2 because

  17. NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary.

    PubMed

    Kiyosu, Chiyo; Tsuji, Takehito; Yamada, Kaoru; Kajita, Shimpei; Kunieda, Tetsuo

    2012-08-01

    Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.

  18. Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos.

    PubMed

    Ozturk, Saffet; Yaba-Ucar, Aylin; Sozen, Berna; Mutlu, Derya; Demir, Necdet

    2016-03-01

    Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.

  19. Cationic Antimicrobial Peptides and Biogenic Silver Nanoparticles Kill Mycobacteria without Eliciting DNA Damage and Cytotoxicity in Mouse Macrophages

    PubMed Central

    Mohanty, Soumitra; Jena, Prajna; Mehta, Ranjit; Pati, Rashmirekha; Banerjee, Birendranath; Patil, Satish

    2013-01-01

    With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template. PMID:23689720

  20. Comparative analysis of cell killing and autosomal mutation in mouse kidney epithelium exposed to 1 GeV protons in vitro or in vivo.

    PubMed

    Kronenberg, Amy; Gauny, Stacey; Kwoh, Ely; Grossi, Gianfranco; Dan, Cristian; Grygoryev, Dmytro; Lasarev, Michael; Turker, Mitchell S

    2013-05-01

    Human exposure to high-energy protons occurs in space flight scenarios or, where necessary, during radiotherapy for cancer or benign conditions. However, few studies have assessed the mutagenic effectiveness of high-energy protons, which may contribute to cancer risk. Mutations cause cancer and most cancer-associated mutations occur at autosomal loci. This study addresses the cytotoxic and mutagenic effects of 1 GeV protons in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events. Results for kidneys irradiated in vivo are compared with the results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3-4 months postirradiation in vivo. Incubation in vivo for longer periods (8-9 months) further attenuates proton-induced cell killing. Protons are mutagenic to cells in vitro and for in vivo irradiated kidneys. The dose-response for Aprt mutation is curvilinear after in vitro or in vivo exposure, bending upward at the higher doses. While the absolute mutant fractions are higher in vivo, the fold-increase over background is similar for both in vitro and in situ exposures. Results are also presented for a limited study on the effect of dose fractionation on the induction of Aprt mutations in kidney epithelial cells. Dose-fractionation reduces the fraction of proton-induced Aprt mutants in vitro and in vivo and also results in less cell killing. Taken together, the mutation burden in the epithelium is slightly reduced by dose-fractionation. Autosomal mutations accumulated during clinical exposure to high-energy protons may contribute to the risk of treatment-associated neoplasms, thereby highlighting the need for rigorous treatment planning to reduce the dose to normal tissues. For low dose exposures that occur during most space flight scenarios, the mutagenic effects of protons appear to be modest.

  1. Comparative analysis of cell killing and autosomal mutation in mouse kidney epithelium exposed to 1 GeV/nucleon iron ions in vitro or in situ.

    PubMed

    Kronenberg, Amy; Gauny, Stacey; Kwoh, Ely; Connolly, Lanelle; Dan, Cristian; Lasarev, Michael; Turker, Mitchell S

    2009-11-01

    Astronauts receive exposures to high-energy heavy ions from galactic cosmic radiation. Although high-energy heavy ions are mutagenic and carcinogenic, their mutagenic potency in epithelial cells, where most human cancers develop, is poorly understood. Mutations are a critical component of human cancer, and mutations involving autosomal loci predominate. This study addresses the cytotoxic and mutagenic effects of 1 GeV/nucleon iron ions in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events contributing to human cancer. Results for kidneys irradiated in situ are compared with results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3-4 months postirradiation in situ, but in situ exposures were less likely to result in cell death than in vitro exposures. Prolonged incubation in situ (8-9 months) further attenuated cell killing at lower doses. Iron ions were mutagenic to cells in vitro and for irradiated kidneys. No sparing was seen for mutant frequency with a long incubation period in situ. In addition, the degree of mutation induction (relative increase over background) was similar for cells exposed in vitro or in situ. We speculate that the latent effects of iron-ion exposure contribute to the maintenance of an elevated mutation burden in an epithelial tissue.

  2. Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C.

    PubMed

    Alexandre, H; Mulnard, J

    1988-12-01

    A passive erratic movement of the germinal vesicle (GV), already visible in small incompetent oocytes, is followed by an active scalloping of the nuclear membrane soon before GV breakdown (GVBD) in cultured competent oocytes. Maturation can be inhibited by activators of protein kinase A (PK-A) and protein kinase C (PK-C). Our time-lapse cinematography analysis allowed us to describe an unexpected behaviour of the GV when PK-C, but not PK-A, is activated: GV undergoes a displacement toward the cortex according to the same biological clock which triggers the programmed translocation of the spindle in control oocytes. It is concluded that, when oocytes become committed to undergo maturation, the cytoplasm acquires a PK-A-controlled "centrifugal displacement property" which is not restricted to the spindle.

  3. Shortened estrous cycle length, increased FSH levels, FSH variance, oocyte spindle aberrations, and early declining fertility in aging senescence-accelerated mouse prone-8 (SAMP8) mice: concomitant characteristics of human midlife female reproductive aging.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Kraemer, Duane C; Morley, John E; Farr, Susan; Chaffin, Charles L; Merchenthaler, István

    2014-06-01

    Women experience a series of specific transitions in their reproductive function with age. Shortening of the menstrual cycle begins in the mid to late 30s and is regarded as the first sign of reproductive aging. Other early changes include elevation and increased variance of serum FSH levels, increased incidences of oocyte spindle aberrations and aneuploidy, and declining fertility. The goal of this study was to investigate whether the mouse strain senescence-accelerated mouse-prone-8 (SAMP8) is a suitable model for the study of these midlife reproductive aging characteristics. Midlife SAMP8 mice aged 6.5-7.85 months (midlife SAMP8) exhibited shortened estrous cycles compared with SAMP8 mice aged 2-3 months (young SAMP8, P = .0040). Midlife SAMP8 mice had high FSH levels compared with young SAMP8 mice, and mice with a single day of high FSH exhibited statistically elevated FSH throughout the cycle, ranging from 1.8- to 3.6-fold elevation on the days of proestrus, estrus, metestrus, and diestrus (P < .05). Midlife SAMP8 mice displayed more variance in FSH than young SAMP8 mice (P = .01). Midlife SAMP8 ovulated fewer oocytes (P = .0155). SAMP8 oocytes stained with fluorescently labeled antitubulin antibodies and scored in fluorescence microscopy exhibited increased incidence of meiotic spindle aberrations with age, from 2/126 (1.59%) in young SAMP8 to 38/139 (27.3%) in midlife SAMP8 (17.2-fold increase, P < .0001). Finally, SAMP8 exhibited declining fertility from 8.9 pups/litter in young SAMP8 to 3.5 pups/litter in midlife SAMP8 mice (P < .0001). The age at which these changes occur is younger than for most mouse strains, and their simultaneous occurrence within a single strain has not been described previously. We propose that SAMP8 mice are a model of midlife human female reproductive aging.

  4. Epidermal growth factor receptor signaling-dependent calcium elevation in cumulus cells is required for NPR2 inhibition and meiotic resumption in mouse oocytes.

    PubMed

    Wang, Yakun; Kong, Nana; Li, Na; Hao, Xiaoqiong; Wei, Kaiwen; Xiang, Xi; Xia, Guoliang; Zhang, Meijia

    2013-09-01

    In preovulatory ovarian follicles, the oocyte is maintained in meiotic prophase arrest by natriuretic peptide precursor C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2). LH treatment results in the decrease of NPR2 guanylyl cyclase activity that promotes resumption of meiosis. We investigated the regulatory mechanism of LH-activated epidermal growth factor (EGF) receptor signaling on NPR2 function. Cumulus cell-oocyte complex is cultured in the medium with 30 nM NPPC to prevent oocyte spontaneous maturation. In this system, EGF could stimulate oocyte meiotic resumption after 4 hours of incubation. Further study showed that EGF elevated intracellular calcium concentrations of cumulus cells and decreased cGMP levels in cumulus cells and oocytes, and calcium-elevating reagents ionomycin and sphingosine-1-phosphate mimicked the effects of EGF on oocyte maturation and cGMP levels. EGF-mediated cGMP levels and meiotic resumption could be reversed by EGF receptor inhibitor AG1478 and the calcium chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester. EGF also decreased the expression of Npr2 mRNA in cumulus cells, which may not be involved in meiotic resumption, because the block of NPR2 protein de novo synthesis by cycloheximide had no effect on NPPC and EGF-mediated oocyte maturation. However, EGF had no effect on oocyte maturation when meiotic arrest was maintained in the present of cGMP analog 8-bromoadenosine-cGMP. These results suggest that EGF receptor signaling induces meiotic resumption by elevating calcium concentrations of cumulus cells to decrease NPR2 guanylyl cyclase activity.

  5. 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.

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

    PubMed Central

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

    2016-01-01

    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

  7. S100A8, An Oocyte-Specific Chemokine, Directs the Migration of Ovarian Somatic Cells During Mouse Primordial Follicle Assembly.

    PubMed

    Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua; Xia, Guoliang

    2015-12-01

    In the mammalian ovaries, the primordial follicle pool determines the reproductive capability over the lifetime of a female. The primordial follicle is composed of two cell members, namely the oocyte and the pre-granulosa cells that encircle the oocyte. However, it is unclear what factors are involved in the reorganization of the two distinct cells into one functional unit. This study was performed to address this issue. Firstly, in an in vitro reconstruction system, dispersed ovarian cells from murine fetal ovaries at 19.0 days post coitum (dpc) reassembled into follicle-like structures, independent of the physical distance between the cells, implying that either oocytes or ovarian somatic cells (OSCs) were motile. We then carried out a series of transwell assay experiments, and determined that it was in fact 19.0 dpc OSCs (as opposed to oocytes), which exhibited a significant chemotactic response to both fetal bovine serum and oocytes themselves. We observed that S100A8, a multi-functional chemokine, may participate in the process as it is mainly expressed in oocytes within the cysts/plasmodia. S100A8 significantly promoted the number of migrating OSCs by 2.5 times in vitro, of which 66.9% were FOXL2 protein-positive cells, implying that the majority of motile OSCs were pre-granulosa cells. In addition, an S100A8-specific antibody inhibited the formation of follicle-like reconstruction cell mass in vitro. And, the primordial follicle formation was reduced when S100a8-specific siRNA was applied onto in vitro cultured 17.5 dpc ovary. Therefore, S100A8 could be a chemokine of oocyte origin, which attracts OSCs to form the primordial follicles. © 2015 Wiley Periodicals, Inc.

  8. Effects of nicotine administration on elemental concentrations in mouse granulosa cells, maturing oocytes and oviduct epithelium studied by X-ray microanalysis.

    PubMed

    Jin, Z; Jin, M; Nilsson, B O; Roomans, G M

    1998-10-01

    A normal maturation of the oocytes is dependent upon, among other things, normally functioning granulosa and corona radiata cells. Analyses performed during human in vitro fertilization programs have revealed that, in smokers, ovarian functions are affected and that smokers have a decreased fertilization rate. Further, animal studies have indicated that nicotine can reach the genital tractus, and that nicotine administration interferes with oocyte maturation, fertilization and early pregnancy. We applied X-ray microanalysis to monitor whether nicotine administration changed the ionic balance of cells in the reproductive tract (granulosa cells, oocytes and oviduct epithelial cells). The animals were given nicotine in the drinking water at a concentration of 108 mumol/l. After 15 days the animals were superovulated, ovaries and oviducts were frozen, and thick cryosections were prepared for energy-dispersive X-ray microanalysis. In the granulosa cells, the concentrations of Na and Cl increased after nicotine treatment, while the K concentrations decreased resulting in an increased Na/K ratio. The treated oocytes had a higher K concentration and a decreased Na/K ratio compared to the controls. In the epithelial cells of the oviduct, the concentrations of Na and K decreased after nicotine treatment without any changes in the Na/K ratio. Thus, heavy nicotine administration to mice causes significant changes in the ionic composition of the granulosa cells, the ovarian oocytes and the oviduct epithelium.

  9. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage

    PubMed Central

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

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1Ser137 and pPlk1Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1Ser137 was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1Ser137 and pPlk1Thr210, as well as the subcellular distribution of pPlk1Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1Ser137 is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  10. Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells.

    PubMed

    Solc, Petr; Schultz, Richard M; Motlik, Jan

    2010-09-01

    Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic

  11. 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."…

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

    PubMed

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

    1987-08-01

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

  13. 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

  14. Inter-alpha-inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes.

    PubMed

    Hess, K A; Chen, L; Larsen, W J

    1999-08-01

    This report characterizes the effects of excess hyaluronan (HA) upon the expansion of the cumulus oocyte complex (COC) within intact follicles and upon ovulation and oocyte viability in mice. Covalent linkage between heavy chains of the inter-alpha-inhibitor (IalphaI) family of serum glycoproteins and HA is necessary for optimal cumulus extracellular matrix (cECM) stabilization and cumulus expansion. Intravenous administration of HA oligosaccharides inhibited the binding of IalphaI to endogenous HA, disrupting the process of expansion and resulting in a reduction in the size of the cumulus mass. Western blot and immunocytochemical analyses of COCs from HA-treated animals demonstrated a reduction of IalphaI heavy chains within the cECM. Additionally, HA-treated immature animals ovulated 56.3% fewer COCs compared to control animals. The developmental potential of COCs in HA-treated animals was also tested. Extended periods of oviductal storage of COCs ovulated by HA-injected adult mice resulted in a reduction of normal embryos and a significant increase in the proportion of fragmented oocytes/embryos. These observations support the view that covalent binding of IalphaI heavy chains to HA is required for optimal cumulus expansion, extrusion of the COCs from the follicle at ovulation, and maintenance of oocyte viability within the oviduct.

  15. Improvements in oocyte competence in superovulated mice following treatment with cilostazol: Ovulation of immature oocytes with high developmental rates.

    PubMed

    Taiyeb, Ahmed M; Muhsen-Alanssari, Saeeda A; Dees, William L; Hiney, Jill; Kjelland, Michael E; Kraemer, Duane C; Ridha-Albarzanchi, Mundhir T

    2017-08-01

    Exogenous administration of superovulatory hormones negatively affects oocyte competence in mammals. Phosphodiesterase 3A inhibitors were found to improve competence of oocytes matured in vitro in several species, including humans. This study was therefore designed to define oocyte maturation synchronization and competence, in vivo, using superovulated mice treated with cilostazol, a selective phosphodiesterase 3A inhibitor. Swiss Webster mice were superovulated and treated orally with 7.5mg cilostazol once or twice to result in ovulation of immature oocytes at the metaphase I (MI) or germinal vesicle (GV) stage, respectively. Control immature oocytes were recovered from preovulatory follicles of superovulated mice not treated with cilostazol. Treated GV oocytes had significantly higher rates of synchronized and advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had a moderate increase in cAMP levels and consequently higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P<0.0001). Treated MI oocytes had higher rates of normal spindles and chromosomes aligned at the metaphase plate than did control MI oocytes (P<0.003). Treated mice ovulating MI oocytes produced litter sizes larger than those observed in control mice ovulating mature oocytes (P<0.002). This study reveals that synchronization of oocyte maturation in superovulated mice improves oocyte development and competence. The capability of cilostazol, a clinically approved medication, to improve mouse oocyte competence suggests the potential benefit of including this compound in ovarian hyperstimulation programs to improve in vitro fertilization outcomes in infertile women. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Site specific mutation of the Zic2 locus by microinjection of TALEN mRNA in mouse CD1, C3H and C57BL/6J oocytes.

    PubMed

    Davies, Benjamin; Davies, Graham; Preece, Christopher; Puliyadi, Rathi; Szumska, Dorota; Bhattacharya, Shoumo

    2013-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing.

  17. Targeted Killings

    DTIC Science & Technology

    2013-03-01

    American territory in history. Two aircraft torpedoed into the Twin Towers in New York City at speeds of over 490mph killing 2,595 people . Shortly...bomb exploded in the World Trade Center in New York City, killing a half-dozen people and wounding over a thousand. Over the next three years Al...executed his most incredible attack killing close to 3,000 people . President Bush announced to the world that, “U.S. troops will hunt down terrorists and

  18. Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes.

    PubMed

    Zhang, John; Liu, Hui

    2015-07-01

    Both the cytoplasmic and nuclear compartments are essential for the acquisition of meiotic competence. This study assessed the role of the cytoplasm in meiosis resumption in meiotically arrested oocytes at the germinal vesicle (GV) stage. Mouse oocytes at GV stage were meiotically arrested with 3-isobutyl-1-methylxanthine (IBMX). GV transfer was performed between IBMX-treated and non-treated (control) mouse oocytes, and between control mouse and human GV oocytes. Extrusion of first polar body (PB) was examined as an indication of nuclear maturation. Meiotic spindle assembly and chromosome alignment were examined by immunostaining. Results indicated that oocytes arrested with IBMX for 24 and 48 h exhibited reduced ability for meiotic maturation and for extruding the first PB when compared with controls (P < 0.01). IBMX-treated oocytes reconstituted with cytoplasm, but not GV, of control oocytes restored the assembly of meiotic spindle and meiotic maturation. Mouse oocytes reconstituted with GV of human oocytes underwent meiosis similar to that observed in mice, but not humans. Additionally, human oocytes reconstituted by mouse GV underwent meiosis similar to that observed in humans, but not mice. These findings suggest that cytoplasm replacement by GV transfer could represent a potential therapeutic option for women who do not produce mature oocytes during IVF. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  19. 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.

    PubMed

    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 (D0 = 0.50 Gy) than FELC (D0 = 0.65 Gy) and V79 cells (D0 = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0 degrees 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 degrees C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5 degrees 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 degrees 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.

  20. 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

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

    PubMed

    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; Liang, Cheng-Guang

    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.

  2. 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

  3. Killing fetuses and killing newborns.

    PubMed

    Di Nucci, Ezio

    2013-05-01

    The argument for the moral permissibility of killing newborns is a challenge to liberal positions on abortion because it can be considered a reductio of their defence of abortion. Here I defend the liberal stance on abortion by arguing that the argument for the moral permissibility of killing newborns on ground of the social, psychological and economic burden on the parents recently put forward by Giubilini and Minerva is not valid; this is because they fail to show that newborns cannot be harmed and because there are morally relevant differences between fetuses and newborns.

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

    PubMed Central

    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 (DC8,9PC). 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

  5. Aging of oocyte, ovary, and human reproduction.

    PubMed

    Ottolenghi, Chris; Uda, Manuela; Hamatani, Toshio; Crisponi, Laura; Garcia, Jose-Elias; Ko, Minoru; Pilia, Giuseppe; Sforza, Chiarella; Schlessinger, David; Forabosco, Antonino

    2004-12-01

    We review age-related changes in the ovary and their effect on female fertility, with particular emphasis on follicle formation, follicle dynamics, and oocyte quality. The evidence indicates that the developmental processes leading to follicle formation set the rules determining follicle quiescence and growth. This regulatory system is maintained until menopause and is directly affected in at least some models of premature ovarian failure (POF), most strikingly in the Foxl2 mouse knockout, a model of human POF with monogenic etiology (blepharophimosis/ptosis/epicanthus inversus syndrome). Several lines of evidence indicate that if the ovarian germ cell lineage maintains regenerative potential, as recently suggested in the mouse, a role in follicle dynamics for germ stem cells, if any, is likely indirect or secondary. In addition, age-related variations in oocyte quality in animal models suggest that reproductive competence is acquired progressively and might depend on parallel growth and differentiation of follicle cells and stroma. Genomewide analyses of the mouse oocyte transcriptome have begun to be used to systematically investigate the mechanisms of reproductive competence that are altered with aging. Investigative and therapeutic strategies can benefit from considering the role of continuous interactions between follicle cells and oocytes from the beginning of histogenesis to full maturation.

  6. Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression

    PubMed Central

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

    2008-01-01

    RNA interference (RNAi) is an effective tool for studying gene function in oocytes, but no studies have targeted somatic cells of primary cultured cumulus cell-oocyte complexes (COCs). This is probably due to difficulty in introducing RNAi-inducing molecules, such as a short-hairpin RNA (shRNA) gene, into COCs by commonly used transfection reagents. We therefore tested whether a developmental process of intact COCs could be suppressed by adenovirus-mediated shRNA expression. Has2, encoding hyaluronan synthase 2, was selected as the target transcript, because the process of cumulus expansion depends upon expression of Has2 mRNA and this process is easily evaluated in vitro. Intact COCs were infected with replication-incompetent adenoviruses containing an expression sequence of shRNA targeting either Has2 (Has2 shRNA) or a control transcript not expressed in cumulus cells, and the effects on epidermal growth factor (EGF)-stimulated cumulus expansion were determined. Has2 shRNA expression suppressed Has2 mRNA levels in COCs by more than 70%, without affecting expression levels of Ptgs2, Ptx3, Tnfaip6 mRNAs, which are also required for cumulus expansion, or other transcripts not related to expansion. Interestingly, levels of Areg and Ereg mRNAs were decreased in COCs expressing Has2 shRNA when compared with those in controls, while Btc mRNA levels remained unaffected. Furthermore, the degree of cumulus expansion by Has2 shRNA-expressing COCs was significantly less than that of controls. Thus adenovirus-mediated introduction of shRNA produces specific gene silencing and a phenotype in intact COCs, providing proof of principle that this method will be a helpful tool for understanding mechanisms of COC development. PMID:18951380

  7. 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.

  8. [Mitochondrial and oocyte development].

    PubMed

    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  9. Testicular oocytes in MRL/MpJ mice possess similar morphological, genetic, and functional characteristics to ovarian oocytes.

    PubMed

    Otsuka-Kanazawa, Saori; Ichii, Osamu; Kon, Yasuhiro

    2015-08-01

    In general, mammalian males produce only spermatozoa in their testes and females produce only oocytes in their ovaries. However, newborn MRL/MpJ male mice produce oocytes within their testes. In this study, we examined the initiation and progression of oogenesis in fetal and neonatal MRL/MpJ mouse testes and evaluated the characteristics of testicular oocytes. Germ cells with positive reactions to oogenesis markers such as NOBOX oogenesis homeobox and synaptonemal complex protein 3 were observed in the MRL/MpJ fetal testes on embryonic day 18.5. These fetal testicular oocytes possessed maternal-specific methylation patterns of histone and DNA. The level of DNA methylation was still low in postnatal testicular oocytes at day 14 after birth. Additionally, the postnatal testicular oocytes contained both X and Y chromosomes and had the ability to fuse with sperm. These results suggest that some XY germ cells in fetal testes of MRL/MpJ mice enter meiosis prematurely, undergo oogenesis, and differentiate into oocytes. In addition, MRL/MpJ testicular oocytes have the ability to carry on oogenesis before and shortly after birth until they obtain some of the morphological, epigenetic, and functional characteristics of oocytes.

  10. Reconstruction of ooplasm recipient oocytes with frozen-thawed donor microcytoplasts and influence on the microtubular spindle.

    PubMed

    Goud, Anuradha P; Goud, Pravin T; Diamond, Michael P; Gonik, Bernard; Dozortsev, Dmitri I; Hughes, Mark R

    2007-04-01

    To cryopreserve micromanipulated ooplast segments (microcytoplasts) from mouse oocytes, compare microcytoplast and parent or recipient oocyte fusion performed within or without the zona pellucida, compare electrofusion of fresh or frozen oocyte with frozen-thawed microcytoplasts, and assess spindle integrity after reconstruction of oocytes. Prospective experimental study. University-based experimental laboratory. Mouse (MII) oocytes obtained after superovulation (n = 363). Micromanipulation of oocytes (n = 363) into microcytoplasts (n = 181), cryopreservation of microcytoplasts along with parent and sibling control oocytes (n = 182), reconstruction by electrofusion of microcytoplast and parent or recipient oocyte performed with (group A, n = 35) or without a zona pellucida (group B, n = 32), comparison of electrofusion of fresh oocyte (group C, n = 40) or frozen oocyte (group D, n = 36) with frozen-thawed microcytoplasts fused within zona, and assessment of spindle morphology of reconstructed oocyte. Post-thaw survival, success of fusion, and spindle integrity as assessed by immunostaining. Higher success of post-thaw fusion was seen in group A (91.4%) compared with group B (56.2%). The post-thaw fusion of microcytoplasts with either fresh or frozen oocytes was not significantly different. Spindle integrity was 82.5% in group C as compared with 47.2% in group D. Microcytoplasts created from oocytes can be successfully cryopreserved, thawed, and used to reconstruct oocytes with intact spindles.

  11. 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

  12. Functional Topography of the Fully Grown Human Oocyte

    PubMed Central

    Monti, Manuela; Calligaro, Alberto; Behr, Barry; Pera, Renee Rejo; Redi, Carlo Alberto; Wossidlo, Mark

    2017-01-01

    In vivo maturation (IVM) of human oocytes is a technique used to increase the number of usable oocytes for in vitro fertilization (IVF) and represents a necessity for women with different ovarian pathologies. During IVM the oocytes progress from the germinal vesicle stage (GV) through the metaphase II and during this journey both nuclear and cytoplasmic rearrangements must be obtained to increase the probability to get viable and healthy zygotes/embryos after IVF. As the successful clinical outcomes of this technique are a reality, we wanted to investigate the causes behind oocytes maturation arrest. For obvious ethical reasons, we were able to analyze only few human immature oocytes discarded and donated to research by transmission electron microscopy showing that, as in the mouse, they have different chromatin and cytoplasmic organizations both essential for further embryo development. PMID:28348419

  13. Combined vaccination of live 1B Chlamydophila abortus and killed phase I Coxiella burnetii vaccine does not destroy protection against chlamydiosis in a mouse model

    PubMed Central

    2004-01-01

    Abstract Q fever and chlamydiosis often affect ovine and caprine flocks simultaneously or successively. Combination vaccines effective against these 2 diseases would be of great value in veterinary medicine. Unfortunately, the current effective vaccines are a live vaccine for chlamydiosis and killed vaccine for Q fever. Vaccination of mice with live chlamydiosis vaccine 1B and killed phase I vaccine against Q fever at 2 points on the back at the same time produced good protection against chlamydial abortion. This suggests that it may be possible to vaccinate ewes and goats against chlamydiosis and Q fever simultaneously. PMID:15352550

  14. pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue.

    PubMed

    Kietz, D; Bartel, D; Lepke, S; Passow, H

    1991-04-26

    The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4'-diisothiocyanodihydrostilbene-2,2'-disfulfonate (H2DIDS) turns slowly into irreversible inhibition. This is due to covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys a and Lys b. In the red cell membrane, the pK value of Lys a is about 2.5 pK units lower than the pK value of Lys b. Hence the susceptibility of Lys a to irreversible modification by H2DIDS far exceeds the susceptibility to Lys b. In the present paper, we have expressed in Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl- exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H2DIDS at a concentration (20 microM) which saturates all H2DIDS binding sites with reversibly bound H2DIDS (KI = 0.3 microM and 1.1 microM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H2DIDS from the extracellular phase. Subsequent measurements of Cl- efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, pK values were calculated. These calculations could be made since in the wild-type, in which Lys a and Lys b are present, the exposure to H2DIDS could be confined to a pH range in which little if any covalent binding to Lys b takes place. The data could be represented by a single pK value of 8.3. In the mutant, Lys a is missing. Hence

  15. The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes.

    PubMed

    Bellani, Marina A; Boateng, Kingsley A; McLeod, Dianne; Camerini-Otero, R Daniel

    2010-09-01

    Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11alpha (exon 2 skipped) and SPO11beta. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11beta. Estimation of the number of SPO11 dimers (betabeta/alphabeta/alphaalpha) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11beta-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11alpha in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11alpha in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

  16. Mutagenic effects of ionizing radiation on immature rat oocytes.

    PubMed

    Asakawa, Jun-ichi; Kamiguchi, Yujiroh; Kamiya, Kenji; Nakamura, Nori

    2014-10-01

    Estimates of genetic risks from radiation delivered to humans are derived largely from mouse studies. In males, the target is spermatogonia and a large amount of information is available. In contrast, in females, immature oocytes are the target, but extrapolations from mice to humans are not very definitive because immature mouse oocytes are highly sensitive to radiation and die by apoptosis, which is not the case in humans. Since mouse offspring derived from surviving immature oocytes have to date not shown any signs of mutation induction, two alternative hypotheses are proposed: 1. Apoptotic death effectively eliminates damaged oocytes in mice and therefore human immature oocytes may be highly mutable; and 2. Immature oocytes are inherently resistant to mutation induction and apoptotic death is not relevant to mutagenesis. To test these hypotheses, rat immature oocytes, which are not as sensitive as those in mice to radiation-induced apoptosis were exposed to 2.5 Gy of gamma rays and the offspring were examined using a two-dimensional DNA analysis method. Screening of a total of 2.26 million DNA fragments, we identified 32 and 18 mutations in the control and exposed groups, respectively. Of these, in the two groups, 29 and 14 mutations were microsatellite mutations, two and one were base changes, and one and three were deletions. Among the four deletions most relevant to radiation exposure, only one was possibly derived from the irradiated dam (but not determined) and three were paternal in origin. Although the number of mutations was small, the results appear to support the second hypothesis and indicate that immature oocytes are generally less sensitive than mature oocytes to mutation induction.

  17. Evaluation of Mouse Oocyte In Vitro Maturation Developmental Competency in Dynamic Culture Systems by Design and Construction of A Lab on A Chip Device and Its Comparison with Conventional Culture System

    PubMed Central

    Sadeghzadeh Oskouei, Behnaz; Pashaiasl, Maryam; Heidari, Mohammad Hasan; Salehi, Mohammad; Veladi, Hadi; Ghaderi Pakdel, Firuz; Shahabi, Parviz; Novin, Marefat Ghaffari

    2016-01-01

    Objective In conventional assisted reproductive technology (ART), oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione (GSH) contents in each experimental group. Materials and Methods In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute (NMRI) mice. Oocytes were randomly placed in static (passive) and dynamic (active) in vitro maturation (IVM) culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus (PN) stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane (MCB) staining. Results We observed significantly higher percentages of mature metaphase II oocytes (MII) in the passive and active dynamic culture systems (DCS) compared to the static group (P<0.01). There were significantly less mean numbers of germinal vesicle (GV) and degenerated oocytes in the passive and active dynamic groups compared to the static group (P<0.01). Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures (P<0.01). There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes (P<0.01). Conclusion Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions. PMID:27540525

  18. Mouse Slc4a11 expressed in Xenopus oocytes is an ideally selective H+/OH- conductance pathway that is stimulated by rises in intracellular and extracellular pH.

    PubMed

    Myers, Evan J; Marshall, Aniko; Jennings, Michael L; Parker, Mark D

    2016-12-01

    The SLC4A11 gene encodes the bicarbonate-transporter-related protein BTR1, which is mutated in syndromes characterized by vision and hearing loss. Signs of these diseases [congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome] are evident in mouse models of Slc4a11 disruption. However, the intrinsic activity of Slc4a11 remains controversial, complicating assignment of its (patho)physiological role. Most studies concur that Slc4a11 transports H(+) (or the thermodynamically equivalent species OH(-)) rather than HCO3(-), but disparities have arisen as to whether the transport is coupled to another species such as Na(+) or NH3/NH4(+) Here for the first time, we examine the action of mouse Slc4a11 in Xenopus oocytes. We simultaneously monitor changes in intracellular pH, membrane potential, and conductance as we alter extracellular pH, revealing the electrical and chemical driving forces that underlie the observed ion fluxes. We find that mSlc4a11 is an ideally selective H(+)/OH(-) conductive pathway, the action of which is uncoupled from the cotransport of any other ion. We also find that the activity of mSlc4a11 is independently enhanced by both extracellular and intracellular alkalinization, suggesting OH(-) as the most likely substrate and providing a novel explanation for the apparent NH3-dependence of Slc4a11-mediated currents reported by others. We suggest that the unique properties of Slc4a11 action underlie its value as a pH regulator in corneal endothelial cells. Copyright © 2016 the American Physiological Society.

  19. Beyond killing

    PubMed Central

    Vale, Pedro F.; McNally, Luke; Doeschl-Wilson, Andrea; King, Kayla C.; Popat, Roman; Domingo-Sananes, Maria R.; Allen, Judith E.; Soares, Miguel P.; Kümmerli, Rolf

    2016-01-01

    The antibiotic pipeline is running dry and infectious disease remains a major threat to public health. An efficient strategy to stay ahead of rapidly adapting pathogens should include approaches that replace, complement or enhance the effect of both current and novel antimicrobial compounds. In recent years, a number of innovative approaches to manage disease without the aid of traditional antibiotics and without eliminating the pathogens directly have emerged. These include disabling pathogen virulence-factors, increasing host tissue damage control or altering the microbiota to provide colonization resistance, immune resistance or disease tolerance against pathogens. We discuss the therapeutic potential of these approaches and examine their possible consequences for pathogen evolution. To guarantee a longer half-life of these alternatives to directly killing pathogens, and to gain a full understanding of their population-level consequences, we encourage future work to incorporate evolutionary perspectives into the development of these treatments. PMID:27016341

  20. Cell Cycle-dependent Regulation of Structure of Endoplasmic Reticulum and Inositol 1,4,5-Trisphosphate-induced Ca2+ Release in Mouse Oocytes and Embryos

    PubMed Central

    FitzHarris, Greg; Marangos, Petros; Carroll, John

    2003-01-01

    The organization of endoplasmic reticulum (ER) was examined in mouse eggs undergoing fertilization and in embryos during the first cell cycle. The ER in meiosis II (MII)-arrested mouse eggs is characterized by accumulations (clusters) that are restricted to the cortex of the vegetal hemisphere of the egg. Monitoring ER structure with DiI18 after egg activation has demonstrated that ER clusters disappear at the completion of meiosis II. The ER clusters can be maintained by inhibiting the decrease in cdk1-cyclin B activity by using the proteasome inhibitor MG132, or by microinjecting excess cyclin B. A role for cdk1-cyclin B in ER organization is further suggested by the finding that the cdk inhibitor roscovitine causes the loss of ER clusters in MII eggs. Cortical clusters are specific to meiosis as they do not return in the first mitotic division; rather, the ER aggregates around the mitotic spindle. Inositol 1,4,5-trisphosphate-induced Ca2+ release is also regulated in a cell cycle-dependent manner where it is increased in MII and in the first mitosis. The cell cycle dependent effects on ER structure and inositol 1,4,5-trisphosphate-induced Ca2+ release have implications for understanding meiotic and mitotic control of ER structure and inheritance, and of the mechanisms regulating mitotic Ca2+ signaling. PMID:12529444

  1. Mutations in TUBB8 and Human Oocyte Meiotic Arrest

    DOE PAGES

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; ...

    2016-01-21

    BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

  2. In vitro maturation of oocytes.

    PubMed

    Smith, G D

    2001-10-01

    In vitro maturation (IVM) of human oocytes is an emerging assisted reproductive technology with great promise. To be successful, this process must entail both nuclear and cytoplasmic maturation. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. Although nuclear maturation during human oocyte IVM progresses normally, cytoplasmic maturation is significantly lacking, as exemplified by poor embryonic developmental competence and pregnancy rates. Advances made in immature oocyte isolation and oocyte and embryo culture conditions have increased the clinical feasibility of IVM. However, in order to achieve acceptable birth rates, future studies should focus on characterization and regulation of oocyte cytoplasmic maturation, and how oocyte-derived factors influence zygotic genome activation and embryonic developmental competence.

  3. Imbalance between the expression dosages of X-chromosome and autosomal genes in mammalian oocytes.

    PubMed

    Fukuda, Atsushi; Tanino, Motohiko; Matoba, Ryo; Umezawa, Akihiro; Akutsu, Hidenori

    2015-09-15

    Oocytes have unique characteristics compared with other cell types. In mouse and human oocytes, two X chromosomes are maintained in the active state. Previous microarray studies have shown that the balance of the expression state is maintained in haploid oocytes. Here, we investigated transcripts using RNA-sequence technology in mouse and human oocytes. The median expression ratio between X chromosome and autosomal genes (X:A) in immature mouse oocytes increased as the gene expression levels increased, reaching a value of 1. However, the ratio in mature oocytes was under 1 for all expression categories. Moreover, we observed a markedly low ratio resulting from the bimodal expression patterns of X-linked genes. The low X:A expression ratio in mature oocyte was independent of DNA methylation. While mature human oocytes exhibited a slightly low X:A expression ratio, this was the result of the skewed high frequency of lowly expressed X-linked genes rather than the bimodal state. We propose that this imbalance between the expression dosages of X-chromosome and autosomal genes is a feature of transcripts in mammalian oocytes lacking X-chromosome inactivation.

  4. Imbalance between the expression dosages of X-chromosome and autosomal genes in mammalian oocytes

    PubMed Central

    Fukuda, Atsushi; Tanino, Motohiko; Matoba, Ryo; Umezawa, Akihiro; Akutsu, Hidenori

    2015-01-01

    Oocytes have unique characteristics compared with other cell types. In mouse and human oocytes, two X chromosomes are maintained in the active state. Previous microarray studies have shown that the balance of the expression state is maintained in haploid oocytes. Here, we investigated transcripts using RNA-sequence technology in mouse and human oocytes. The median expression ratio between X chromosome and autosomal genes (X:A) in immature mouse oocytes increased as the gene expression levels increased, reaching a value of 1. However, the ratio in mature oocytes was under 1 for all expression categories. Moreover, we observed a markedly low ratio resulting from the bimodal expression patterns of X–linked genes. The low X:A expression ratio in mature oocyte was independent of DNA methylation. While mature human oocytes exhibited a slightly low X:A expression ratio, this was the result of the skewed high frequency of lowly expressed X-linked genes rather than the bimodal state. We propose that this imbalance between the expression dosages of X-chromosome and autosomal genes is a feature of transcripts in mammalian oocytes lacking X-chromosome inactivation. PMID:26370379

  5. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice.

    PubMed

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A; Cohen, Paula E; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë

    2016-03-07

    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women's fertility depends on the longevity of cohesin proteins that established cohesion in utero.

  6. Glucocorticoids impair oocyte developmental potential by triggering apoptosis of ovarian cells via activating the Fas system.

    PubMed

    Yuan, Hong-Jie; Han, Xiao; He, Nan; Wang, Guo-Liang; Gong, Shuai; Lin, Juan; Gao, Min; Tan, Jing-He

    2016-04-04

    Previous studies indicate that stress damages oocytes with increased secretion of glucorticoids. However, although injection of female mice with cortisol decreased oocyte competence, exposure of mouse oocytes directly to physiological or stress-induced concentrations of glucorticoids did not affect oocyte maturation and embryo development. This study has explored the mechanisms by which glucocorticoids impair oocyte competence. Female mice were injected with cortisol and the effects of cortisol-injection on oocyte competence, ovarian cell apoptosis and Fas/FasL activation were observed. The results showed that cortisol-injection decreased (a) oocyte developmental potential, (b) the E2/P4 ratio in serum and ovaries, and (c) expression of insulin-like growth factor 1, brain-derived neurotrophic factor and glucocorticoid receptor in mural granulosa cells (MGCs), while increasing levels of (a) cortisol in serum and ovaries, (b) apoptosis in MGCs and cumulus cells (CCs), (c) FasL secretion in ovaries and during oocyte maturation in vitro, and (d) Fas in MGCs, CCs and oocytes. The detrimental effects of cortisol-injection on oocyte competence and apoptosis of MGCs and CCs were significantly relieved when the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations were observed. Together, the results suggested that glucocorticoids impair oocyte competence by triggering apoptosis of ovarian cells via activating the Fas system.

  7. Glucocorticoids impair oocyte developmental potential by triggering apoptosis of ovarian cells via activating the Fas system

    PubMed Central

    Yuan, Hong-Jie; Han, Xiao; He, Nan; Wang, Guo-Liang; Gong, Shuai; Lin, Juan; Gao, Min; Tan, Jing-He

    2016-01-01

    Previous studies indicate that stress damages oocytes with increased secretion of glucorticoids. However, although injection of female mice with cortisol decreased oocyte competence, exposure of mouse oocytes directly to physiological or stress-induced concentrations of glucorticoids did not affect oocyte maturation and embryo development. This study has explored the mechanisms by which glucocorticoids impair oocyte competence. Female mice were injected with cortisol and the effects of cortisol-injection on oocyte competence, ovarian cell apoptosis and Fas/FasL activation were observed. The results showed that cortisol-injection decreased (a) oocyte developmental potential, (b) the E2/P4 ratio in serum and ovaries, and (c) expression of insulin-like growth factor 1, brain-derived neurotrophic factor and glucocorticoid receptor in mural granulosa cells (MGCs), while increasing levels of (a) cortisol in serum and ovaries, (b) apoptosis in MGCs and cumulus cells (CCs), (c) FasL secretion in ovaries and during oocyte maturation in vitro, and (d) Fas in MGCs, CCs and oocytes. The detrimental effects of cortisol-injection on oocyte competence and apoptosis of MGCs and CCs were significantly relieved when the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations were observed. Together, the results suggested that glucocorticoids impair oocyte competence by triggering apoptosis of ovarian cells via activating the Fas system. PMID:27040909

  8. The Effects of Voluntary Exercise on Oocyte Quality in a Diet-Induced Obese Murine Model

    PubMed Central

    Boudoures, Anna L.; Chi, Maggie; Thompson, Alysha; Zhang, Wendy; Moley, Kelle H.

    2016-01-01

    Obesity negatively affects many aspects of the human body, including reproductive function. In females, the root of the decline in fertility is linked to problems in the oocyte. Problems seen in oocytes that positively correlate with increasing BMI include changes to the metabolism, lipid accumulation, meiosis, and metaphase II (MII) spindle structure. Studies in mice indicate dietary interventions fail to reverse these problems [4]. How exercise affects the oocytes has not been addressed. Therefore, we hypothesized an exercise intervention would improve oocyte quality. Here we show in a mouse model of an exercise intervention can improve lipid metabolism in germinal vesicle (GV) stage oocytes. Oocytes significantly increased activity and transcription of the β-oxidation enzyme Hadha (Hydroxyacyl-CoA-dehydrogenase) in response to exercise training only if the mice had been fed a high fat diet (HFD). An exercise intervention also reversed the lipid accumulation seen in GV stage oocytes of HFD females. However, delays in meiosis and disorganized MII spindles remained present. Therefore, exercise is able to improve, but not reverse, damage imparted on oocytes as a result of a high fat diet and obesity. By utilizing an exercise intervention on a HFD, we determined only lipid content and lipid metabolism is changed in GV oocytes. Moving forward, interventions to improve oocyte quality may need to be more targeted to the oocyte specifically. Because of the HFD induced deficiency in β-oxidation, dietary supplementation with substrates to improve lipid utilization may be more beneficial. PMID:26700938

  9. From fresh heterologous oocyte donation to autologous oocyte banking

    PubMed Central

    Stoop, D.

    2012-01-01

    Introduction: Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. Methods: We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Results: Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women <37 years old remains constant with a mean of 4.47%. A significant proportion of young women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. Discussion and Conclusion: The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock. PMID:24753920

  10. Antimalarial Pyrido[1,2-a]benzimidazoles: Lead Optimization, Parasite Life Cycle Stage Profile, Mechanistic Evaluation, Killing Kinetics, and in Vivo Oral Efficacy in a Mouse Model.

    PubMed

    Singh, Kawaljit; Okombo, John; Brunschwig, Christel; Ndubi, Ferdinand; Barnard, Linley; Wilkinson, Chad; Njogu, Peter M; Njoroge, Mathew; Laing, Lizahn; Machado, Marta; Prudêncio, Miguel; Reader, Janette; Botha, Mariette; Nondaba, Sindisiwe; Birkholtz, Lyn-Marie; Lauterbach, Sonja; Churchyard, Alisje; Coetzer, Theresa L; Burrows, Jeremy N; Yeates, Clive; Denti, Paolo; Wiesner, Lubbe; Egan, Timothy J; Wittlin, Sergio; Chibale, Kelly

    2017-02-23

    Further structure-activity relationship (SAR) studies on the recently identified pyrido[1,2-a]benzimidazole (PBI) antimalarials have led to the identification of potent, metabolically stable compounds with improved in vivo oral efficacy in the P. berghei mouse model and additional activity against parasite liver and gametocyte stages, making them potential candidates for preclinical development. Inhibition of hemozoin formation possibly contributes to the mechanism of action.

  11. A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys

    PubMed Central

    Smith, Eric J.; Olson, Kara; Haber, Lauric J.; Varghese, Bindu; Duramad, Paurene; Tustian, Andrew D.; Oyejide, Adelekan; Kirshner, Jessica R.; Canova, Lauren; Menon, Jayanthi; Principio, Jennifer; MacDonald, Douglas; Kantrowitz, Joel; Papadopoulos, Nicholas; Stahl, Neil; Yancopoulos, George D.; Thurston, Gavin; Davis, Samuel

    2015-01-01

    Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies. PMID:26659273

  12. High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes.

    PubMed

    Shishova, Kseniya V; Khodarovich, Yuriy M; Lavrentyeva, Elena A; Zatsepina, Olga V

    2015-10-01

    Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The "internal" rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5'ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin.

  13. Oocyte Maturation and Development

    PubMed Central

    Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary. PMID:26998245

  14. Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation

    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

    Background: L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods: The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results: L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions: It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification. PMID:27503023

  15. How do oocytes disappear?

    PubMed

    Bonilla-Musoles, F; Renau, J; Hernandez-Yago, J; Torres, J

    1975-07-29

    It has been study using transmission and scanner electron microscopy the mean procedures of dessaparence of the oocytes. On described three methods: 1. The necrosis of the oocytes. 2. The autolysis and fagocitosis by granulosa cells. 3. The migration of those to the superphicie and fall into the peritoneal cavity. Using the scanner electron microscopy in ovaries of fetus and newborn it seems the latest method to bee the most important during the intrauterine life. After the birth, this last phenomenon seems to disappear.

  16. Mining the oocyte transcriptome.

    PubMed

    Andreu-Vieyra, Claudia; Lin, Yi-Nan; Matzuk, Martin M

    2006-01-01

    Mammalian folliculogenesis and oocyte physiology are complex and not fully understood. However, major advances over the past 15 years in our ability to create and study in vivo models have improved our understanding of these essential physiological processes. More recently, the availability of vast arrays of DNA sequence information in the forms of "complete" genomes, expressed sequence tag libraries and microarray data from reproductive tissues have stimulated the discovery of new information through genome scanning, prediction programs and in silico screening techniques. These technological improvements will help to expand our understanding of folliculogenesis and oocyte physiology and improve human reproductive health.

  17. Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: a model for utilization of primordial oocytes.

    PubMed

    Kaneko, Hiroyuki; Nakai, Michiko; Tanihara, Fuminori; Noguchi, Junko; Kikuchi, Kazuhiro

    2013-11-01

    Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1(nu)). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to 45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion.

    PubMed

    Coonrod, S A; Naaby-Hansen, S; Shetty, J; Shibahara, H; Chen, M; White, J M; Herr, J C

    1999-03-15

    The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster

  19. Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis.

    PubMed

    Schmelcher, Mathias; Powell, Anne M; Camp, Mary J; Pohl, Calvin S; Donovan, David M

    2015-10-01

    Bovine mastitis results in billion dollar losses annually in the USA alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca(2+) concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 μg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis.

  20. Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis

    PubMed Central

    Schmelcher, Mathias; Powell, Anne M.; Camp, Mary J.; Pohl, Calvin S.; Donovan, David M.

    2015-01-01

    Bovine mastitis results in billion dollar losses annually in the United States alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca2+ concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 μg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis. PMID:25895090

  1. Reactive oxygen species and oocyte aging: role of superoxide, hydrogen peroxide, and hypochlorous acid.

    PubMed

    Goud, Anuradha P; Goud, Pravin T; Diamond, Michael P; Gonik, Bernard; Abu-Soud, Husam M

    2008-04-01

    Aging of the unfertilized oocyte inevitably occurs following ovulation, limiting its fertilizable life span. However, the mechanisms that regulate oocyte aging are still unclear. We hypothesize that reactive oxygen species such as superoxide (O2-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are likely candidates that may initiate these changes in the oocyte. In order to test this hypothesis, we investigated direct effects of O2- [hypoxanthine/xanthine oxidase system generating 0.12 (n=42) and 0.25 (n=45) microM O2-/min], H2O2 (20 or 100 microM, n=60), and HOCl, (1, 10, and 100 microM, n=50) on freshly ovulated or relatively old mouse oocytes, while their sibling oocytes were fixed immediately or cultured under physiological conditions (n=96). The aging process was assessed by the zona pellucida dissolution time (ZPDT), ooplasm microtubule dynamics (OMD), and cortical granule (CG) status. The ZPDT increased 2-fold in relatively old, compared to young, untreated oocytes (P<0.0001). Exposure to O2- increased it even further (P<0.0001). Similarly, more O2- exposed oocytes exhibited increased OMD and major CG loss, with fewer having normal OMD and intact CG compared to untreated controls. Interestingly, young oocytes resisted "aging," when exposed to 20 microM H2O2, while the same enhanced the aging phenomena in relatively old oocytes (P<0.05). Exposure to even very low levels of HOCl induced the aging phenomena in young and relatively old oocytes, and higher concentrations of HOCl compromised oocyte viability. Overall, O2-, H2O2, and HOCl each augment oocyte aging, more so in relatively old oocytes, suggesting compromised antioxidant capacity in aging oocytes.

  2. Inhibitory effect of heat-killed Lactobacillus strain on immunoglobulin E-mediated degranulation and late-phase immune reactions of mouse bone marrow-derived mast cells.

    PubMed

    Kawahara, Takeshi

    2010-12-01

    This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)- and antigen-induced mast cell degranulation and subsequent gene expression. Bone marrow-derived mast cells (BMMCs) from DBA/2 mice were cultured with heat-killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE- and antigen-induced β-hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)-4, IL-13, tumor necrosis factor-α, and cyclooxygenase-2 expression levels that was induced by 1-2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll-like receptor (TLR)2-neutralizing antibody. In addition, downregulation of cell surface FcεRIα expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE-mediated mast cell degranulation and subsequent late-phase reactions involving mast cells via a TLR2-dependent mechanism with FcεRIα downregulation.

  3. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

    EPA Science Inventory

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  4. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

    EPA Science Inventory

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  5. DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes.

    PubMed

    Sun, Ming-Hong; Yang, Mo; Xie, Feng-Yun; Wang, Wei; Zhang, Lili; Shen, Wei; Yin, Shen; Ma, Jun-Yu

    2017-01-01

    As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.

  6. DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

    PubMed Central

    Sun, Ming-Hong; Yang, Mo; Xie, Feng-Yun; Wang, Wei; Zhang, Lili; Shen, Wei; Yin, Shen

    2017-01-01

    As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells. PMID:28099474

  7. p27(Kip1) negatively regulates the activation of murine primordial oocytes.

    PubMed

    Hirashima, Yumiko; Moniruzzaman, Mohammad; Miyano, Takashi

    2011-04-01

    In mice, small oocytes (primordial oocytes) are enclosed within flattened granulosa cells to form primordial follicles around birth. A small number of primordial oocytes enter the growth phase, whereas others are quiescent. The mechanism regulating this selection of primordial oocytes is not well understood. The objective of the present study was to understand the role of p27(Kip1), which regulates cell cycle progression in somatic cells, in the growth initiation of primordial oocytes in neonatal mice. We studied the localization of p27(Kip1) in 0-, 3-, 5-, 7- and 21-day-old mouse ovaries by immunohistochemistry. Ovaries from 3-day-old mice were treated with p27(Kip1) siRNAs (small interfering RNAs), and knockdown of p27(Kip1) was determined by immunohistochemistry and Western blotting. Ovaries treated with siRNAs were organ-cultured for 6 days, and oocyte growth was estimated histologically. Expression of p27(Kip1) was undetectable in the primordial oocytes of newborn mice. In the 3-day-old ovaries (n=3), p27(Kip1) was demonstrated in the nucleus of 36 ± 6% primordial oocytes. The percentage of p27(Kip1)-positive primordial oocytes increased to 72 ± 8 (n=3), 85 ± 7 (n=3) and 93 ± 5 (n=3) in the 5-, 7- and 21-day-old mouse ovaries, respectively. After knockdown of the p27(Kip1) protein by siRNAs, a higher proportion of oocytes entered the growth phase in cultured ovaries than those in the control. These results suggest that p27(Kip1) negatively regulates primordial oocyte growth and that knockdown of p27(Kip1) leads primordial oocytes to enter the growth phase in vitro.

  8. Truths and myths of oocyte sensitivity to controlled rate freezing.

    PubMed

    Coticchio, G; Bonu, M A; Sciajno, R; Sereni, E; Bianchi, V; Borini, A

    2007-07-01

    The mammalian oocyte is especially sensitive to cryopreservation. Because of its size and physiology, it can easily undergo cell death or sub-lethal damage as a consequence of intracellular ice formation, increase in the concentration of solutes and other undesired effects during the conversion of extracellular water into ice. This has generated the belief that oocyte storage cannot be achieved with the necessary efficiency and safety. However, many concerns raised by oocyte freezing are the result of unproven hypotheses or observations conducted under sometimes inappropriate conditions. For instance, spindle organization can undergo damage under certain freezing conditions but not with other protocols. The controversial suggestion that cryopreservation induces cortical granule discharge and zona pellucida hardening somehow questions the routine use of sperm microinjection. Damage to mouse oocytes caused by solute concentration is well documented but, in the human, there is no solid evidence that modifications of freezing mixtures, to prevent this problem, provide an actual advantage. The hope of developing oocyte cryopreservation as a major IVF option is becoming increasingly realistic, but major efforts are still required to clarify the authentic implications of oocyte cryopreservation at the cellular level and identify freezing conditions compatible with the preservation of viability and developmental ability.

  9. Oocyte freezing: here to stay?

    PubMed

    Van der Elst, Josiane

    2003-01-01

    Oocyte freezing is an established technology but, in contrast to embryo freezing, it has very limited application in clinical IVF programmes. Is there a chance that oocyte freezing will become an integrated routine in assisted reproductive technology? The delicate cytological architecture of the oocyte with a cold-sensitive spindle and a hardening zona have made the frozen oocyte 'unwanted' in assisted reproductive technology. Nevertheless, empirical improvements in freezing protocols and the use of ICSI for fertilization have led to an increasing number of live births. This mitigates against a simple ban on oocyte freezing. While efficiency of oocyte freezing can certainly be further improved by basic research, it is clear that there are humanitarian reasons for considering oocyte freezing as a future fully utilized assisted reproductive technology. The storage of the female genome as a particulate entity can provide an alternative in case of moral, ethical, legal or religious concerns about embryo freezing. Oocyte freezing can also offer hope for oocyte donation and preservation of fertility for women facing ovarian loss. The message is one of cautious optimism when looking for a place for oocyte freezing in routine assisted reproductive technology.

  10. [Pregnancy following oocyte donation].

    PubMed

    Boks, D E; Braat, D D

    1997-08-23

    Five women, aged 31, 26, 31, 34, and 28 years, became pregnant after oocyte donation and in-vitro fertilization. One was a carrier of Leber's optical atrophy, three had had an early menopause (in two because of chromosomal abnormalities), and one had had bilateral ovarian extirpation because of a cystadenoma and endometriosis. Three developed (pre-)eclampsia during pregnancy and one had a serious fluxus post partum. One twin died in utero, the other children were healthy. In the Netherlands in-vitro fertilization (with or without egg-donation) takes place up to the age of about 40. Regarding the high incidence of obstetrical complications in women under 40, raising the age limit could lead to even more pregnancy problems. Candidates for oocyte donation should be informed about these risks, furthermore they should not deliver at home.

  11. Natriuretic peptides stimulate oocyte meiotic resumption in bovine.

    PubMed

    De Cesaro, Matheus P; Macedo, Mariana P; Santos, Joabel T; Rosa, Paulo R A; Ludke, Charles A; Rissi, Vitor B; Gasperin, Bernardo G; Gonçalves, Paulo B D

    2015-08-01

    The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

  12. Optimization of cryoprotectant loading into murine and human oocytes.

    PubMed

    Karlsson, Jens O M; Szurek, Edyta A; Higgins, Adam Z; Lee, Sang R; Eroglu, Ali

    2014-02-01

    Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Planning a dynamic kill

    SciTech Connect

    Abel, L.W.

    1996-05-01

    This article discusses the methodology, design philosophy, and guidelines for planning a dynamic-kill operation for a wild well. The topics covered are two methods of computer analysis for designing dynamic-kill requirements, the design process, determining the pumping spread, and the pitfalls that a designer faces in planning a dynamic kill.

  14. Combinatory antibiotic therapy increases rate of bacterial kill but not final outcome in a novel mouse model of Staphylococcus aureus spinal implant infection

    PubMed Central

    Hu, Yan; Johansen, Daniel; Loftin, Amanda H.; Dworsky, Erik; Zoller, Stephen D.; Park, Howard Y.; Hamad, Christopher D.; Nelson, George E.; Francis, Kevin P.; Scaduto, Anthony; Bernthal, Nicholas M.

    2017-01-01

    Background Management of spine implant infections (SII) are challenging. Explantation of infected spinal hardware can destabilize the spine, but retention can lead to cord compromise and biofilm formation, complicating management. While vancomycin monotherapy is commonly used, in vitro studies have shown reduced efficacy against biofilm compared to combination therapy with rifampin. Using an established in vivo mouse model of SII, we aim to evaluate whether combination therapy has increased efficacy compared to both vancomycin alone and infected controls. Methods An L-shaped, Kirschner-wire was transfixed into the L4 spinous process of 12-week-old C57BL/6 mice, and inoculated with bioluminescent Staphylococcus aureus. Mice were randomized into a vancomycin group, a combination group with vancomycin plus rifampin, or a control group receiving saline. Treatment began on post-operative day (POD) 7 and continued through POD 14. In vivo imaging was performed to monitor bioluminescence for 35 days. Colony-forming units (CFUs) were cultured on POD 35. Results Bioluminescence peaked around POD 7 for all groups. The combination group had a 10-fold decrease in signal by POD 10. The vancomycin and control groups reached similar levels on POD 17 and 21, respectively. On POD 25 the combination group dropped below baseline, but rebounded to the same level as the other groups, demonstrating a biofilm-associated infection by POD 35. Quantification of CFUs on POD 35 confirmed an ongoing infection in all three groups. Conclusions Although both therapies were initially effective, they were not able to eliminate implant biofilm bacteria, resulting in a rebound infection after antibiotic cessation. This model shows, for the first time, why histologic-based, static assessments of antimicrobials can be misleading, and the importance of longitudinal tracking of infection. Future studies can use this model to test combinations of antibiotic therapies to see if they are more effective in

  15. Oocyte cryopreservation in oncological patients.

    PubMed

    Porcu, Eleonora; Fabbri, Raffaella; Damiano, Giuseppe; Fratto, Rosita; Giunchi, Susanna; Venturoli, Stefano

    2004-04-05

    The use of chemotherapy and radiotherapy in oncological patients may reduce their reproductive potential. Sperm cryopreservation has been already used in men affected by neoplastic disease. Oocyte cryopreservation might be an important solution for these patients at risk of losing ovarian function. A program of oocyte cryopreservation for oncological patients is also present in our center. From June 1996 to January 2000, 18 patients awaiting chemotherapy and radiotherapy for neoplastic disease were included in our oocyte cryopreservation program. Our experience documents that oocyte storage may be a concrete and pragmatic alternative for oncological patients. The duration of oocyte storage does not seem to interfere with oocyte survival as pregnancies occurred even after several years of gamete cryopreservation in liquid nitrogen.

  16. Is the mouse a clinically relevant model for human fertilization failures?

    PubMed

    Neuber, E; Powers, R D

    2000-01-01

    This study compares failed fertilization oocytes from patients participating in an in-vitro fertilization (IVF) programme with failed fertilization oocytes from B6SJLF(1)/J mice, in order to characterize and describe the distribution of DNA in oocytes that do not undergo normal fertilization. Our goal is to evaluate the mouse IVF system as a model to gain insight into reasons for human fertilization failures. All oocytes were stained with the vital fluorescent dye, Hoechst 33342, which rapidly stains double-stranded DNA. Of the 237 human oocytes that had been scored as failed fertilization by brightfield microscopy, 61 (25.7%) showed the presence of at least one spermatozoon within the oocyte cytoplasm. In contrast, out of 69 failed fertilization mouse oocytes, only one oocyte showed the presence of a spermatozoon within its cytoplasm. Mouse failed fertilization oocytes exhibited a significantly lower internal sperm rate (P < 0.0001) than human failed fertilization oocytes. Human failed fertilization oocytes show a higher incidence of sperm penetration, but the cytoplasm fails to support pronuclear development, whereas, at least in this strain, mouse failed fertilization oocytes arise from an inability of the spermatozoa to penetrate the oocyte. This study suggests that the mouse is not a clinically relevant model for human fertilization failures.

  17. Oocyte quality in mice is affected by a mycotoxin-contaminated diet.

    PubMed

    Hou, Yan-Jun; Xiong, Bo; Zheng, Wei-Jiang; Duan, Xing; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Qiang; Xu, Yin-Xue; Sun, Shao-Chen

    2014-05-01

    Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 μg/kg), and AF (806 μg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality.

  18. Melamine negatively affects oocyte architecture, oocyte development and fertility in mice.

    PubMed

    Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Sun, Shao-Chen

    2015-07-01

    Does melamine have a toxic effect on oocyte development and fertility in vivo? Melamine had toxic effects on oocyte quality and fertility due to its effects on the oocyte cytoskeleton, apoptosis and autophagy induction, and epigenetic modifications in an in vivo mouse model. Melamine is a chemical compound that is widely used during the manufacture of amino resins and plastics. In 2008, melamine was reported to adulterate milk and infant formulas in China, which sparked food safety concerns worldwide. Ingesting melamine may result in reproductive damage, and bladder or kidney stones, which can lead to bladder cancer. Mice were randomly assigned to three groups and fed a diet that included melamine (0, 10 and 50 mg/kg/day) for 8 weeks. The in vivo effect of melamine on female reproduction was examined. We used immunofluorescent staining, western blotting and qRT-PCR to examine the effect of melamine on oocyte quality. Our results showed the following effects of this melamine-containing diet. (i) Ovary weights were reduced in melamine fed mice. Oocyte developmental competence was also reduced, as shown by reduced polar body extrusion rates. (ii) Melamine feeding resulted in abnormal oocyte cytoskeletons, as shown by increased rates of aberrant spindles and reduced actin microfilament expression. (iii) Melamine exposed oocytes had higher rates of abnormal mitochondrial distributions and early stage apoptosis/autophagy, which were shown by increased microtubule-associated protein 1 light chain 3 (LC3) protein expression level and caspase 9, autophagy-related protein 14 (atg14), and lc3 mRNA levels. (iv) Fluorescence intensity analysis showed that DNA methylation levels were reduced in the oocytes of melamine fed mice. Histone methylation levels were also altered, as Di-methyl-Histone H3 (Lys4) (H3K4me2) level was increased and Tri-methyl-Histone H3 (Lys9) (H3K9me3), Di-methyl-Histone H3 (Lys9) (H3K9me2), and Tri-methyl-Histone H3 (Lys27) (H3K27me3) levels were reduced

  19. The Crucial Role of Zona Pellucida in Cryopreservation of Oocytes by Vitrification

    PubMed Central

    Choi, Jung Kyu; Yue, Tao; Huang, Haishui; Zhao, Gang; Zhang, Mingjun; He, Xiaoming

    2015-01-01

    Mammalian oocytes have a proteinaceous hydrogel-like outer shell known as the zona pellucida (ZP) that semi-encloses their plasma membrane and cytoplasm. In this study, we cryopreserved mouse oocytes either with or without ZP by vitrification. Our results show that the presence of an intact ZP could significantly improve the post-vitrification survival of oocytes to 92.1% from 13.3% for oocytes without ZP. Moreover, there was no significant difference in embryonic development between fresh and cryopreserved oocytes with ZP after in vitro fertilization (IVF). Further atomic force microscopy (AFM) analysis showed that the intact oocytes with ZP have an elastic modulus that is more than 85 times higher than that of oocytes without ZP. This may partially explain the important role of ZP in protecting the oocytes by resisting the mechanical stress due to possible ice formation during cryopreservation by vitrification. Collectively, this study reveals a new biophysical role of ZP during vitrification of oocytes and suggests microencapsulation of the many mammalian cells without a ZP in ZP-like hydrogel is an effective strategy to improve their survival post cryopreservation by vitrification. PMID:26297946

  20. The crucial role of zona pellucida in cryopreservation of oocytes by vitrification.

    PubMed

    Choi, Jung Kyu; Yue, Tao; Huang, Haishui; Zhao, Gang; Zhang, Mingjun; He, Xiaoming

    2015-10-01

    Mammalian oocytes have a proteinaceous hydrogel-like outer shell known as the zona pellucida (ZP) that semi-encloses their plasma membrane and cytoplasm. In this study, we cryopreserved mouse oocytes either with or without ZP by vitrification. Our results show that the presence of an intact ZP could significantly improve the post-vitrification survival of oocytes to 92.1% from 13.3% for oocytes without ZP. Moreover, there was no significant difference in embryonic development between fresh and cryopreserved oocytes with ZP after in vitro fertilization (IVF). Further atomic force microscopy (AFM) analysis showed that the intact oocytes with ZP have an elastic modulus that is more than 85 times higher than that of oocytes without ZP. This may partially explain the important role of ZP in protecting the oocytes by resisting the mechanical stress due to possible ice formation during cryopreservation by vitrification. Collectively, this study reveals a new biophysical role of ZP during vitrification of oocytes and suggests microencapsulation of the many mammalian cells without a ZP in ZP-like hydrogel is an effective strategy to improve their survival post cryopreservation by vitrification.

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

    SciTech Connect

    Yoneda, Akihiro; Watanabe, Tomomasa

    2015-05-01

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

  2. Epigenetic profile of developmentally important genes in bovine oocytes.

    PubMed

    Heinzmann, J; Hansmann, T; Herrmann, D; Wrenzycki, C; Zechner, U; Haaf, T; Niemann, H

    2011-03-01

    Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM-matured oocytes, 215 alleles/4,725 CpGs in mSOF-matured oocytes, and 78 alleles/1,672 CpGs from in vivo-matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real-time PCR. We found different mRNA expression profiles between in vivo-matured oocytes versus their in vitro-matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation.

  3. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-01-01

    Only a fraction of oocytes present in the ovaries at birth are ever ovulated during the lifetime of a female mammal. In vitro maturation (IVM) offers the possibility to exploit what is a largely untapped biological resource. Although IVM is used routinely for the in vitro production of embryos in domestic species, especially cattle, its clinical use in human-assisted reproduction is still evolving. The successful recapitulation in vitro of the events associated with successful oocyte maturation is not always achieved, with the majority of immature oocytes typically failing to develop to the blastocyst stage. Evidence suggests that although culture conditions throughout in vitro embryo production may have a modest influence on the developmental potential of the early embryo, the quality of the oocyte at the start of the process is the key factor determining the proportion of oocytes developing to the blastocyst stage.

  4. Null Killing vectors

    NASA Astrophysics Data System (ADS)

    Lukács, B.; Perjés, Z.; Sebestyén, Á.

    1981-06-01

    Space-times admitting a null Killing vector are studied, using the Newman-Penrose spin coefficient formalism. The properties of the eigenrays (principal null curves of the Killing bivector) are shown to be related to the twist of the null Killing vector. Among the electrovacs, the ones containing a null Maxwell field turn out to belong to the twist-free class. An electrovac solution is obtained for which the null Killing vector is twisting and has geodesic and shear-free eigenrays. This solution is parameterless and appears to be the field of a zero-mass, spinning, and charged source.

  5. [Motivations of oocytes donors].

    PubMed

    Cauvin, P

    2009-01-01

    Oocyte donation is a complex situation that requires the applicant couple to deal with the presence of the donor in the history of the child conception. Accepting the eggs is not the same thing than accepting the donor. Her place in the child's life depends on how his parents will accept her phantasmal reality beyond her real person. Paying attention to the story told by the donors on their motivations may help parents internalize this conception to three. We show from two clinical observations, that the generosity of donors is connected to personal issues that do not relate to unborn child or its parents. If there are two mothers in oocyte donation, they are not really in competition because there are also two children: the child conceived through donation is that of the project of the couple, the child to which the donor thinks, is and will remain in phantasmal domain, i.e. linked to the personal history of the donor. We also show that the psychological interview fully responds to the donor expectations when it seeks to highlight her motives.

  6. Ion-kill dosimetry

    NASA Technical Reports Server (NTRS)

    Katz, R.; Cucinotta, F. A.; Fromm, M.; Chambaudet, A.

    2001-01-01

    Unanticipated late effects in neutron and heavy ion therapy, not attributable to overdose, imply a qualitative difference between low and high LET therapy. We identify that difference as 'ion kill', associated with the spectrum of z/beta in the radiation field, whose measurement we label 'ion-kill dosimetry'.

  7. Killing tensors on tori

    NASA Astrophysics Data System (ADS)

    Heil, Konstantin; Moroianu, Andrei; Semmelmann, Uwe

    2017-07-01

    We show that Killing tensors on conformally flat n-dimensional tori whose conformal factor only depends on one variable, are polynomials in the metric and in the Killing vector fields. In other words, every first integral of the geodesic flow polynomial in the momenta on the sphere bundle of such a torus is linear in the momenta.

  8. Ion-kill dosimetry

    NASA Technical Reports Server (NTRS)

    Katz, R.; Cucinotta, F. A.; Fromm, M.; Chambaudet, A.

    2001-01-01

    Unanticipated late effects in neutron and heavy ion therapy, not attributable to overdose, imply a qualitative difference between low and high LET therapy. We identify that difference as 'ion kill', associated with the spectrum of z/beta in the radiation field, whose measurement we label 'ion-kill dosimetry'.

  9. Cloning, killing, and identity.

    PubMed Central

    McMahan, J

    1999-01-01

    One potentially valuable use of cloning is to provide a source of tissues or organs for transplantation. The most important objection to this use of cloning is that a human clone would be the sort of entity that it would be seriously wrong to kill. I argue that entities of the sort that you and I essentially are do not begin to exist until around the seventh month of fetal gestation. Therefore to kill a clone prior to that would not be to kill someone like you or me but would be only to prevent one of us from existing. And even after one of us begins to exist, the objections to killing it remain comparatively weak until its psychological capacities reach a certain level of maturation. These claims support the permissibility of killing a clone during the early stages of its development in order to use its organs for transplantation. PMID:10226909

  10. On the killing of mycobacteria by macrophages.

    PubMed

    Jordao, Luisa; Bleck, Christopher K E; Mayorga, Luis; Griffiths, Gareth; Anes, Elsa

    2008-02-01

    Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis, the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.

  11. Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

    PubMed

    Lin, Fei; Ma, Xue-Shan; Wang, Zhen-Bo; Wang, Zhong-Wei; Luo, Yi-Bo; Huang, Lin; Jiang, Zong-Zhe; Hu, Meng-Wen; Schatten, Heide; Sun, Qing-Yuan

    2014-01-01

    In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

  12. Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

    SciTech Connect

    Bahouth, S.W.; Malbon, C.C.

    1987-05-01

    Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in the control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.

  13. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    PubMed

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

  14. Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility.

    PubMed

    Nicholas, Cory R; Haston, Kelly M; Grewall, Amarjeet K; Longacre, Teri A; Reijo Pera, Renee A

    2009-11-15

    Ten to 15% of couples are infertile, with the most common causes being linked to the production of few or no oocytes or sperm. Yet, our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility.

  15. RAD51 plays a crucial role in halting cell death program induced by ionizing radiation in bovine oocytes.

    PubMed

    Kujjo, Loro L; Ronningen, Reg; Ross, Pablo; Pereira, Ricardo J G; Rodriguez, Ramon; Beyhan, Zeki; Goissis, Marcelo D; Baumann, Thomas; Kagawa, Wataru; Camsari, Cagri; Smith, George W; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki; Cibelli, Jose B; Perez, Gloria I

    2012-03-01

    Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

  16. Evolution of coalitionary killing.

    PubMed

    Wrangham, R W

    1999-01-01

    Warfare has traditionally been considered unique to humans. It has, therefore, often been explained as deriving from features that are unique to humans, such as the possession of weapons or the adoption of a patriarchal ideology. Mounting evidence suggests, however, that coalitional killing of adults in neighboring groups also occurs regularly in other species, including wolves and chimpanzees. This implies that selection can favor components of intergroup aggression important to human warfare, including lethal raiding. Here I present the principal adaptive hypothesis for explaining the species distribution of intergroup coalitional killing. This is the "imbalance-of-power hypothesis," which suggests that coalitional killing is the expression of a drive for dominance over neighbors. Two conditions are proposed to be both necessary and sufficient to account for coalitional killing of neighbors: (1) a state of intergroup hostility; (2) sufficient imbalances of power between parties that one party can attack the other with impunity. Under these conditions, it is suggested, selection favors the tendency to hunt and kill rivals when the costs are sufficiently low. The imbalance-of-power hypothesis has been criticized on a variety of empirical and theoretical grounds which are discussed. To be further tested, studies of the proximate determinants of aggression are needed. However, current evidence supports the hypothesis that selection has favored a hunt-and-kill propensity in chimpanzees and humans, and that coalitional killing has a long history in the evolution of both species.

  17. Oocyte development, meiosis and aneuploidy.

    PubMed

    MacLennan, Marie; Crichton, James H; Playfoot, Christopher J; Adams, Ian R

    2015-09-01

    Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes' prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes.

  18. Tri-directional anaphases as a novel chromosome segregation defect in human oocytes.

    PubMed

    Haverfield, Jenna; Dean, Nicola L; Nöel, Diana; Rémillard-Labrosse, Gaudeline; Paradis, Veronique; Kadoch, Isaac-Jacques; FitzHarris, Greg

    2017-06-01

    What are the chromosome segregation errors in human oocyte meiosis-I that may underlie oocyte aneuploidy? Multiple modes of chromosome segregation error were observed, including tri-directional anaphases, which we attribute to loss of bipolar spindle structure at anaphase-I. Oocyte aneuploidy is common and associated with infertility, but mechanistic information on the chromosome segregation errors underlying these defects is scarce. Lagging chromosomes were recently reported as a possible mechanism by which segregation errors occur. Long-term confocal imaging of chromosome dynamics in 50 human oocytes collected between January 2015 and May 2016. Germinal vesicle (GV) stage oocytes were collected from women undergoing intracytoplasmic sperm injection cycles and also CD1 mice. Oocytes were microinjected with complementary RNAs to label chromosomes, and in a subset of oocytes, the meiotic spindle. Oocytes were imaged live through meiosis-I using confocal microscopy. 3D image reconstruction was used to classify chromosome segregation phenotypes at anaphase-I. Segregation phenotypes were related to spindle dynamics and cell cycle timings. Most (87%) mouse oocytes segregated chromosomes with no obvious defects. We found that 20% of human oocytes segregated chromosomes bi-directionally with no lagging chromosomes. The rest were categorised as bi-directional anaphase with lagging chromosomes (20%), bi-directional anaphase with chromatin mass separation (34%) or tri-directional anaphase (26%). Segregation errors correlated with chromosome misalignment prior to anaphase. Spindles were tripolar when tri-directional anaphases occurred. Anaphase phenotypes did not correlate with meiosis-I duration (P = 0.73). Not applicable. Oocytes were recovered at GV stage after gonadotrophin-stimulation, and the usual oocyte quality caveats apply. Whilst the possibility that imaging may affect oocyte physiology cannot be formally excluded, detailed controls and justifications are presented

  19. Mutations in TUBB8 and Human Oocyte Meiotic Arrest

    SciTech Connect

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L.; Cowan, Nicholas J.; Wang, Lei

    2016-01-21

    BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.

  20. Report Bee Kills

    EPA Pesticide Factsheets

    EPA uses incident report data to help inform our pesticide regulatory decisions. Information from these reports helps us identify patterns of bee kills associated with the use of specific pesticides or active ingredients. Here's how to report incidents.

  1. Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.

    PubMed

    Zhang, Hua; Risal, Sanjiv; Gorre, Nagaraju; Busayavalasa, Kiran; Li, Xin; Shen, Yan; Bosbach, Benedikt; Brännström, Mats; Liu, Kui

    2014-11-03

    The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Survival of mouse oocytes after being cooled in a vitrification solution to −196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification☆

    PubMed Central

    Mazur, Peter; Seki, Shinsuke

    2011-01-01

    There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196°C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95°C/min to 69,250°C/min to −196°C and for each cooling rate, subjecting them to five warming rates back above 0°C at rates ranging from 610°C/min to 118,000°C/min. In samples warmed at the highest rate (118,000°C/min), survivals were 70 to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610°C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95°C/min to 69,000°C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming. PMID:21055397

  3. HORMAD1-dependent checkpoint/surveillance mechanism eliminates asynaptic oocytes.

    PubMed

    Kogo, Hiroshi; Tsutsumi, Makiko; Ohye, Tamae; Inagaki, Hidehito; Abe, Takaya; Kurahashi, Hiroki

    2012-06-01

    Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1-deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1-deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11-deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11-deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11-deficient testis and ovary, suggesting an involvement of novel DNA damage-independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1-dependent checkpoint in response to asynapsis in mammalian meiosis.

  4. Ultrastructure of in vitro Matured Human Oocytes.

    PubMed

    Shahedi, Abbas; Khalili, Mohammad Ali; Soleimani, Mehrdad; Morshedizad, Shekoufeh

    2013-12-01

    Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures. These oocytes represent a potential resource for both clinical and basic science application. The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission electron microscopy (TEM). A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary fresh in vivo matured oocytes (n = 10) were used as control. The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle complexes (M-VC). Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions and improve the implantation rate of in-vitro matured oocytes.

  5. PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

    PubMed

    Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

    2016-06-02

    Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.

  6. Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging

    PubMed Central

    Ben-Meir, Assaf; Burstein, Eliezer; Borrego-Alvarez, Aluet; Chong, Jasmine; Wong, Ellen; Yavorska, Tetyana; Naranian, Taline; Chi, Maggie; Wang, Ying; Bentov, Yaakov; Alexis, Jennifer; Meriano, James; Sung, Hoon-Ki; Gasser, David L; Moley, Kelle H; Hekimi, Siegfried; Casper, Robert F; Jurisicova, Andrea

    2015-01-01

    Female reproductive capacity declines dramatically in the fourth decade of life as a result of an age-related decrease in oocyte quality and quantity. The primary causes of reproductive aging and the molecular factors responsible for decreased oocyte quality remain elusive. Here, we show that aging of the female germ line is accompanied by mitochondrial dysfunction associated with decreased oxidative phosphorylation and reduced Adenosine tri-phosphate (ATP) level. Diminished expression of the enzymes responsible for CoQ production, Pdss2 and Coq6, was observed in oocytes of older females in both mouse and human. The age-related decline in oocyte quality and quantity could be reversed by the administration of CoQ10. Oocyte-specific disruption of Pdss2 recapitulated many of the mitochondrial and reproductive phenotypes observed in the old females including reduced ATP production and increased meiotic spindle abnormalities, resulting in infertility. Ovarian reserve in the oocyte-specific Pdss2-deficient animals was diminished, leading to premature ovarian failure which could be prevented by maternal dietary administration of CoQ10. We conclude that impaired mitochondrial performance created by suboptimal CoQ10 availability can drive age-associated oocyte deficits causing infertility. PMID:26111777

  7. Oocyte--granulosa cell interactions.

    PubMed

    Canipari, R

    2000-01-01

    In the past, different protocols of ovulation induction, aimed to overcome problems of anovulatory infertility in humans, have been developed during IVF programmes. However, administration of exogenous hormones may cause severe health problems, e.g. ovarian hyperstimulation syndrome. To overcome this problem an attractive alternative is to develop in-vitro systems that allow follicle and oocyte growth and maturation. This paper reviews the current status of research on oocyte-granulosa cell interactions and on the autocrine and paracrine factors involved in follicle development. The ovarian follicle is a morphological and functional unit in which the somatic and germ cell components are intimately associated and interdependent. The co-ordinate development of follicle and oocyte leads to a number of modifications in the growing oocyte necessary for the acquisition of competence to mature correctly and to undergo fertilization and embryo development. The search for the optimal culture conditions and the correct balance of hormones necessary to obtain a fertilizable oocyte in vitro is extremely important for clinical and agricultural applications.

  8. Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

    PubMed Central

    Ross, Pablo J; Beyhan, Zeki; Iager, Amy E; Yoon, Sook-Young; Malcuit, Christopher; Schellander, Karl; Fissore, Rafael A; Cibelli, Jose B

    2008-01-01

    Background During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes. Results Mouse and bovine PLCZ1 cRNAs were injected into matured bovine oocytes at different concentrations. Within the concentrations tested, mouse PLCZ1 injection activated bovine oocytes at a maximum rate when the pipette concentration of cRNA ranged from 0.25 to 1 μg/μL, while bovine PLCZ1 was optimal at 0.1 μg/μL. At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP. Injection of mouse and bovine PLCZ1 cRNA induced dose-dependent sperm-like calcium oscillations whose frequency increased over time. Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart. Conclusion Injection of PLCZ1 cRNA efficiently activated bovine oocytes by inducing a sperm-like calcium oscillatory pattern. Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos. PMID:18284699

  9. Oocyte maturation and fertilization in marine nemertean worms: using similar sorts of signaling pathways as in mammals, but often with differing results.

    PubMed

    Stricker, Stephen A; Cline, Cory; Goodrich, David

    2013-08-01

    In marine worms belonging to the phylum Nemertea, oocyte maturation and fertilization are regulated by the same general kinds of signals that control such processes in mammals. However, unlike mammalian oocytes that develop within follicles, nemertean oocytes characteristically lack a surrounding sheath of follicle cells and often respond differently to maturation-related cues than do mammalian oocytes. For example, elevators of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) levels promote the resumption of meiotic maturation (=germinal vesicle breakdown, GVBD) in nemertean oocytes, whereas increasing intraoocytic cAMP and cGMP typically blocks GVBD in mammals. Similarly, AMP-activated kinase (AMPK) signaling keeps nemertean oocytes from maturing, but in mouse oocytes, AMPK activation triggers GVBD. In addition, protein kinase C (PKC) activity is required for seawater-induced GVBD in nemerteans, whereas some PKCs have been shown to inhibit GVBD in mammals. Furthermore, although fertilization causes both types of oocytes to reorganize their endoplasmic reticulum and generate calcium oscillations that can involve soluble sperm factor activity and inositol 1,4,5-trisphosphate signaling, some discrepancies in the spatiotemporal patterns and underlying mechanisms of fertilization are also evident in nemerteans versus mammals. Thus, to characterize differences and similarities in gamete biology more fully, aspects of oocyte maturation and fertilization in marine nemertean worms are reviewed and briefly compared with related findings that have been published for mammalian oocytes. In addition, possible causes of the alternative responses displayed by oocytes in these two animal groups are addressed.

  10. Meiosis and Maternal Aging: Insights from Aneuploid Oocytes and Trisomy Births

    PubMed Central

    Herbert, Mary; Kalleas, Dimitrios; Cooney, Daniel; Lamb, Mahdi; Lister, Lisa

    2015-01-01

    In most organisms, genome haploidization requires reciprocal DNA exchanges (crossovers) between replicated parental homologs to form bivalent chromosomes. These are resolved to their four constituent chromatids during two meiotic divisions. In female mammals, bivalents are formed during fetal life and remain intact until shortly before ovulation. Extending this period beyond ∼35 years greatly increases the risk of aneuploidy in human oocytes, resulting in a dramatic increase in infertility, miscarriage, and birth defects, most notably trisomy 21. Bivalent chromosomes are stabilized by cohesion between sister chromatids, which is mediated by the cohesin complex. In mouse oocytes, cohesin becomes depleted from chromosomes during female aging. Consistent with this, premature loss of centromeric cohesion is a major source of aneuploidy in oocytes from older women. Here, we propose a mechanistic framework to reconcile data from genetic studies on human trisomy and oocytes with recent advances in our understanding of the molecular mechanisms of chromosome segregation during meiosis in model organisms. PMID:25833844

  11. Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

    PubMed Central

    Dupré, Aude; Jessus, Catherine; Ozon, René; Haccard, Olivier

    2002-01-01

    In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes. PMID:12145203

  12. Mos is not required for the initiation of meiotic maturation in Xenopus oocytes.

    PubMed

    Dupré, Aude; Jessus, Catherine; Ozon, René; Haccard, Olivier

    2002-08-01

    In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic