Hormesis of low-dose inhibition of pAkt1 (Ser473) followed by a great increase of proline-rich inositol polyphosphate 5-phosphatase (PIPP) level in oocytes.

Hormesis describes a biphasic dose-response relationship generally characterized by a low-dose excitement and a high-dose inhibition. This phenomenon has been observed in the regulation of cell, organ, and organismic level. However, hormesis has not reported in oocytes. In this study, we observed, for the first time, hormetic responses of PIPP levels in oocytes by inhibitor of Akt1 or PKCδ. The expression of PIPP was detected by qPCR, immunofluorescent (IF), and Western Blot (WB). To observe the changes of PIPP levels, we used the inhibitors against pAkt1 (Ser473) or PKCδ, SH-6 or sotrastaurin with low and/or high-dose, treated GV oocytes and cultured for 4 h, respectively. The results showed that PIPP expression was significantly enhanced when oocytes were treated with SH-6 or sotrastaurin 10 µM, but decreased with SH-6 or sotrastaurin 100 µM. We also examined the changes of PIPP levels when GV oocytes were treated with exogenous PtdIns(3,4,5)P3 or LY294002 for 4 h. Our results showed that PIPP level was enhanced much higher under the treatment of 0.1 µM PtdIns(3,4,5)P3 than that of 1 µM PtdIns(3,4,5)P3, which is consistent with the changes of PIPP when oocytes were treated with inhibitors of pAkt1 (Ser473) or PKCδ. In addition, with PIPP siRNA, we detected that down-regulated PIPP may affect distributions of Akt, Cdc25, and pCdc2 (Tyr15). Taken together, these results show that the relationships between PIPP and Akt may follow the principle of hormesis and play a key role during release of diplotene arrest in mouse oocytes.

Cilostamide and rolipram prevent spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro.

The involvement of specific phosphodiesterases (PDEs) in the modulation of cAMP and thereby spontaneous meiotic resumption remains poorly understood. This work aims to evaluate the effects of cilostamide and rolipram (PDE 3A and PDE 4D inhibitors) on spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro. For this purpose, diplotene-arrested cumulus oocyte complexes (COCs) were collected from rat ovary. The COCs and denuded oocytes were exposed to various concentrations of cilostamide (0.0, 2.5, 5.0 and 10 µM) and rolipram (0, 10, 50 and 100 µM) for various times (0, 3, 5, 7, 14, 16, 18, 20, 22 and 24 h). Cilostamide inhibited spontaneous meiotic resumption in a concentration- and time-dependent manner in COCs and denuded oocytes. Although rolipram showed inhibition of spontaneous meiotic resumption up to some extent, cilostamide was more potent to prevent spontaneous meiotic resumption in both COCs and denuded oocytes. Cilostamide significantly reduced PDE 3A expression, increased cAMP level and prevented spontaneous meiotic resumption in COCs and denuded oocytes. Although rolipram inhibited PDE 4D expression in cumulus cells, increased cAMP level but was not sufficient to prevent spontaneous meiotic resumption. We conclude that both drugs prevent spontaneous resumption from diplotene-arrest through PDE 3A/PDE 4D-cAMP mediated pathway. However, as compare to rolipram, cilostamide was more potent in preventing spontaneous resumption from diplotene-arrest in rat oocytes cultured in vitro. Thus, cilostamide could be used as a potential candidate for the development of female contraceptive drug in future.

Hydrogen Sulfide Impairs Meiosis Resumption in Xenopuslaevis Oocytes.

The role of hydrogen sulfide (H2S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H2S metabolism, cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H2S. On another hand, a dose-dependent inhibition was obtained using the H2S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H2S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H2S signaling in the timing of amphibian oocytes meiosis resumption.

Molecular analysis of the effects of steroid hormones on mouse meiotic prophase I progression.

BACKGROUND: Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis. METHODS: Fetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique. RESULTS: MSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage. CONCLUSIONS: We set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.

Dibutyl phthalate exposure disrupts the progression of meiotic prophase I by interfering with homologous recombination in fetal mouse oocytes.

Dibutyl phthalate (DBP), one of the most widely used plasticizers, is a known environmental endocrine disruptor that impairs male and female fertility. In this study, oral administration of DBP was given to pregnant mice on 14.5 days post coitus (dpc) for 3 days; and additionally, DBP was added into the culture of 14.5 dpc fetal ovaries for 3 days. DBP exposure during gestation disturbed the progression of meiotic prophase I of mouse oocytes, specifically from the zygotene to pachytene stages. Meanwhile, the DBP-exposed pachytene oocytes showed increased homologous recombination sites and unrepaired DNA damage. Furthermore, DBP caused DNA damage by increasing oxidative stress, decreased the expression of multiple critical meiotic regulators, and consequently induced oocyte apoptosis. Moreover, the effect of DBP on meiosis I prophase involved estrogen receptors α and ß. Collectively, these results demonstrated a set of meiotic defects in DBP-exposed fetal oocytes. As aberrations in homologous recombination can result in aneuploid gametes and embryos, this study provides new support for the deleterious effects of phthalates.

Reduction of nitric oxide level results in maturation promoting factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro.

Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured in vitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3 h in vitro and downstream factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to maturation promoting factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured in vitro.

Carbenoxolone reduces cyclic nucleotides level, destabilizes maturation promoting factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro.

BACKGROUND: Disruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing maturation promoting factor (MPF) in rat oocytes cultured in vitro. METHODS: Diplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3h in vitro. The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. RESULTS: CBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. CONCLUSIONS: Our results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs.

The impact of Zearalenone on the meiotic progression and primordial follicle assembly during early oogenesis.

Zearalenone (ZEA) is a mycotoxin produced by fusarium graminearum. It can cause abnormal reproductive function by acting as an environmental estrogen. Research has traditionally focused on acute and chronic injury on mammalian reproductive capacity after ZEA treatment. Little research has been done studying the effects of ZEA exposure on early oogenesis. In this study, we investigate the effects of ZEA exposure on meiotic entry, DNA double-strand breaks (DSBs), and primordial follicle assembly during murine early oogenesis. The results show that ZEA exposure significantly decreased the percentage of diplotene stage germ cells, and made more germ cells remain at zygotene or pachytene stages. Moreover, the mRNA expression level of meiosis-related genes was significantly reduced after ZEA treatment. ZEA exposure significantly increased DNA-DSBs at the diplotene stage. Meanwhile, DNA damage repair genes such as RAD51 and BRCA1 were activated. Furthermore, maternal exposure to ZEA significantly decreased the number of primordial follicles in newborn mouse ovaries. In conclusion, ZEA exposure impairs mouse female germ cell meiotic progression, DNA-DSBs, and primordial follicle assembly.

Arrest at the diplotene stage of meiotic prophase I is delayed by progesterone but is not required for primordial follicle formation in mice.

BACKGROUND: In mammalian females, reproductive capacity is determined by the size of the primordial follicle pool. During embryogenesis, oogonia divide mitotically but cytokinesis is incomplete so oogonia remain connected in germ cell cysts. Oogonia begin to enter meiosis at 13.5 days postcoitum in the mouse and over several days, oocytes progress through the stages of meiotic prophase I arresting in the diplotene stage. Concurrently, germ cell cysts break apart and individual oocytes become surrounded by granulosa cells forming primordial follicles. In rats, inhibition of a synaptonemal complex protein caused premature arrival at the diplotene stage and premature primordial follicle assembly suggesting diplotene arrest might trigger primordial follicle formation. Cyst breakdown and primordial follicle formation are blocked by exposure to steroid hormones but hormone effects on the timing of diplotene arrest are unclear. Here, we asked: (1) if oocytes were required to arrest in diplotene before follicles formed, (2) if all oocytes within a germ cell cyst arrested at diplotene synchronously, and (3) if steroid hormones affected progression through prophase I. METHODS: Meiotic stage and follicle formation were assessed in histological sections. Statistical differences over time were determined using one-way ANOVA followed by Newman-Keuls multiple comparisons test. To determine if steroid hormones affect the rate of progression to the diplotene stage, 17.5 dpc ovaries were placed in organ culture with media containing estradiol, progesterone or both hormones. In this case, differences were determined using one-way ANOVA followed by Dunnett's multiple comparisons test. RESULTS: We found primordial follicles containing oocytes at the diplotene stage as well as follicles containing oocytes at pre-diplotene stages. We also found individual germ cell cysts containing oocytes at both diplotene and pre-diplotene stages. Progesterone but not estradiol reduced the number of diplotene oocytes in ovary organ culture. CONCLUSIONS: Our results suggest that meiotic progression and primordial follicle formation are independent events. In addition, oocytes in germ cell cysts do not synchronously proceed through meiosis. Finally, only progesterone delayed transit though meiotic prophase I.

Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis.

Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4-PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels.

Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway.

Moderate increase of reactive oxygen species triggers meiotic resumption in rat follicular oocytes.

AIM: The mammalian ovary generates reactive oxygen species (ROS) on an extraordinary scale; however, the role of ROS during meiotic cell cycle progression in follicular oocytes remains poorly understood. The present study was aimed to determine whether a moderate increase of ROS level in the ovary is beneficial for meiotic resumption from diplotene arrest in follicular oocytes. METHODS: Cumulus oocyte complexes were collected from the ovaries of female rats that had been treated with either: (i) pregnant mare's serum gonadotrophin; or (ii) pregnant mare's serum gonadotrophin + human chorionic gonadotrophin. We analyzed morphological changes, ROS and hydrogen peroxide levels, catalase activity, 3',5'-cyclic adenosine monophosphate and 3',5'-cyclic guanosine monophosphate levels, Thr14/Tyr15, Th-161, total cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels. RESULTS: Human chorionic gonadotrophin treatment induced meiotic resumption from diplotene arrest and extrusion of first polar body in cumulus oocyte complexes collected from ovaries and cultured for 3 h in vitro. Meiotic resumption from diplotene arrest was associated with increased ROS and hydrogen peroxide levels but decreased 3',5'-cyclic adenosine monophosphate as well as 3',5'-cyclic guanosine monophosphate levels. The reduced cyclic nucleotide levels were associated with decreased Thr161 phosphorylated Cdk1 and cyclin B1 level but increased Thr14/Tyr15 phosphorylated Cdk1 level leading to maturation promoting factor destabilization. Destabilized maturation-promoting factor triggered meiotic resumption from diplotene arrest and progression to metaphase-I as well as metaphase-II stage in follicular oocytes. CONCLUSION: Our findings suggest that a moderate increase of ROS in the ovary is beneficial for meiotic resumption from diplotene arrest and extrusion of first polar body in follicular oocytes.

Silver nanoparticles cause complications in pregnant mice.

BACKGROUND: Silver nanoparticles (AgNPs) have attracted much interest and have been used for antibacterial, antifungal, anticancer, and antiangiogenic applications because of their unique properties. The increased usage of AgNPs leads to a potential hazard to human health. However, the potential effects of AgNPs on animal models are not clear. This study was designed to investigate the potential impact of AgNPs on pregnant mice. METHODS: The synthesis of AgNPs was performed using culture extracts of Bacillus cereus. The synthesized AgNPs were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy. AgNPs were administrated into pregnant mice via intravenous infusion at 1.0 mg/kg doses at 6.5 days postcoitum (dpc). At 13.5, 15.5, and 17.5 dpc, the pregnant mice were euthanized, and the embryo and placenta were isolated. The meiotic status of oocytes was evaluated. DNA methylation studies were performed, and aberrant imprinting disrupted fetal, placental, and postnatal development. Quantitative real-time polymerase chain reaction analysis and Western blot were used to analyze various gene expressions. RESULTS: The synthesized AgNPs were uniformly distributed and were spherical in shape with an average size of 8 nm. AgNPs exposure increased the meiotic progression of female germ cells in the fetal mouse ovaries, and maternal AgNP exposure significantly disrupted imprinted gene expression in 15.5 dpc embryos and placentas, such as Ascl2, Snrpn, Kcnq1ot1, Peg3, Zac1, H19, Igf2r, and Igf2; DNA methylation studies revealed that AgNPs exposure significantly altered the methylation levels of differentially methylated regions of Zac1. CONCLUSION: The results from this study indicated that early exposure to AgNPs has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta. These results can contribute to research involved in the safe use of various biomedical applications of AgNPs and improves the understanding of the development of AgNPs in biomedical applications.

Zygotic chromosomal structural aberrations after paternal drug treatment.

In recent years, the field of male-mediated reproductive toxicology has received growing attention. It is now well-established that many drugs, chemicals, and environmental factors can harm male germ cells by inducing DNA damage. Male germ cells have extensive repair mechanisms that allow detection and repair of damaged DNA during the early phases of spermatogenesis. However, during the later phase of spermiogenesis, when the haploid spermatids undergo chromatin condensation and become transcriptionally quiescent, their ability to repair damaged DNA is lost. [1] ,[2] It is also thought that the highly compacted chromatin of the sperm can protect DNA against damage. [3] Therefore, it is expected that late spermatids will be most susceptible to DNA damaging agents. Unrepaired or misrepaired damage in the germ cells leads to the generation of spermatozoa with DNA damage that can be transmitted to the next generation. Fortunately, the maternal DNA repair machinery is capable of recognizing and repairing, at least to some degree, damaged paternal DNA after fertilization in the zygote. Therefore, the efficiency of the maternal repair machinery will greatly influence the risk of transmitting paternal DNA damage to offspring. [4].

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

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

Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway.

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.

The effect of taxol microinjection on the microtubular structure in polar body formation of starfish oocytes.

In starfish oocytes, microtubules (MTs) form a spindle, which plays an important role in contributing to the selective loss of chromosomes and centrosomes to the polar bodies (PBs) during meiosis. When Taxol was locally injected near the germinal vesicle (GV) or the mitotic apparatus during meiosis I, PB formation was inhibited as mentioned below. In the oocytes, which were injected with Taxol after spindle formation, the spindle became large, and then the volume of the first PB also increased more than that of the control. In contrast, in the oocytes injected with Taxol before the spindle formation, chromosome capture and alignment were inhibited. These oocytes did not form PB, but only a bulge at the cell cortex was occasionally observed. Moreover, in the oocytes injected with Taxol before GV breakdown, the chromosomes did not gather in one place, and then two asters were observed at distant positions from the cell cortex. These results suggested that MTs lost not only the ability to obtain the bipolar attachment of chromosomes by Taxol injection but also the aster closer to the cell cortex lost its interaction with the cell cortex of the animal pole.

Fetal exposure to bisphenol A affects the primordial follicle formation by inhibiting the meiotic progression of oocytes.

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Frequent human exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on germ cell cyst breakdown and primordial follicle formation. Pregnant mice were treated with BPA at doses of 0, 0.02, 0.04, 0.08 mg/kg body weight/day from 12.5 day postcoitum. BPA was delivered orally to pregnant female mice. A dose-response relationship was observed with increased BPA exposure level associated with more oocytes in germ cell cyst and less primordial follicle at postnatal day 3 (P < 0.01). Progression to meiosis prophase I of oocytes was delayed in the 0.08 mg/kg bw/day treated group (P < 0.01). Decreased mRNA expression of specific meiotic genes including Stra8, Dmc1, Rec8 and Scp3 were observed. In conclusion, BPA exposure can affect the formation of primordial follicle by inhibiting meiotic progression of oocytes.