Polar Body Emission Research Articles

Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes

Asymmetric meiotic divisions in mammalian oocytes rely on the eccentric positioning of the spindle and the remodeling of the overlying cortex, resulting in the formation of small polar bodies. The mechanism of this cortical polarization, exemplified by the formation of a thick F-actin cap, is poorly understood. Cdc42 is a major player in cell polarization in many systems; however, the spatio-temporal dynamics of Cdc42 activation during oocyte meiosis, and its contribution to mammalian oocyte polarization, have remained elusive. In this study, we investigated Cdc42 activation (Cdc42–GTP), dynamics and role during mouse oocyte meiotic divisions. We show that Cdc42–GTP accumulates in restricted cortical regions overlying meiotic chromosomes or chromatids, in a Ran–GTP-dependent manner. This polarized activation of Cdc42 is required for the recruitment of N-WASP and the formation of F-actin-rich protrusions during polar body formation. Cdc42 inhibition in MII oocytes resulted in the release of N-WASP into the cytosol, a loss of the polarized F-actin cap, and a failure to protrude the second polar body. Cdc42 inhibition also resulted in central spindle defects in activated MII oocytes. In contrast, emission of the first polar body during oocyte maturation could occur in the absence of a functional Cdc42/N-WASP pathway. Therefore, Cdc42 is a new protagonist in chromatin-induced cortical polarization in mammalian oocytes, with an essential role in meiosis II completion, through the recruitment and activation of N-WASP, downstream of the chromatin-centered Ran–GTP gradient.

The Mos-MAPK pathway regulates Diaphanous-related formin activity to drive cleavage furrow closure during polar body emission in starfish oocytes

Maintenance of spindle attachment to the cortex and formation of the cleavage furrow around the protruded spindle are essential for polar body extrusion (PBE) during meiotic maturation of oocytes. Although spindle movement to the cortex has been well-studied, how the spindle is maintained at the cortex during PBE is unknown. Here, we show that activation of Diaphanous-related formin mediated by mitogen-activated protein kinase (MAPK) is required for tight spindle attachment to the cortex and cleavage furrow closure during PBE in starfish (Asterina pectinifera) oocytes. A. pectinifera Diaphanous-related formin (ApDia) had a distinct localization in immature oocytes and was localized to the cleavage furrow during PBE. Inhibition of the Mos-MAPK pathway or the actin nucleating activity of formin homology 2 domain prevented cleavage furrow closure and resulted in PBE failure. In MEK/MAPK-inhibited oocytes, activation of ApDia by relief of its intramolecular inhibition restored PBE. In summary, this study elucidates a link between the Mos-MAPK pathway and Diaphanous-related formins, that is responsible for maintaining tight spindle attachment to the cortex and cleavage furrow closure during PBE.

Chimeric embryos—potential mechanism of avian offspring sex manipulation

Environmental and social effects on offspring sex ratio bias in birds are among the most studied topics in evolutionary biology. Although it is established that offspring sex is determined at the stage of the first meiotic division, there is no direct evidence for the common belief that the bias happens via nonrandom sex chromosome segregation. In this paper, we suggest an alternative mechanism based on the failure of polar body emission, followed by fertilization of multiple haploid nuclei through polyspermy and subsequent competition of Z and W bearing cells within chimeric embryos. Although the occurrence of failure of polar body extrusion is obvious from the observations of chimeric birds, the idea that it could be the first step of offspring sex ratio bias has been entirely overlooked. We review the evidence in support of that idea, demonstrate that it is consistent with the observations of biased offspring sex ratio reported so far, and suggest a way to verify it.

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

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.

Rho GTPases in animal cell cytokinesis: An occupation by the one percent

Rho GTPases are molecular switches that elicit distinct effects on the actomyosin cytoskeleton to accurately promote cytokinesis. Although they represent less than 1% of the human genome, Rho GTPases exert disproportionate control over cell division. Crucial to this master regulatory role is their localized occupation of specific domains of the cell to ensure the assembly of a contractile ring at the proper time and place. RhoA occupies the division plane and is the central positive Rho family regulator of cytokinesis. Rac1 is a negative regulator of cytokinesis and is inactivated within the division plane while active Rac1 occupies the cell poles. Cdc42 regulation during cytokinesis is less studied, but thus far a clear role has only been shown during polar body emission. Here we review what is known about the function of Rho family GTPases during cell division, as well as their upstream regulators and known downstream cytokinetic effectors.

Strain Specific Spontaneous Activation During Mouse Oocyte Maturation.

Objective To examine whether spontaneous oocyte activation is determined by genetic differences and interacts with culture environment. Design Experimental study. Setting Temple University School of Medicine. Animal(s) C57BL/6, DBA/2, C3H/HeJ, and A/J strains, along with reciprocal F1 hybrid female mice (5–6 weeks). Intervention(s) Immature oocytes from different mouse strains collected and cultured in different maturation conditions, including different serum, serum replacement, bovine serum albumin (BSA), and follicle-stimulation hormone (FSH). Main Outcome Measure(s) The emission of first polar body, pronucleus formation, meiotic arrest, spontaneous activation, and expression of maturation regulators. Result(s) Oocytes from C57BL/6 mice display a high rate of delayed first meiotic division and spontaneous activation after the first meiotic division with in vitro maturation (IVM), and the second meiotic division with in vivo maturation (VVM) after superovulation. Spontaneous activation with IVM is sensitive to culture environment. Oocytes that spontaneously activated during the first meiotic division with IVM have unusual replicated sister chromatid pairs with slight connections at centromeres at first mitosis, whereas oocytes that activated in vivo display haploidization from the second meiotic division. Spontaneous activation is also seen in F1 hybrid oocytes, indicating a dominant trait from C57BL/6. Delayed meiosis was associated with reduced cyclin B and securin expression. Conclusion(s) Both mouse strain and culture environment have a statistically significant effect on the incidence of meiotic defects and spontaneous activation. Reduced expression of meiotic regulators may underlie this effect.

Strain-specific spontaneous activation during mouse oocyte maturation

To examine whether spontaneous oocyte activation is determined by genetic differences and interacts with culture environment. Experimental study. Temple University School of Medicine. C57BL/6, DBA/2, C3H/HeJ, and A/J strains, along with reciprocal F1 hybrid female mice (5-6 weeks). Immature oocytes from different mouse strains collected and cultured in different maturation conditions, including different serum, serum replacement, bovine serum albumin (BSA), and follicle-stimulation hormone (FSH). The emission of first polar body, pronucleus formation, meiotic arrest, spontaneous activation, and expression of maturation regulators. Oocytes from C57BL/6 mice display a high rate of delayed first meiotic division and spontaneous activation after the first meiotic division with in vitro maturation (IVM), and the second meiotic division with in vivo maturation (VVM) after superovulation. Spontaneous activation with IVM is sensitive to culture environment. Oocytes that spontaneously activated during the first meiotic division with IVM have unusual replicated sister chromatid pairs with slight connections at centromeres at first mitosis, whereas oocytes that activated in vivo display haploidization from the second meiotic division. Spontaneous activation is also seen in F1 hybrid oocytes, indicating a dominant trait from C57BL/6. Delayed meiosis was associated with reduced cyclin B and securin expression. Both mouse strain and culture environment have a statistically significant effect on the incidence of meiotic defects and spontaneous activation. Reduced expression of meiotic regulators may underlie this effect.

Translation of incenp During Oocyte Maturation Is Required for Embryonic Development in Xenopus laevis1

The chromosome passenger complex (CPC) consists of Aurora-B kinase and several other subunits. One of these, incenp, binds Aurora-B and regulates its kinase activity. During Xenopus oocyte maturation, incenp accumulates through translation, contributing to aurora-b activation. A previous study has demonstrated that inhibition of incenp translation during oocyte maturation diminishes aurora-b activation but does not interfere with oocyte maturation, characterized by normal maturation-specific cyclin-b phosphorylation, degradation, and resynthesis. Here we have extended these findings, showing that inhibition of incenp translation during oocyte maturation did not interfere with meiosis I or II, as indicated by the normal emission of the first polar body and metaphase II arrest, followed by the successful emission of the second polar body upon parthenogenetic egg activation. Most importantly, however, when transferred to host frogs and subsequently ovulated, the incenp-deficient eggs were fertilized but failed to undergo mitotic cleavage. Thus, translation of incenp during oocyte maturation appears to be part of oocyte cytoplasmic maturation, preparing the egg for the rapid mitosis following fertilization.

Calcium influx-mediated signaling is required for complete mouse egg activation

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

The role of Akt signalling in the mammalian ovary

The serine/threonine protein kinase Akt is involved in many cellular processes including cell growth, survival, proliferation and metabolism. Akt activity is modulated downstream of phosphatidylinositol-3-kinase (PI3K) in response to different extracellular stimuli. In the mammalian ovary, Akt collaborates with other kinases in the regulation of coordinate follicle and oocyte development. Akt determines the pool of primordial follicles and the transition from quiescent to growing phase. In addition, the kinase modulates granulosa cell apoptosis throughout folliculogenesis. In oocytes Akt participates in the control of meiosis resumption and, at metaphase II stage, regulates polar body emission and spindle organization. Its inhibition negatively affects preimplantation embryo development. As a consequence of such a central role, Akt dysregulation is associated with several human diseases including infertility and ovarian cancer.

Temporary Inhibition of Germinal Vesicle Breakdown by Rho Kinase Inhibitor Y-27632 is Detrimental to Oocyte Maturation

Abstract: In the present study, we investigated the effects of a specific Rho kinase (ROCK) inhibitor, Y-27632, on germinal vesicle breakdown (GVBD), changes of cytoskeletal organization, and the resumption of porcine oocyte maturation after its removal. In Experiment 1, cumulus-oocyte complexes (COCs) cultured in medium containing Y-27632 for 24 h showed GVBD of 98, 88, 84, and 4% (P<0.01) of the oocytes treated with 0, 1, 10, and 100 µM of the drug, respectively. In addition, Y-27632 treatment from 24 to 44 h of incubation significantly inhibited maturation of the oocytes. The metaphase II (MII) rates were 84, 78, 65, and 10% (P<0.01) for the corresponding groups, and 0, 2, 8, and 45% of the oocytes failed to show emission of the first polar body (PB1). In Experiment 2, resumption of meiotic maturation was examined after removal of Y-27632. COCs incubated with Y-27632 for 18 h and then cultured in Y-27632-free medium for an additional 44 h showed a significantly lower MII rate (86 vs. 33%, P<0.01). Afte...

The spatial and mechanical challenges of female meiosis.

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.

WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.

The small GTPase Cdc42 promotes membrane protrusion during polar body emission via ARP2-nucleated actin polymerization

Polar body emission is a specialized cell division throughout the animal kingdom, serving to reduce chromosome ploidy while preserving the egg cytoplasm. Critical to polar body emission are the asymmetric positioning of the meiotic spindle prior to anaphase, with one pole attached to the oocyte cortex, and the simultaneous membrane protrusion during subsequent cytokinesis. We have shown that, during Xenopus oocyte maturation, the small GTPase Cdc42 promotes membrane protrusion while a classical RhoA contractile ring forms and constricts at the base of the protrusion. We report here that treating oocytes with low concentrations of nocodazole diminished the size of metaphase I spindles and prevented polar body emission, and yet an active Cdc42 cap of correspondingly diminished size still developed, on time, atop of the spindle pole. Conversely, treating oocytes with low concentrations of taxol resulted in a spindle with multiple poles attached to the cortex, but still each of these poles were associated with activated cortical Cdc42 at the appropriate time. Therefore, the asymmetric positioning of the meiotic spindle with one pole anchored to the cortex is a prerequisite for Cdc42 activation. Furthermore, we demonstrated that the Cdc42-regulated F-actin nucleator ARP2/3 complex was similarly localized at the cortex of the protruding polar body membrane, suggesting that Cdc42 promotes membrane protrusion through an F-actin meshwork mechanism. Finally, we demonstrated that Cdc42 and RhoA formed similarly complementary activity zones during egg activation and that inhibition of Cdc42 prevented second polar body emission. Therefore, Cdc42 activation likely promotes membrane protrusion during polar body emission in widespread systems.

The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development

Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24h after IVM, respectively) nor the accelerated polar body emission (at 18h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Short-term Preservation of Porcine Oocytes in Ambient Temperature: Novel Approaches

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

Survivin is a critical regulator of spindle organization and chromosome segregation during rat oocyte meiotic maturation

Survivin is a novel member of the inhibitor of apoptosis gene family that bear baculoviral IAP repeats (BIRs), whose physiological roles in regulating meiotic cell cycle need to be determined. Confocal microscopy was employed to observe the localization of survivin in rat oocytes. At the germinal vesicle (GV) stage, survivin was mainly concentrated in the GV. At the prometaphase I (pro-MI) and metaphase I (MI) stage, survivin was mainly localized at the kinetochores, with a light staining detected on the chromosomes. After transition to anaphase I or telophase I stage, survivin migrated to the midbody, and signals on the kinetochores and chromosomes disappeared. At metaphase II (MII) stage, survivin became mainly localized at the kinetochores again. Microinjection of oocytes with anti-survivin antibodies at the beginning of the meiosis, thus blocking the normal function of survivin, resulted in abnormal spindle assembly, chromosome segregation and first polar body emission. These results suggest that survivin is involved in regulating the meiotic cell cycle in rat oocytes.

Localization and function of mSpindly during mouse oocyte meiotic maturation

Spindly was first identified in Drosophila; its homologues are termed SPDL-1 in Caenorhabditis elegans and Hs Spindly/hSpindly in humans. In all species, Spindly and its homologues function by recruiting dynein to kinetochores and silencing SAC in mitosis of somatic cells. Depletion of Spindly causes an extensive metaphase arrest during somatic mitoses in Drosophila, C. elegans and humans. In Drosophila, Spindly is required for shedding of Rod and Mad2 from the kinetochores in metaphase; in C. elegans, SPDL-1 presides over the recruitment of dynein and MDF-1 to the kinetochores; in humans, Hs Spindly is required for recruiting both dynein and dynactin to kinetochores but it is dispensable for removal of checkpoint proteins from kinetochores. The present study was designed to investigate the localization and function of the Spindly homologue (mSpindly) during mouse oocyte meiotic maturation by immunofluorescent analysis, and by overexpression and knockdown of mSpindly. We found that mSpindly was typically localized to kinetochores when chromatin condensed into chromosomes after GVBD. In metaphase of both first meiosis and second meiosis, mSpindly was localized not only to kinetochores but also to the spindle poles. Overexpression of mSpindly did not affect meiotic progression, but its depletion resulted in an arrest of the pro-MI/MI stage, failure of anaphase entry and subsequent polar body emission, and in abnormal spindle morphology and misaligned chromosomes. Our data suggest that mSpindly participates in SAC silencing and in spindle formation as a recruiter and/or a transporter of kinetochore proteins in mouse oocytes, but that it needs to cooperate with other factors to fulfill its function.

Inhibition of the Ubiquitin-proteasome System Leads to Delay of the Onset of ZGA Gene Expression

In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.