Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation.

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 µM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

Early rescue oocyte activation for activation-impaired oocytes with no second polar body extrusion after intracytoplasmic sperm injection.

PURPOSE: When rescue artificial oocyte activation (ROA) is performed on the day after intracytoplasmic sperm injection (ICSI) or later, embryonic development is poor and seldom results in live births. The efficacy of an early ROA after ICSI is unclear. Is early ROA effective in rescuing unfertilized oocytes that have not undergone second polar body extrusion several hours after ICSI? METHODS: We performed retrospective cohort study between October 2016 and September 2019, targeting 2891 oocytes in 843 cycles when ICSI was performed. We performed ROA with calcium ionophore on 395 of the 475 oocytes with no second polar extrusion 2.5-6 h after ICSI. RESULTS: The normal fertilization rate of ROA oocytes was significantly higher than non-ROA oocytes (65.8% vs 6.7%, P < 0.001). The blastocyst development rate in ROA oocytes was significantly lower than spontaneously activated oocytes (48.9% vs 67.2%, P < 0.001). The ROA oocyte implantation rate did not significantly differ from the spontaneously activated oocytes (36.0% vs 41.2%). We observed no differences in the implantation rates and blastocyst development rates over the 2.5-6 h from ICSI until ROA. CONCLUSION: Early ROA is effective, and the optimal timing appears to be 2.5-6 h after ICSI.

Production of mouse androgenetic embryos using spindle perturbation.

To study the functional differences between maternal and paternal genomes in mammalian development, embryos with only one parental genome are often used. Androgenetic embryos are produced by the removal of maternal chromosomes before or after fertilization by techniques that require specialized skills and are associated with high risk of cellular damage. Here, we developed a novel method for producing androgenetic mouse embryos without the invasive enucleation process. We found that during in vitro fertilization in the presence of low-dose nocodazole, a microtubule destabilizing drug, whole oocyte chromosomes were extruded into the second polar body resulting in the production of androgenetic embryos. We further demonstrated that low-dose nocodazole decreased the spindle size and prevented chromosome segregation but did not compromise oocyte meiotic resumption. This led to the formation of a protrusion around the chromosomes, accumulation of protein regulator of cytokinesis 1 (PRC1) to the microtubules around the chromosomes, and assembly of a contractile ring at the neck region of the protrusion. Our method uses the intrinsic cytokinetic mechanism to exclude maternal chromatin from zygotes and may be applicable to other mammals.

Effects of high calcium levels on the disturbed extrusion of the second polar body during in vitro fertilization in C3H/He mouse substrains.

We previously reported that high concentrations (≥3.42 mM) of calcium during in vitro fertilization (IVF) disturbed the extrusion of the second polar body (PBII) in C3H/He inbred mice. In this study, the substrain specificity of this phenomenon was examined under 1.71-6.84 mM calcium concentration in ova from six C3H/He mouse commercially available substrains in Japan. PBII extrusion in ova from J substrains was not affected by calcium concentrations (<10% at any calcium level), but was grossly disturbed at high calcium levels in the ova of other substrains. This result has practical applications for the efficient production of normal zygotes by IVF, therefore contributing to the reduction in the numbers of donor animals for further zygote or embryo manipulation. Care must be taken in choosing IVF medium for particular strains and substrains.

Vitamin C protects against defects induced by juglone during porcine oocyte maturation.

Juglone, a naphthoquinone isolated from many species of the Juglandaceae family, has been used in traditional Chinese medicine for centuries because of its antiviral, antibacterial, and antitumor activities. However, the toxicity of juglone has also been demonstrated. Here, we used porcine oocytes as a model to explore the effects of juglone on oocyte maturation and studied the impact of vitamin C (VC) administration on juglone exposure-induced meiosis defects. Exposure to juglone significantly restricted cumulus cell expansion and decreased the first polar body extrusion. In addition, juglone exposure disturbed spindle organization, actin assembly, and the distribution of mitochondria during oocyte meiosis, while the acetylation level of α-tubulin was also reduced. These defects were all ameliorated by VC administration. Our findings indicate that juglone exposure induced meiotic failure in porcine oocytes, while VC protected against these defects during porcine oocyte maturation by ameliorating the organization of the cytoskeleton and mitochondrial distribution.

Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro.

Previous studies suggest that hydrogen sulfide (H2S) and ammonia (NH3) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na2S and/or NH4Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na2S treatment but not after NH4Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3 B expression level was higher in oocytes treated with Na2S and/or NH4Cl, which might be caused by ROS elevation. Additionally, exposure to Na2S and/or NH4Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H2S and/or NH3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy.

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

Comparison of the effects of BPA and BPAF on oocyte spindle assembly and polar body release in mice.

Bisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 µg/ml) for 18 h. At concentrations of 50 and 100 µg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 µg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 µg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 µg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.

Roscovitine inhibits extrusion of second polar body and induces apoptosis in rat eggs cultured in vitro.

BACKGROUND: Inhibition of cyclin-dependent kinases (Cdks) may result in meiotic cell cycle arrest and apoptosis in rat eggs in vitro. We aimed to find out whether roscovitine, a Cdk inhibitor, inhibits extrusion of second polar body (II PB) and induced egg apoptosis in vitro. METHODS: The metaphase-II (M-II) arrested eggs were collected from oviduct and exposed to various concentrations of roscovitine for 3h in vitro. The morphological changes, phosphorylation status of Cdk1, cyclin B1 level, hydrogen peroxide (H2O2), p53, Bax, Bcl2 and cytochrome c expressions, caspase-3 activity and DNA fragmentation were analyzed. RESULTS: We showed that the lower concentrations of roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level and inhibited extrusion of II PB. The higher concentrations of roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level but total Cdk as well as cyclin B1 levels remained high. Higher concentrations of roscovitine increased H2O2 level and expressions of p53, Bax and cytochrome c in treated eggs. The increased proapoptotic factors induced capsase-3 activity and thereby DNA fragmentation that finally resulted in cytoplasmic fragmentation, a morphological apoptotic feature. CONCLUSION: Our data suggest that roscovitine inhibited II PB extrusion possibly by reducing Thr-161 phosphorylated Cdk1 level and induced apoptosis through mitochondria-caspase-mediated apoptotic pathway in rat eggs cultured in vitro.

Sirtuin Inhibition Adversely Affects Porcine Oocyte Meiosis.

Sirtuins have been implicated in diverse biological processes, including oxidative stress, energy metabolism, cell migration, and aging. Here, we employed Sirtuin inhibitors, nicotinamide (NAM) and Sirtinol, to investigate their effects on porcine oocyte maturation respectively. The rate of polar body extrusion in porcine oocytes decreased after treatment with NAM and Sirtinol, accompanied with the failure of cumulus cell expansion. We further found that NAM and Sirtinol significantly disrupted oocyte polarity, and inhibited the formation of actin cap and cortical granule-free domain (CGFD). Moreover, the abnormal spindles and misaligned chromosomes were readily detected during porcine oocyte maturation after treatment with NAM and Sirtinol. Together, these results suggest that Sirtuins are involved in cortical polarity and spindle organization in porcine oocytes.

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

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.

TGN38 is required for the metaphase I/anaphase I transition and asymmetric cell division during mouse oocyte meiotic maturation.

The cellular functions of the trans-Golgi network protein TGN38 remain unknown. In this research, we studied the expression, localization and functions of TGN38 in the meiotic maturation of mouse oocytes. TGN38 was expressed at every stage of oocyte meiotic maturation and colocalized with γ-tubulin at metaphase I and metaphase II. The spindle microtubule disturbing agents nocodazole and taxol did not affect the colocalization of TGN38 and γ-tubulin. Depletion of TGN38 with specific siRNAs resulted in increased metaphase I arrest, accompanied with spindle assembly checkpoint activation and decreased first polar extrusion (PB1). In the oocytes that had extruded the PB1 after the depletion of TGN38, symmetric division occurred, leading to the production of 2 similarly sized cells. Moreover, the peripheral migration of metaphase I spindle and actin cap formation were impaired in TGN38-depleted oocytes. Our data suggest that TGN38 may regulate the metaphase I/anaphase I transition and asymmetric cell division in mouse oocytes.

The effects of cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression in mice.

Cigarette smoking can harm fertility, but the existing research has targeted primarily on ovarian follicles, embryos or sex hormone. In this study, we tested cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression associated with inhibition of oxidative stress using C57BL/6 mice. Mice in the experimental group were administered a cigarette smoke extract (CSE) solution (2 mg/ml) orally daily, while the blank control group was given dimethylsulfoxide (DMSO). A positive control group (menadione) was used that received an intraperitoneal injection of 15 mg/kg menadione in oil solution daily. We found that the CSE group manifested a reduced diameter of zona pellucida-free oocyte (ZP-free OD) and a morphologically misshapen first polar body (PB). Our results suggest that CSE exposure is associated with a shrink size and poor quality of oocytes. Quitting smoking is a wise choice to ensure good fertility.

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

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.

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

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.

MKlp2 inhibitor paprotrain affects polar body extrusion during mouse oocyte maturation.

BACKGROUND: Mammalian oocyte meiotic maturation involves a number of important processes, including spindle assembly and migration, cortical reorganization and polar body extrusion. Numerous proteins contribute to these processes, but it is unknown whether MKlp2 (mitotic kinesin-like protein 2; also called KIF20A), a microtubule-associated protein that regulates cytokinesis during mitosis, is involved in oocyte maturation. METHODS: Confocal microscopy, time lapse microscopy, inhibitor treatment were adopted to examine the roles of MKlp2 in mouse oocyte. RESULTS: Immunostaining results showed that MKlp2 localized to oocyte microtubules. Time-lapse microscopy showed that disrupting MKlp2 expression with paprotrain, a specific inhibitor of MKlp2, resulted in polar body extrusion failure. This could be rescued after rescuing oocytes from paprotrain in fresh medium. Cell cycle analysis showed that most oocytes were arrested at metaphase I or telophase I. However, oocyte spindle structure and chromosome alignment were not disrupted after the inhibition of MKlp2 by paprotrain. CONCLUSIONS: This study demonstrated that MKlp2 is crucial for oocyte maturation by regulating polar body extrusion.

Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei.

Oocytes with germinal vesicles (GVs) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments, whereas PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments and not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.

Effect of phosphodiesterase type 3 inhibitor on nuclear maturation and in vitro development of ovine oocytes.

The present study aims to investigate if prematuration culture (PMC) of ovine oocytes in the presence of a phosphodiesterase type 3 (PDE3) inhibitor cilostamide can improve the shortcomings of conventional in vitro maturation (IVM) system. Therefore, a two-step culture system consisting of 22 hours culture in the presence of 1, 10, and 20 µM cilostamide (PMC medium), followed by 22 hours culture in maturation medium, was designed. The effect of cilostamide on gap junction communications and nuclear status was studied. The variables assessed were chromosome organization, spindle pattern, polar body extrusion, and embryonic development. According to the results, inhibition of PDE3 could not permanently block nuclear maturation in ovine oocytes but it delayed the process of nuclear maturation. Elevation of intra-oocyte cAMP concentration could inhibit cumulus cells expansion and maintain gap junction communications between oocyte and cumulus cells. Deletion of cilostamide and refreshing maturation medium after 22 hours culture revealed that cumulus cells were completely expanded. The inhibitory effect induced by 1 µM cilostamide was reversible, and it increased the number of mature oocytes with aligned chromosomes and normal spindle. However, the inhibitory effects of 10 and 20 µM cilostamide was not fully reversible and was associated with deleterious effects on chromosome organization and spindle pattern. Investigation of embryonic development via parthenogenetic activation and in vitro fertilization revealed that the blastocyst rate of oocytes that were prematured with 1 µM cilostamide was not significantly different from oocytes that underwent conventional IVM but it was significantly reduced in oocytes that were prematured with 10 and 20 µM cilostamide. Our results provide the evidence that reduced cAMP via PDE3 is not the only mechanism that controls the progress of nuclear maturation in sheep oocytes, and that alternative or additional mechanisms may also exist.

The effects of DNA double-strand breaks on mouse oocyte meiotic maturation.

Both endogenous and exogenous factors can induce DNA double-strand breaks (DSBs) in oocytes, which is a potential risk for human-assisted reproductive technology as well as animal nuclear transfer. Here we used bleomycin (BLM) and laser micro-beam dissection (LMD) to induce DNA DSBs in germinal vesicle (GV) stage oocytes and compared the germinal vesicle breakdown (GVBD) rates and first polar body extrusion (PBE) rates between DNA DSB oocytes and untreated oocytes. Employing live cell imaging and immunofluorescence labeling, we observed the dynamics of DNA fragments during oocyte maturation. We also determined the cyclin B1 expression pattern in oocytes to analyze spindle assembly checkpoint (SAC) activity in DNA DSB oocytes. We used parthenogenetic activation to determine if the DNA DSB oocytes could be activated. As a result, we found that the BLM- or LMD-induced DSB oocytes showed lower GVBD rates and took a longer time to undergo GVBD compared with untreated oocytes. PBE was also delayed in DSB oocytes, but once GVBD had occurred, PBE was not affected, even in oocytes with severe DSBs. Compared with control oocytes, the DSB oocytes showed higher SAC activity, as indicated by less Ccnb1-GFP degradation during metaphase I to anaphase I transition. Parthenogenetic activation could activate the metaphase to interphase transition in the DNA DSB mature oocytes, but many oocytes contained multiple pronuclei or numerous micronuclei. These data suggest that DNA damage inhibits or delays the G2/M transition, but once GVBD occurs, DNA-damaged oocytes can complete chromosome separation and polar body extrusion even under a higher SAC activity, causing the formation of numerous micronuclei in early embryos.

Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence.

In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 µM of milrinone, the enucleation rate was significantly improved by 100 µM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 µM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 µM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.