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Podophyllotoxin (PPT) is a lignan extracted from podophyllum genera and it shows potent antitumor activity since it could effectively inhibit the assembly of microtubule in tumor cells. However, the effects of podophyllotoxin exposure on porcine oocyte quality is still unclear. In present study we tried to examine whether podophyllotoxin exposure was toxic to porcine oocyte maturation. Our results showed that podophyllotoxin exposure inhibited porcine oocyte maturation, showing with the failure of polar body extrusion, and the inhibitory effects of podophyllotoxin on porcine oocytes was dose-depended. Moreover, the meiotic spindle formation was disturbed and the chromosomes were misaligned in the podophyllotoxin-treated porcine oocytes. However, there was no different expression for p-MAPK and ace-tubulin between the control and podophyllotoxin treatment group. In addition, after 0.01 μM podophyllotoxin treatment, the intracellular reactive oxygen species (ROS) levels and the Annexin-V signal at MI stage significantly increased compared to the control group, indicating the occurrence of oxidative stress and early apoptosis. Taken together, our results suggested that the toxic effects of podophyllotoxin exposure on porcine oocyte maturation might be through its effects on spindle formation and the induction of oxidative stress-mediated early apoptosis.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l -carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

Ubiquinol-10, the reduced form of coenzyme Q10, protects mammalian cells from oxidative damage and enhances mitochondrial activity. However, the protective effect of ubiquinol-10 on mammalian oocytes is not well understood. In this study, we investigated the effect of ubiquinol-10 on porcine oocytes during postovulatory aging. Metaphase II oocytes were selected as fresh oocytes and further cultured for 48 h with different concentrations of ubiquinol-10 (0–400 μM) in vitro as a postovulatory aging model. After choosing the optimal concentration of ubiquinol-10 (100 μM) that maintained oocyte morphology and developmental competence during the progression of aging, the oocytes were randomly divided into five groups: fresh, control-24 h, ubiquinol-24 h, control-48 h, and ubiquinol-48 h. The results revealed that ubiquinol-10 significantly prevented aging-induced oxidative stress, GSH reduction, cytoskeleton impairment, apoptosis, and autophagy. Mitochondrial biogenesis (SIRT1 and PGC-1α) and mitophagy (PINK1 and PARKIN)-related proteins were decreased during aging. Addition of ubiquinol-10 prevented the aging-induced reduction of these proteins. Consequently, although mitochondrial content was decreased, the number of active mitochondria and ATP level were significantly increased upon treatment with ubiquinol-10. Thus, ubiquinol-10 has beneficial effects on porcine postovulatory aging oocytes owing to its antioxidant properties and ability to promote mitochondrial renewal.

Citrinin (CTN) is a polyketide mycotoxin produced by several strains of Penicillium, Monascus, and Aspergillus. While CTN poses various toxic effects on the female reproductive system in animals, its direct effects on germ cell development are unclear. This study aimed to evaluate the effects of increasing concentrations of CTN (0,20,40,80,100 μM) on porcine oocyte in vitro maturation. Our results indicate that CTN supplementation inhibited polar body extrusion in a dose-dependent manner. Actin and spindle assembly were also disrupted after treatment, indicating that CTN affects the cytoskeleton of porcine oocytes. Oxidative stress and apoptosis were observed under CTN treatment to explore the cause of meiotic maturation failure in porcine oocytes. The results showed that reactive oxygen species levels, cathepsin B activity, and caspase-3 activity were increased in the treated group, indicating that CTN induced oxidative stress and apoptosis. In conclusion, CTN exposure could reduce porcine oocyte maturation by affecting cytoskeletal dynamics, oxidative stress, and apoptosis.

L-carnitine (LC) is well known for its antioxidant activity. In this study, we explored the potential mechanistic effects of LC supplementation on aged bovine oocytes in vitro. We showed that in-vitro maturation could enhance the subsequent developmental capacity of aging oocytes, when supplemented with LC. After in vitro fertilization, the blastocyst formation rate in the aged oocytes post-LC treatment significantly increased compared to that in untreated aged oocytes (29.23 ± 2.20% vs. 20.90 ± 3.05%). Furthermore, after LC treatment, the level of intracellular reactive oxygen species in aged oocytes significantly decreased, and glutathione levels significantly increased, compared to those in untreated aged oocytes. Mitochondrial membrane potential, the percentage of early apoptotic oocytes, and caspase-3 activity were significantly reduced in LC-treated aged oocytes compared to those in untreated aged oocytes. Furthermore, during in vitro aging, the mRNA levels of the anti-apoptotic genes, Bcl-xl and survivin in LC-treated aged oocytes were significantly higher than those in untreated aged oocytes. Overall, these results indicate that at least in in vitro conditions, LC can prevent the aging of bovine oocytes and improve the developmental capacity of bovine embryo.

// Yu Wu 1 , Na Zhang 1 , Ying-Hua Li 2 , Liang Zhao 3 , Mo Yang 1 , Yimei Jin 1 , Yong-Nan Xu 2 and Hongyan Guo 1 1 Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, P.R. China 2 College of Agriculture, Yanbian University, Yanji 133002, P.R. China 3 Department of Obstetrics and Gynecology, Beijing Jishuitan Hospital, Beijing 100035, P.R. China Correspondence to: Hongyan Guo, email: bysyghy@163.com Yong-Nan Xu, email: xuyongnan@ybu.edu.cn Keywords: citrinin, oocyte, cytoskeleton, apoptosis, oxidative stress Received: November 30, 2016 Accepted: January 11, 2017 Published: February 28, 2017 ABSTRACT Citrinin is one of the mycotoxins and has been shown to have various toxic effects in animals and humans. Although previous study showed the toxic effects of citrinin on the female reproductive system, especially on oocyte maturation, however, the causes or mechanism of citrinin on oocyte quality is unclear. In present study we deeply investigated this topic. We found thatcitrinin toxin exposure inhibited mouse oocyte maturation and early embryo development. Further investigation showed that the actin distribution in oocytes and embryos was disrupted, and the reduced expression of actin nucleator ARP2 expression in the oocyte cortex further confirmed this. We also found that meiotic spindle morphology was abnormal after citrinin treatment. These results indicated that citrinin toxin exposure could disrupt cytoskeleton dynamics to affect oocyte maturation and early embryo development. We also examined the ROS level and early apoptosis marker Annexin signals, and the results showed that both levels increased, indicating that citrinin induced oxidative stress and further resulted in oocyte early apoptosis. Taken together, our results indicated that citrinin toxin exposure could reduce mouse oocyte maturation and early embryo development capability by affecting cytoskeletal dynamics, which may be due to the oxidative stress induced early apoptosis.

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti-inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging-related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 μM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE-treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency-related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE-treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.

Objective To investigate the molecular mechanism of mouse oocyte polarity loss during aging. Design Experimental study. Setting Academic basic research laboratory. Animal(s) Mice. Intervention(s) Oocytes were collected 16 hours after injection of hCG and cultured in M16 medium for an additional 14 hours with or without caffeine. Main Outcome Measure(s) Expression and localizations of actin nucleators actin-related protein 2/3 complex, JMY, and WAVE2 were examined by immunofluorescence staining, and their messenger RNA levels were examined by real-time reverse transcription–polymerase chain reaction. Result(s) The protein and messenger RNA levels of actin-related protein 2/3 complex, JMY, and WAVE2 were decreased in aged oocytes, but the levels were normal in caffeine-treated aged oocytes. Conclusion(s) Our data indicated that the loss of oocyte polarity may be due to the degradation of actin nucleators in aged oocytes.

Ndc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

To evaluate DNA synthesis and epigenetic modification in mouse oocytes during the first cell cycle following the injection of human or hamster sperm.Mouse oocytes following the injection of human and hamster sperm and cultured in M16 medium.Male and female pronucleus formation, DNA synthesis, histone protein modification, and heterochromatin formation were similar in mouse oocytes injected with human or hamster sperm. However, DNA methylation patterns were altered in mouse oocytes following human sperm injection. Immunocytochemical staining with a histone H3-MeK9 antibody revealed that human and hamster sperm chromatin associated normally with female mouse chromatin, then entered into the metaphase and formed normal, two-cell stage embryos.Although differences in epigenetic modification of DNA were observed, fertilization and cleavage occurred in a species non-specific manner in mouse oocytes.

Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation.

Junction-mediating and regulatory protein(JMY) is a multifunctional protein with roles in the transcriptional co-activation of p53 and the regulation of actin nucleation promoting factors and, hence, cell migration; however, its role in the maturation of porcine oocytes is unclear. In the current study, we investigated functional roles of JMY in porcine oocytes. Porcine oocytes expressed JMY mRNA and protein, and the mRNA expression level decreased during oocyte maturation. Knockdown of JMY by RNA interference decreased the rate of polar body extrusion, validating its role in the asymmetric division of porcine oocytes. JMY knockdown also down-regulated the mRNA and protein levels of actin and Arp2/3. Furthermore, JMY accumulated in the nucleus in response to DNA damage, and JMY knockdown suppressed DNA damage-mediated p53 activation. In conclusion, our results show that JMY has important roles in oocyte maturation as a regulator of actin nucleation-promoting factors and an activator of p53 during DNA damage during DNA damages in porcine oocytes.

Meiotic maturation in many species is initiated by the activation of maturation-promoting factor (MPF) with concomitant inactivation of counteracting phosphatases, most notably protein phosphatase 2A (PP2A). Recently, Greatwall (GWL) has been identified as a cell cycle regulator that inhibits PP2A activity. In this study, we demonstrate that GWL is required for meiotic maturation in porcine oocytes. GWL expression increases from germinal vesicle (GV) to metaphase II (MII) stages of porcine oocytes and dramatically decreases with progression of the meiotic cell cycle. GWL is initially localized in the nucleus of GV oocytes and is associated with spindle fibers following GV breakdown. Depletion of GWL inhibited or delayed meiotic maturation secondary to defects in chromosome congression and spindle formation. Conversely, overexpression of GWL overcame meiotic arrest and initiated progression to MII stage. However, these oocytes had severe spindle defects. Furthermore, MII oocytes depleted of GWL progressed to pronuclear formation. Taken together, our data demonstrate that GWL is required not only for meiotic maturation but also for maintenance of MII arrest in porcine oocytes.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein (EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5%±2.2% v.s. 22.9%±2.9%). Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer (NT). Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.

SummaryInterspecies intracytoplasmic sperm injection has been carried out to understand species-specific differences in oocyte environments and sperm components during fertilization. While sperm aster organization during cat fertilization requires a paternally derived centriole, mouse and hamster fertilization occur within the maternal centrosomal components. To address the questions of where sperm aster assembly occurs and whether complete fertilization is achieved in cat oocytes by interspecies sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse, or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at 6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent, and that fertilization processes and cleavage occur in a non-species specific manner in cat 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.

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment and the spindle assembly checkpoint in mitosis. The roles of Spc25 in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 by mRNA injection resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi by siRNA injection resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and proper spindle checkpoint signaling during meiosis.

The use of intracytoplasmic sperm injection (ICSI) in model animals is a powerful approach for the study of species-specific fertilization processes and multiploidy embryogenesis. In this study, we examined the fertilization process in mouse oocytes following injection of a single mouse or cat sperm, two mouse spermatozoa or mouse and cat spermatozoa. These treatments did not affect histone H3K9 acetylation or methylation, although the pattern of DNA methylation differed following the injection of cat sperm. Immunocytochemical staining revealed that sperm chromatin was normally incorporated with female mouse chromatin following any of the four injection scenarios. Furthermore, metaphase was successfully entered to reach a normal two-cell stage, and cell division could even persist to produce blastocyst stage embryos. In addition, both mouse and cat Pou5l and Nanog mRNA were expressed in the hybrid embryos. These results suggest that, although epigenetic modification of DNA is affected by the sperm injection treatment, fertilization and cleavage occur in a non-species-specific manner. In addition, despite abnormal division of the chromosomes, intra- and inter-species ICSI produced embryos that could develop into blastocysts. J. Exp. Zool. 315:349–357, 2011. © 2011 Wiley-Liss, Inc.