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Mammalian oocyte quality is critical for fertilization and early embryo development. The type B trichothecene nivalenol (NIV) is a mycotoxin produced by Fusarium oxysporum, and it is commonly found with deoxynivalenol in contaminated food or feed. NIV has been shown to affect the immune system and female reproductive system, cause emesis and growth retardation. Here, we investigated the toxicity of NIV on mouse oocyte quality, as well as the protective effects of melatonin on the NIV-exposed oocytes. We found NIV exposure caused meiotic arrest and further induced the failure of polar body extrusion in mouse oocytes. Transcriptome analysis data showed that NIV exposure altered the expression of multiple pathway-related genes in oocytes, indicating its wide toxicity on oocyte maturation. Based on the RNA-seq data, we showed that NIV exposure induced oxidative stress and caused DNA damage in oocytes. Besides, autophagy, and early apoptosis were also found in NIV-exposed oocytes. Treatment with melatonin significantly ameliorated these defects through its effects on ROS level. Thus, our results demonstrated that exposure to NIV affected oocyte quality and melatonin treatment could reduce the defects caused by NIV in mouse oocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sudan I is one of the industry dyes and widely used in cosmetics, wax agent, solvent and textile. Sudan I has multiple toxicity such as carcinogenicity, mutagenicity, genotoxicity and oxidative damage. However, Sudan I has been illegally used as colorant in food products, triggering worldwide attention about food safety. Nevertheless, the toxicity of Sudan I on reproduction, particularly on oocyte maturation is still unclear. In the present study, using mouse in vivo models, we report the toxicity effects of Sudan I on mouse oocyte. The results reflect that Sudan I exposure disrupts spindle organization and chromosomes alignment as well as cortical actin distribution, thus leading to the failure of polar body extrusion. Based on the transcriptome results, it is found that the exposure of Sudan I leads to the change in expression of 764 genes. Moreover, it's further reflected that the damaging effects of Sudan I are mediated by the destruction of mitochondrial functions, which induces the accumulated ROS to stimulate oxidative stress-induced apoptosis. As an endogenous hormone, melatonin within the ovarian follicle plays function on improving oocyte quality and female reproduction by efficiently suppressing oxidative stress. Moreover, melatonin supplementation also improves oocyte quality and increases fertilization rate during in vitro culture. Consistent with these, we find that in vivo supplementation of melatonin efficaciously suppresses mitochondrial dysfunction and the accompanying apoptosis, thus reverses oocyte meiotic deteriorations. Collectively, our results prove the reproduction toxicity of Sudan I for the exposure of Sudan I reduces the oocyte quality, and demonstrate the protective effects of melatonin against Sudan I-induced meiotic deteriorations., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Citrinin (CTN) is a mycotoxin, which is isolated from Penicillium citrinum and widely existed in the contaminated feeds. It is reported that CTN is toxic to heart, liver, and reproductive system. Previous studies indicated that CTN induced apoptosis in oocytes and embryos. In this study, we reported the potential causes of CTN on embryo development. Our results showed that 40 μM CTN exposure significantly reduced the first cleavage of mouse embryos, showing with the low rate of 2-cell embryos. We found that CTN induced DNA damage, showing the higher positive γH2A.X signals. Autophagy was occurred since more LC3 positive autophagosomes were found in the cytoplasm. This could be confirmed by the enhanced lysosome function, since higher accumulated lysosome distribution were found and LAMP2 was also increased under CTN exposure. Besides, we showed that mitochondria distribution was disturbed, indicating that CTN could disrupt mitochondria function, which could be the possible reason for the oxidative stress and apoptosis in CTN-exposed embryos. In conclusion, our study showed that CTN exposure had adverse effects on the early embryo development during first cleavage through its effects on the induction of DNA damage, autophagy, and mitochondria dysfunction., (© 2021 Wiley Periodicals LLC.)

Background: RAB GTPases constitute the largest family of small GTPases and are found in all eukaryotes. RAB GTPases regulate components of the endomembrane system, the nucleus and the plasma membrane, and are involved in intracellular actin/tubulin-dependent vesicle movement, membrane fusion and cell growth in mitosis., Objective and Rationale: RAB GTPases play multiple critical roles during both female and male meiosis. This review summarizes the progress made in our understanding of the role of RAB GTPases in female and male meiosis in different species. We also discuss the potential relationship between RAB GTPases and oocyte/sperm quality, which may help in understanding the mechanisms underlying oogenesis and spermatogenesis and potential genetic causes of infertility., Search Methods: The PubMed database was searched for articles published between 1991 and 2020 using the following terms: 'RAB', 'RAB oocyte', 'RAB sperm' and 'RAB meiosis'., Outcomes: An analysis of 126 relevant articles indicated that RAB GTPases are present in all eukaryotes, and ten subfamilies (almost 70 members) are expressed in human cells. The roles of 25 RAB proteins and orthologues in female meiosis and 12 in male meiosis have been reported. RAB proteins are essential for the accurate continuity of genetic material, successful fertilization and the normal growth of offspring. Distinct and crucial functions of RAB GTPases in meiosis have been reported. In oocytes, RAB GTPases are involved in spindle organization, kinetochore-microtubule attachment, chromosome alignment, actin filament-mediated spindle migration, cytokinesis, cell cycle and oocyte-embryo transition. RAB GTPases function in mitochondrial processes and Golgi-mediated vesicular transport during female meiosis, and are critical for cortical granule transport during fertilization and oocyte-embryo transition. In sperm, RAB GTPases are vital for cytoskeletal organization and successful cytokinesis, and are associated with Golgi-mediated acrosome formation, membrane trafficking and morphological changes of sperm cells, as well as the exocytosis-related acrosome reaction and zona reaction during fertilization., Wider Implications: Abnormal expression of RAB GTPases disrupts intracellular systems, which may induce diverse diseases. The roles of RAB proteins in female and male reproductive systems, thus, need to be considered. The mechanisms underlying the function of RAB GTPases and the binding specificity of their effectors during oogenesis, spermatogenesis and fertilization remain to be studied. This review should contribute to our understanding of the molecular mechanisms of oogenesis and spermatogenesis and potential genetic causes of infertility., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Oocyte quality is critical for fertilization and early embryo development. Fumonisin B1 (FB1) is a Fusarium mycotoxin and it is commonly found in contaminated food and feedstuff, posing a potential health hazard to both animals and human. FB1 is reported to have hepatotoxicity, neurotoxicity, nephrotoxicity, immunotoxicity and embryotoxicity. However, the effects of FB1 on mouse oocyte quality are still unknown. Here, we explored the toxic effects and potential mechanisms of FB1 on oocyte maturation quality in mice. FB1 exposure inhibited the first polar body extrusion at concentrations of 30 μM and 50 μM, which further induced oocyte meiotic arrest. Besides, disrupted spindle structure was found in oocytes after FB1 exposure. Our results also showed that FB1 exposure impaired mitochondria dysfunction, which further induced oxidative stress and early apoptosis. In addition, we reported that FB1 exposure induced the accumulation of lysosome and occurrence of autophagy. Aberrant ER distribution and ER stress were also found in FB1-exposed oocytes. Moreover, DNA damage was also observed. These results together suggested that FB1 exposure affected oocyte quality by destroying spindle structure, leading to mitochondria, lysosome and ER dysfunction, which further induced oxidative stress, apoptosis, autophagy and DNA damage in mouse oocytes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Primordial germ cells (PGCs) are precursors of both male and female gametes as fundamental materials for organism development. The transcriptome, methylome, and chromatin accessibility profiles of PGCs in both mice and humans have been recently reported. However, little is known about the characteristics of PGCs at the protein levels, which directly exert cellular functions. Here, we construct landscapes of both proteome and 3D spatial distribution of mouse PGCs at E11.5, E13.5 and E16.5 days, the three critical developmental windows for PGCs' sex differentiation, female meiosis initiation and male mitotic arrest. In each developmental stage of PGCs, nearly 2,000-3,000 proteins are identified, among which specific functional pathways such as oxidative phosphorylation, DNA damage repair, and meiotic cell cycle are involved for different events during PGCs development. Interestingly, by 3D modeling we find that PGCs spatially cluster into around 1,300 nests in genital ridge at E11.5 and the nest number is not increased by the exponential proliferation of PGCs. Comparative analysis of our proteomic data with published transcriptomic data does not show a close correlation, meaning that the practically executive factors are beyond the transcriptome. Thus, our work offers a valuable resource for the systematic investigations of PGC development at protein level and spatial map.

Podophyllotoxin (POD) is one of the most characterized lignans that is commonly found in podophyllum, and its preparations and derivatives are widely used in clinical treatment due to strong antitumor and antivirus activities. POD has been reported for its neurotoxicity, liver toxicity, and potential reproductive toxicity. In the present study, we investigated the effects of POD on the organelles of mouse oocytes during meiosis. Our results showed that exposure to POD significantly reduced the developmental competence of mouse oocytes. Further analysis revealed that the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, suggesting that POD exposure might affect protein synthesis during oocyte meiotic maturation. Similarly, abnormal Golgi apparatus distribution was found after POD exposure, which could be confirmed by the aberrant localization of Rab11a-related vesicles, indicating that POD induced vesicle-based protein transport disorder. We also found the aberrant accumulation of lysosomes in the cytoplasm of POD-exposed oocytes, which implied that POD might lead to aberrant protein degradation. Moreover, the perinuclear distribution of mitochondria was also significantly disturbed, indicating the mitochondrial dysfunction after POD exposure. In all, our study illustrated that exposure to POD might disrupt protein synthesis, transport, degradation, and ATP production by its effects on the distribution and functions of organelles during mouse oocyte meiotic maturation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Xie, Kong, Xu and Sun.)

GBF1 [Golgi brefeldin A (BFA) resistance factor 1] is a member of the guanine nucleotide exchange factors Arf family. GBF1 localizes at the cis-Golgi and endoplasmic reticulum (ER)-Golgi intermediate compartment where it participates in ER-Golgi traffic by assisting in the recruitment of the coat protein COPI. However, the roles of GBF1 in oocyte meiotic maturation are still unknown. In the present study, we investigated the regulatory functions of GBF1 in mouse oocyte organelle dynamics. In our results, GBF1 was stably expressed during oocyte maturation, and GBF1 localized at the spindle periphery during metaphase I. Inhibiting GBF1 activity led to aberrant accumulation of the Golgi apparatus around the spindle. This may be due to the effects of GBF1 on the localization of GM130, as GBF1 co-localized with GM130 and inhibiting GBF1 induced condensation of GM130. Moreover, the loss of GBF1 activity affected the ER distribution and induced ER stress, as shown by increased GRP78 expression. Mitochondrial localization and functions were affected, as the mitochondrial membrane potential was altered. Taken together, these results suggest that GBF1 has wide-ranging effects on the distribution and functions of Golgi apparatus, ER, and mitochondria as well as normal polar body formation in mouse oocytes.

Infertility in humans at their reproductive age is a world-wide problem. Oocyte in vitro maturation (IVM) is generally used in such cases to acquire the embryo in assisted reproductive technology (ART). However, the differences between an in vivo (IVO) and IVM culture environment in the RNA expression profile in oocytes, remains unclear. In this study, we compared the global RNA transcription pattern of oocytes from in vitro and in vivo maturation. Our results showed that 1,864 genes differentially expressed between the IVO and IVM oocytes. Among these, 1,638 genes were up-regulated, and 226 genes were down-regulated, and these changes were mainly divided into environmental adaption, metabolism, and genetic expression. Our detailed analysis showed that the expression of genes that belonged to metabolism-related processes such as energy metabolism, nucleotide metabolism, and carbohydrate metabolism was changed; and these genes also belonged to organismal systems including environmental adaptation and the circulatory system; moreover, we also found that the relative gene expression of genetic expression processes, such as protein synthesis, modification, and DNA replication and repair were also altered. In conclusion, our data suggests that in vitro maturation of mouse oocyte resulted in metabolism and genetic expression changes due to environmental changes compared with in vivo matured oocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Xu, Ju, Pan, Liu and Sun.)

Zearalenone (ZEN) is one of the most common mycotoxins produced by fungus in contaminated feed. ZEN has multiple toxicities, including reproductive toxicity of domestic animals, particularly pigs. However, studies on the effects of ZEN on ovary/oocytes have been primarily based on in vitro experiments, and there is still no evidence from porcine in vivo models due to multiple limitations. Moreover, no report has investigated the effect of hydrated sodium calcium aluminosilicate (HSCAS) as a supplement on pig oocyte quality. In the present study, we fed pigs a 1.0 mg/kg ZEN-contaminated diet for 10 days. The results showed that pigs fed ZEN presented reduced oocyte-cumulus cell interactions, an increase in the number of denuded oocytes in ovaries, a decrease in the number of oocytes in each ovary, and an increase in the oocyte death rate. Oocytes from ZEN-exposed pigs exhibited a delayed cell cycle and abnormal cytoskeletal dynamics during meiotic maturation, which could be due to oxidative stress-induced autophagy. Moreover, we also show that supplementing the ZEN-contaminated diet with modified HSCAS effectively protected porcine oocyte quality. Taken together, our study provides in vivo data demonstrating the protective effects of HSCAS against ZEN toxicity in porcine oocytes., (© 2020 Environmental Mutagen Society.)

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development., (© 2020 Wiley Periodicals LLC.)

Nonylphenol (NP) is a chemical raw material and intermediate which is mainly used in the production of surfactants, lubricating oil additives and pesticide emulsifiers. NP is reported to be toxic on the immune system, nervous system and reproductive system due to its binding to estrogen receptors. However, the toxicity of NP on mammalian oocyte quality remains unclear. In present study, we explored the effects of NP exposure on mouse oocyte maturation. Our results showed that 4 weeks of NP exposure increased the number of atresia follicles and decreased oocyte developmental competence. Transcriptomic analysis indicated that NP exposure altered the expression of more than 800 genes in oocytes, including multiple biological pathways. Subcellular structure examination indicated that NP exposure disrupted meiotic spindle organization and caused chromosome misalignment. Moreover, aberrant mitochondrial distribution and decreased membrane potential were also observed, indicating that NP exposure caused mitochondria dysfunction. Further analysis showed that NP exposure resulted in the accumulation of reactive oxygen species (ROS), which causes oxidative stress; and the NP-exposed oocytes showed positive Annexin-V signal, indicating the occurrence of early apoptosis. In summary, our results indicated that NP exposure reduced oocyte quality by affecting cytoskeletal dynamics and mitochondrial function, which further induced oxidative stress and apoptosis in mice., Competing Interests: Declaration of competing interest There are no potential conflicts of interests for all authors to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

2, 2', 4, 4'-Tetrabrominated diphenyl ether (PBDE47) is the most abundant homologues in polybrominated diphenyl ethers (PBDEs) and is used widely in daily life as a brominated flame retardant. It has been shown that PBDE47 has neurotoxicity, thyroid toxicity, and also is reported to have reproductive toxicity, which can impair the follicular function of female rats and activate ovarian endoplasmic reticulum stress, leading to ovarian damage. However, the toxicity of PBDE47 to the quality of mammalian oocyte has not been reported. In this study, we reported that PBDE47 exposure affected the mouse ovarian and uterine. We also showed that oocyte number reduced and meiotic maturation competence decreased. Moreover, PBDE47 exposure disrupted actin filaments distribution, and also changed the level of histone methylation. Based on the genome-wide sequencing we showed that PBDE47 altered multiple gene expression in mouse oocytes. PBDE47 exposure caused mitochondria dysfunction, showing with aberrant distribution and mitochondrial membrane potential. In addition, our results suggested that PBDE47 induced early apoptosis, which was caused by oxidative stress, showing with increased ROS level and positive Annexin-V signal. In conclusion, our results indicated that PBDE47 exposure affected the maturation of mouse oocyte via its effects on mitochondria function, ROS level and its related apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The quality of the early embryo is critical for embryonic development and implantation. Errors during cleavage lead to aneuploidy in embryos. As a cell cycle checkpoint protein, CHK2 participates in DNA replication, cell cycle arrest and spindle assembly. However, the functions of CHK2 in early development of the mouse embryo remain largely unknown. In this study, we show that CHK2 is localized on the spindle in metaphase and mainly accumulates at spindle poles in anaphase/telophase during the first cleavage of the mouse embryo. CHK2 inhibition led to cleavage failure in early embryonic development, accompanied by abnormal spindle assembly and misaligned chromosomes. Moreover, the loss of CHK2 activity increased the level of cellular DNA damage, which resulted in oxidative stress. Then, apoptosis and autophagy were found to be active in these embryos. In summary, our results suggest that CHK2 is an essential regulator of spindle assembly and DNA repair during early embryonic development in mice.

Formin-like 3 (FMNL3) is a member of the formin-likes (FMNLs), which belong to the formin family. As an F-actin nucleator, FMNL3 is essential for several cellular functions, such as polarity control, invasion, and migration. However, the roles of FMNL3 during oocytes meiosis remain unclear. In this study, we investigated the functions of FMNL3 during mouse oocyte maturation. Our results showed that FMNL3 mainly concentrated in the oocyte cortex and spindle periphery. Depleting FMNL3 led to the failure of polar body extrusion, and we also found large polar bodies in the FMNL3-deleted oocytes, indicating the occurrence of symmetric meiotic division. There was no effect of FMNL3 on spindle organization; however, we observed spindle migration defects at late metaphase I, which might be due to the decreased cytoplasmic actin. Microinjecting Fmnl3-EGFP mRNA into Fmnl3-depleted oocytes significantly rescued these defects. In addition, the results of co-immunoprecipitation and the perturbation of protein expression experiments suggested that FMNL3 interacted with the actin-binding protein FASCIN for the regulation of actin filaments in oocytes. Thus, our results provide the evidence that FMNL3 regulates FASCIN for actin-mediated spindle migration and cytokinesis during mouse oocyte meiosis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Melatonin is a hormone which is generated from pineal gland, and it is responsible for the regulation of wake-sleep cycle. Melatonin is a well-known antioxidant and free radical scavenger to protect against multiple type of tissue damage. While ochratoxin A (OTA) is a mycotoxin found widely in contaminated food and foodstuffs, which causes nephrotoxicity, hepatotoxicity, immunotoxicity, and reproductive damage in humans and animals. In present study we report the toxicity of OTA on porcine oocyte quality and the protective effects of melatonin on OTA-exposed oocytes. Using transcriptome analysis our results show that OTA exposure alters the expression of multiple genes in oocytes, indicating its effect on oocyte maturation. The cellular changes following OTA treatment are examined, and the results show that OTA adversely affects oocyte polar body extrusion, which is confirmed by the delay of Cdc2-mediated cell cycle progression. OTA exposure also disrupts meiotic spindle formation, which is confirmed by altered phosphorylated MAPK expression. RNA-seq screening and further fluorescence staining results show that OTA induces aberrant mitochondria distribution and oxidative phosphorylation defects, which then causes oxidative stress, followed by early apoptosis and autophagy. Treatment with melatonin significantly ameliorates oxidative stress and apoptosis, which further protects cell cycle and spindle formation in OTA-exposed oocytes. Collectively, these results show the protective effects of melatonin against defects induced by OTA during porcine meiotic oocyte maturation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mammalian oocyte maturation involves a unique asymmetric cell division, in which meiotic spindle formation and actin filament-mediated spindle migration to the oocyte cortex are key processes. Here, we report that the vesicle trafficking regulator, RAB35 GTPase, is involved in regulating cytoskeleton dynamics in mouse oocytes. RAB35 GTPase mainly accumulated at the meiotic spindle periphery and cortex during oocyte meiosis. Depletion of RAB35 by morpholino microinjection led to aberrant polar body extrusion and asymmetric division defects in almost half the treated oocytes. We also found that RAB35 affected SIRT2 and αTAT for tubulin acetylation, which further modulated microtubule stability and meiotic spindle formation. Additionally, we found that RAB35 associated with RHOA in oocytes and modulated the ROCK-cofilin pathway for actin assembly, which further facilitated spindle migration for oocyte asymmetric division. Importantly, microinjection of Myc-Rab35 cRNA into RAB35-depleted oocytes could significantly rescue these defects. In summary, our results suggest that RAB35 GTPase has multiple roles in spindle stability and actin-mediated spindle migration in mouse oocyte meiosis., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium fungi commonly found in corn, wheat, and other cereals which can infect food and feed commodities, and ZEA mainly has reproductive toxicity which causes widely reproductive disorders in pigs and other animals. However, the toxicity and the functional ways of ZEA on early embryo development is still unclear. In present study we showed that exposure to ZEA (10 μM) significantly decreased the 2-cell and blastocyst developmental rate in porcine early embryos in vitro . ZEA treatment resulted in the occurrence of oxidative stress, showing with increased reactive oxygen species (ROS) level, following with aberrant mitochondrial distribution. Moreover, we found positive signals of γH2A.X in the ZEA-treated embryos, indicating that ZEA induced DNA damage, and the increased autophagy confirmed this. These results suggested that ZEA induced oxidative stress, which further caused mitochondria dysfunction and DNA damage on early embryonic development. We next investigated the effects of melatonin on the ZEA-treated embryo development, and we found that melatonin supplementation could significantly ameliorate ZEA-induced oxidative stress, aberrant mitochondria distribution and DNA damage. In all, our results showed that ZEA was toxic for porcine embryos cultured in vitro and melatonin supplementation could protect their development from the effects of ZEA.

Cytoskeletal dynamics are involved in multiple cellular processes during oocyte meiosis, including spindle organization, actin-based spindle migration and polar body extrusion. Here, we report that the vesicle trafficking protein Rab23, a GTPase, drives the motor protein Kif17, and that this is important for spindle organization and actin dynamics during mouse oocyte meiosis. GTP-bound Rab23 accumulated at the spindle and promoted migration of Kif17 to the spindle poles. Depletion of Rab23 or Kif17 caused polar body extrusion failure. Further analysis showed that depletion of Rab23/Kif17 perturbed spindle formation and chromosome alignment, possibly by affecting tubulin acetylation. Kif17 regulated tubulin acetylation by associating with αTAT and Sirt2, and depletion of Kif17 altered expression of these proteins. Moreover, depletion of Kif17 decreased the level of cytoplasmic actin, which abrogated spindle migration to the cortex. The tail domain of Kif17 associated with constituents of the RhoA-ROCK-LIMK-cofilin pathway to modulate assembly of actin filaments. Taken together, our results demonstrate that the Rab23-Kif17-cargo complex regulates tubulin acetylation for spindle organization and drives actin-mediated spindle migration during meiosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Protein kinase D (PKD) subfamily which includes PKD1, PKD2 and PKD3 is a novel family of serine/threonine kinases. PKD has been widely implicated in the regulation of multiple physiological effects including immune responses, apoptosis and cell proliferation. However, the roles of PKD in oocytes have not been fully clarified. In this study we investigated the regulatory functions of PKD during porcine oocyte maturation. Our results indicated that PKD expressed in porcine oocytes and the inhibition of PKD family activity led to the failure of meiosis resumption and the first polar body extrusion. Further analysis indicated that the spindle assembly and chromosome alignment were disrupted after PKD family inhibition, and this might be through its regulatory role on MAPK phosphorylation. We also found that PKD phosphorylated cofilin for actin assembly, which further affected cortical actin distribution, indicating the roles of PKD family on cytoskeleton. In addition, a decreased expression of PKD in postovulatory aging porcine oocytes was observed, which might connect PKD with cytoskeleton defects in aged oocytes. Taken together, these results suggest that PKD possesses important functions in porcine oocyte maturation by regulating spindle organization and actin assembly.

Histone deacetylases (HDACs) have been shown to deacetylate numerous cellular substrates that govern a wide array of biological processes. HDAC3, a member of the Class I HDACs, is a highly conserved and ubiquitously expressed protein. However, its roles in meiotic oocytes are not known. In the present study, we find that mouse oocytes depleted of HDAC3 are unable to completely progress through meiosis, and are blocked at metaphase I. These HDAC3 knockdown oocytes show spindle/chromosome organization failure, with severely impaired kinetochore-microtubule attachments. Consistent with this, the level of BubR1, a central component of the spindle assembly checkpoint, at kinetochores is dramatically increased in metaphase oocytes following HDAC3 depletion. Knockdown and overexpression experiments reveal that HDAC3 modulates the acetylation status of α-tubulin in mouse oocytes. Importantly, the deacetylation mimetic mutant tubulin-K40R can partly rescue the defective phenotypes of HDAC3 knockdown oocytes. Our data support a model whereby HDAC3, through deacetylating tubulin, promotes microtubule stability and the establishment of kinetochore-microtubule interaction, consequently ensuring proper spindle morphology, accurate chromosome movement and orderly meiotic progression during oocyte maturation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Study Question: Does melamine have a toxic effect on oocyte development and fertility in vivo?, Summary Answer: Melamine had toxic effects on oocyte quality and fertility due to its effects on the oocyte cytoskeleton, apoptosis and autophagy induction, and epigenetic modifications in an in vivo mouse model., What Is Known Already: Melamine is a chemical compound that is widely used during the manufacture of amino resins and plastics. In 2008, melamine was reported to adulterate milk and infant formulas in China, which sparked food safety concerns worldwide. Ingesting melamine may result in reproductive damage, and bladder or kidney stones, which can lead to bladder cancer., Study Design, Size, Duration: Mice were randomly assigned to three groups and fed a diet that included melamine (0, 10 and 50 mg/kg/day) for 8 weeks. The in vivo effect of melamine on female reproduction was examined., Participants/materials, Setting, Methods: We used immunofluorescent staining, western blotting and qRT-PCR to examine the effect of melamine on oocyte quality., Main Results and the Role of Chance: Our results showed the following effects of this melamine-containing diet. (i) Ovary weights were reduced in melamine fed mice. Oocyte developmental competence was also reduced, as shown by reduced polar body extrusion rates. (ii) Melamine feeding resulted in abnormal oocyte cytoskeletons, as shown by increased rates of aberrant spindles and reduced actin microfilament expression. (iii) Melamine exposed oocytes had higher rates of abnormal mitochondrial distributions and early stage apoptosis/autophagy, which were shown by increased microtubule-associated protein 1 light chain 3 (LC3) protein expression level and caspase 9, autophagy-related protein 14 (atg14), and lc3 mRNA levels. (iv) Fluorescence intensity analysis showed that DNA methylation levels were reduced in the oocytes of melamine fed mice. Histone methylation levels were also altered, as Di-methyl-Histone H3 (Lys4) (H3K4me2) level was increased and Tri-methyl-Histone H3 (Lys9) (H3K9me3), Di-methyl-Histone H3 (Lys9) (H3K9me2), and Tri-methyl-Histone H3 (Lys27) (H3K27me3) levels were reduced in oocytes from melamine fed mice. (v) The litter sizes of melamine fed mice were significantly reduced when compared with those of controls., Limitations, Reasons for Caution: Although we examined the possible effects of melamine on oocyte quality and fertility, we did not determine the effect of melamine on offspring development., Wider Implications of the Findings: Our findings indicate that melamine plays a major role in oocyte quality and fertility. This information could contribute to a better understanding of melamine toxicity in female reproduction., Study Funding/competing Interests: This study was supported by the National Basic Research Program of China (2014CB138503) and the Natural Science Foundation of Jiangsu Province (BK20140030). The authors have no conflict of interest to disclose., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

G9A-like protein (GLP) plays an important role in mouse early embryonic development. Glp-deficient embryos exhibit severe growth retardation and defects that lead to lethality at approximately Embryonic Day 9.5. In the present study we investigated the effect of microinjection of Glp-specific short interference (si) RNA into mouse zygotes on in vitro embryonic development. Knockdown of Glp induced abnormal embryonic development and reduced blastocyst formation. Expression of the pluripotency markers octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog was also significantly decreased in Glp-deficient embryos. The apoptotic index and expression of two pro-apoptotic genes, namely Caspase 3 and Caspase 9, were increased in Glp-deficient embryos. Moreover, methylation levels of dimethylated H3K9 (H3K9me2) were decreased in Glp-knockdown embryos. In conclusion, the results of the present study suggest that Glp deficiency suppresses H3K9me2 modification and hinders mouse embryo development in vitro.

Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 μg/kg), and AF (806 μg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality., (Copyright © 2013 Wiley Periodicals, Inc.)

Interspecies 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.

Background: The effect of Doxorubicin, a widely used chemotherapeutic agent, on early mouse embryonic development has not been previously characterized., Materials and Methods: Expression of apoptosis-related genes and poly(ADP-ribose) polymerase (PARP) family genes were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Apoptosis in mouse blastocysts was tested using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Cleaved (c)-PARP was analyzed by western blot., Results: A 20 h exposure to doxorubicin caused rapid cytoplasmic fragmentation, DNA condensation and disruption of the cytoskeleton in mouse embryos. Doxorubicin altered the expression of genes involved in DNA repair and apoptosis and blocked early embryonic development, suggesting that doxorubicin affects DNA synthesis and repair. Furthermore, the effect of doxorubicin on early embryo development was determined by assessing the rates of development to different stages and an apoptosis index. Both assays confirmed that doxorubicin altered embryonic development. In conclusion, doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by PARP.

BACKGROUND Meiosis is a unique form of cell division in which cells divide twice but DNA is duplicated only once. Errors in chromosome segregation during meiosis will result in aneuploidy, followed by loss of the conceptus during pregnancy or birth defects. During mitosis, cells utilize a mechanism called the spindle assembly checkpoint (SAC) to ensure faithful chromosome segregation. A similar mechanism has been uncovered for meiosis in the last decade, especially in the past several years. METHODS For this review, we included data and relevant information obtained through a PubMed database search for all articles published in English from 1991 through 2011 which included the term 'meiosis', 'spindle assembly checkpoint', or 'SAC'. RESULTS There are 91 studies included. Evidence for the existence of SAC functions in meiosis is provided by studies on the SAC proteins mitotic-arrest deficient-1 (Mad1), Mad2, budding uninhibited by benzimidazole-1 (Bub1), Bub3, BubR1 and Mps1; microtubule-kinetochore attachment regulators Ndc80 complex, chromosomal passenger complex, mitotic centromere-associated kinesin (MCAK), kinetochore null 1 (KNL1) and Mis12 complex and spindle stability regulators. CONCLUSIONS SAC and its regulators exist and function in meiosis, and their malfunctions may cause germ cell aneuploidy. However, species and sexual differences exist. Moreover, interaction of SAC components with other regulators is still poorly understood, which needs further study.

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.

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

In mitosis, Borealin is a member of the chromosomal passenger complex (CPC), which plays interaction roles with INCENP and survivin in the complex. Its roles in mammalian meiosis are unknown. Here, we report the expression, localization, and function of Borealin and its relation with survivin in mouse oocyte meiosis. Borealin expression was gradually increased from GV stage to MII. Immunofluorescence results revealed that Borealin accumulated near chromosomes after GVBD, localized at the spindle poles in MI, AI and MII, and at the midbody in TI stage. Taxol and nocodazole treatment showed that the localization of Borealin was dependent on microtubule dynamics, whereas survivin was independent of this. Disruption of Borealin function by antibody injection resulted in severe spindle assembly defects, but did not affect PBE. We also found that depletion of survivin by MO injection had no effect on the localization of Borealin. In conclusion, our data suggest that Borealin is required for bipolar spindle formation, but may not regulate spindle checkpoint activity as a component of the CPC during mouse oocyte meiosis.

P21-activated kinase 1 (PAK1), an effector of Rho GTPase Rac 1 and Cdc42, is required for mitotic progression. However, its functions in meiosis are unclear. In the present study, we examined the expression, localization and function of PAK1 during mouse oocyte meiotic maturation and found that PAK1 was mainly associated with the meiotic spindle microtubules. Taxol treatment resulted in localization of PAK1 on spindle and aster microtubules, while nocodazole treatment induced the dispersion of PAK1 protein into the cytoplasm. Loss-of-function of PAK1 by both inhibitor treatment and morpholino oligonucleotide injection caused disorganized spindles, decreased polar body extrusion and misaligned chromosomes. In addition, inhibition of PAK1 resulted in abnormal localization of mitogen-activated protein kinase kinase (MEK). Taken together, our results suggest that PAK1 plays an important role in spindle assembly and chromosome alignment during mouse oocyte meiotic maturation.

Calcium is one of the most ubiquitous signaling molecules, and controls a wide variety of cellular processes. It is mainly stored in the endoplasmic reticulum (ER), bound to lumenal proteins. Calreticulin is the major Ca(2+)-binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER- calreticulin-Ca(2+) pathway in oocyte maturation and early embryogenesis, we characterized the porcine calreticulin gene and investigated its expression profile during oocyte maturation and early embryonic development. Calreticulin was widely expressed in pig tissues and its transcripts were downregulated during maturation, especially at 44 hr, and were undetectable at the blastocyst stage. We also investigated the effect of increased cytosolic Ca(2+) induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 microM did not inhibit germinal vesicle breakdown, but did result in the arrest of 38.6% oocytes at or before the MI stage. In addition, expression of the maternal genes C-mos, BMP15, GDF9, and Cyclin B1 was significantly increased in CPA-treated MII oocytes compared with control groups. These data were supported by the results of poly(A)-test PCR, which revealed that the cyclin B1 short isoform (CB-S), GDF9, and C-mos underwent more intensive polyadenylation modification in CPA-treated oocytes than control oocytes, suggesting that polyadenylation may influence Ca(2+)-modulated changes in gene expression. Furthermore, CPA treatment decreased the percentage of four-cell parthenotes that developed into blastocysts, suggesting the need for functional SR/ER Ca(2+)-ATPase pumps or Ca(2+) signals during early embryo development after zygotic genome activation. Together, these data indicate that ER-calreticulin-associated Ca(2+) homeostasis plays a role in oocyte and embryo development, and that alterations in maternal gene expression may contribute to the underlying molecular mechanism, at least partially, via polyadenylation.