SIRT6 Maintains Redox Homeostasis to Promote Porcine Oocyte Maturation

SIRT6, the sixth member of the sirtuin family proteins, has been characterized as a crucial regulator in multiple molecular pathways related to aging, including genome stability, DNA damage repair, telomere maintenance, and inflammation. However, the exact roles of SIRT6 during female germ cell development have not yet been fully determined. Here, we assessed the acquisition of meiotic competency of porcine oocytes by inhibition of SIRT6 activity. We observed that SIRT6 inhibition led to the oocyte meiotic defects by showing the impairment of polar body extrusion and cumulus cell expansion. Meanwhile, the compromised spindle/chromosome structure and actin dynamics were also present in SIRT6-inhibited oocytes. Moreover, SIRT6 inhibition resulted in the defective cytoplasmic maturation by displaying the disturbed distribution dynamics of cortical granules and their content ovastacin. Notably, we identified that transcript levels of genes related to oocyte meiosis, oxidative phosphorylation, and cellular senescence were remarkably altered in SIRT6-inhibited oocytes by transcriptome analysis and validated that the meiotic defects caused by SIRT6 inhibition might result from the excessive reactive oxygen species (ROS)-induced early apoptosis in oocytes. Taken together, our findings demonstrate that SIRT6 promotes the porcine oocyte meiotic maturation through maintaining the redox homeostasis.

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Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

Cell Division ◽

10.1186/s13008-021-00071-y ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Ming-Hong Sun ◽

Lin-Lin Hu ◽

Chao-Ying Zhao ◽

Xiang Lu ◽

Yan-Ping Ren ◽

...

Keyword(s):

Golgi Apparatus ◽

Oocyte Maturation ◽

Polar Body ◽

Mouse Oocyte ◽

Actin Dynamics ◽

Mouse Oocytes ◽

Oocyte Meiosis ◽

Ral Gtpase ◽

Polar Body Extrusion ◽

Cytoskeleton Dynamics

Abstract Background Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation. Results Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion. Conclusions In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

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Comparison of the toxic effects of different mycotoxins on porcine and mouse oocyte meiosis

PeerJ ◽

10.7717/peerj.5111 ◽

2018 ◽

Vol 6 ◽

pp. e5111 ◽

Cited By ~ 7

Author(s):

Yujie Lu ◽

Yue Zhang ◽

Jia-Qian Liu ◽

Peng Zou ◽

Lu Jia ◽

...

Keyword(s):

Oocyte Maturation ◽

Animal Feed ◽

Polar Body ◽

Oocyte Quality ◽

Mouse Oocyte ◽

Toxic Effects ◽

Mouse Oocytes ◽

Oocyte Meiosis ◽

Porcine Oocyte ◽

Polar Body Extrusion

Background Aflatoxin B1 (AFB1), deoxynivalenol (DON), HT-2, ochratoxin A (OTA), zearalenone (ZEA) are the most common mycotoxins that are found in corn-based animal feed which have multiple toxic effects on animals and humans. Previous studies reported that these mycotoxins impaired mammalian oocyte quality. However, the effective concentrations of mycotoxins to animal oocytes were different. Methods In this study we aimed to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA for mycotoxin research. We adopted the polar body extrusion rate of mouse and porcine oocyte as the standard for the effects of mycotoxins on oocyte maturation. Results and Discussion Our results showed that 10 μM AFB1 and 1 μM DON significantly affected porcine oocyte maturation compared with 50 μM AFB1 and 2 μM DON on mouse oocytes. However, 10 nM HT-2 significantly affected mouse oocyte maturation compared with 50 nM HT-2 on porcine oocytes. Moreover, 5 μM OTA and 10 μM ZEA significantly affected porcine oocyte maturation compared with 300 μM OTA and 50 μM ZEA on mouse oocytes. In summary, our results showed that porcine oocytes were more sensitive to AFB1, DON, OTA, and ZEA than mouse oocytes except HT-2 toxin.

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WAPL orchestrates porcine oocyte meiotic progression via control of spindle assembly checkpoint activity

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00740-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Changyin Zhou ◽

Yilong Miao ◽

Xue Zhang ◽

Bo Xiong

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Polar Body ◽

Spindle Assembly ◽

Protein Levels ◽

Meiotic Progression ◽

Oocyte Meiosis ◽

Chromosome Alignment ◽

Porcine Oocyte ◽

Polar Body Extrusion

Abstract Background In mitotic cells, WAPL acts as a cohesin release factor to remove cohesin complexes from chromosome arms during prophase to allow the accurate chromosome segregation in anaphase. However, we have recently documented that Wapl exerts a unique meiotic function in the spindle assembly checkpoint (SAC) control through maintaining Bub3 stability during mouse oocyte meiosis I. Whether this noncanonical function is conserved among species is still unknown. Methods We applied RNAi-based gene silencing approach to deplete WAPL in porcine oocytes, validating the conserved roles of WAPL in the regulation of SAC activity during mammalian oocyte maturation. We also employed immunostaining, immunoblotting and image quantification analyses to test the WAPL depletion on the meiotic progression, spindle assembly, chromosome alignment and dynamics of SAC protein in porcine oocytes. Results We showed that depletion of WAPL resulted in the accelerated meiotic progression by displaying the precocious polar body extrusion and compromised spindle assembly and chromosome alignment. Notably, we observed that the protein level of BUB3 was substantially reduced in WAPL-depleted oocytes, especially at kinetochores. Conclusions Collectively, our data demonstrate that WAPL participates in the porcine oocyte meiotic progression through maintenance of BUB3 protein levels and SAC activity. This meiotic function of WAPL in oocytes is highly conserved between pigs and mice.

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LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation

PeerJ ◽

10.7717/peerj.2553 ◽

2016 ◽

Vol 4 ◽

pp. e2553 ◽

Cited By ~ 4

Author(s):

Ru-Xia Jia ◽

Xing Duan ◽

Si-Jing Song ◽

Shao-Chen Sun

Keyword(s):

Oocyte Maturation ◽

Polar Body ◽

Meiotic Spindle ◽

Actin Dynamics ◽

Immunofluorescent Staining ◽

Meiotic Maturation ◽

Spindle Positioning ◽

Oocyte Meiosis ◽

Porcine Oocyte ◽

Oocyte Meiotic Maturation

LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the cortex of porcine oocyte, which co-localized with actin. After LIMKi 3 treatment, the diffusion of COCs became weak and the rate of polar body extrusion was decreased. This could be rescued by moving oocytes to fresh medium. After prolonging the culture time of oocytes, the maturation rate of porcine oocyte increased in LIMKi 3 groups, indicating that LIMKi 3 may suppress the cell cycle during porcine oocyte maturation. We also found that after LIMKi 3 treatment actin distribution was significantly disturbed at porcine oocyte membranes and cytoplasm, indicating the conserved roles of LIMK1/2 on actin dynamics. Next we examined the meiotic spindle positioning in porcine oocyte, and the results showed that a majority of spindles were not attached to the cortex of porcine oocyte, indicating that LIMKi 3 may affect actin-mediated spindle positioning. Taken together, these results showed that LIMK1/2 inhibitor LIMKi 3 had a repressive role on porcine oocyte meiotic maturation.

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FASCIN regulates actin assembly for spindle movement and polar body extrusion in mouse oocyte meiosis

Journal of Cellular Physiology ◽

10.1002/jcp.30443 ◽

2021 ◽

Author(s):

Lin‐Lin Hu ◽

Meng‐Hao Pan ◽

Feng‐Lian Yang ◽

Zi‐Ao Zong ◽

Feng Tang ◽

...

Keyword(s):

Polar Body ◽

Mouse Oocyte ◽

Actin Assembly ◽

Oocyte Meiosis ◽

Polar Body Extrusion

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OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes

10.7287/peerj.preprints.27837 ◽

2019 ◽

Author(s):

Di Xie ◽

Juan Zhang ◽

JinLi Ding ◽

Jing Yang ◽

Yan Zhang

Keyword(s):

Spindle Assembly Checkpoint ◽

Polar Body ◽

Germinal Vesicle ◽

Spindle Assembly ◽

Mouse Oocyte ◽

Immunofluorescent Staining ◽

Germinal Vesicle Breakdown ◽

Oocyte Meiosis ◽

Polar Body Extrusion ◽

Spread Analysis

Background. OLA1 is a member of the GTPase protein family, unlike other members, it can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown. Methods. In this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity. Results. Immunoﬂuorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.

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Vitamin C protects porcine oocytes from microcystin-LR toxicity during maturation

10.21203/rs.3.rs-41138/v1 ◽

2020 ◽

Author(s):

Xue Zhang ◽

Changyin Zhou ◽

Weijian Li ◽

Juan Li ◽

Wangjun Wu ◽

...

Keyword(s):

Dna Damage ◽

Vitamin C ◽

Actin Polymerization ◽

Polar Body ◽

Cumulus Cell ◽

Human Reproduction ◽

Secondary Products ◽

Oocyte Meiosis ◽

First Polar Body ◽

The Impact

Abstract Background: Microcystin-leucine arginine (MC-LR) is the most toxic cyanotoxin found in water bodies. Microcystins are produced as secondary products of cyanobacteria metabolism. They have a stable structure, and can bioaccumulate in living organisms. Humans and livestock who drink fresh water containing MC-LR can be poisoned. However, few studies have reported the effects of MC-LR exposure on livestock or human reproduction.Results: We used porcine oocytes as the model to explore the effects of MC-LR on oocyte maturation, and studied the impact of vitamin C (VC) administration on MC-LR‐induced meiosis defects. Exposure to MC-LR significantly restricted cumulus cell expansion and decreased first polar body extrusion. Further studies showed that MC-LR exposure led to meiosis arrest by disturbing cytoskeleton dynamics with MC-LR exposed oocytes displaying aberrant spindle organization, low levels of acetylate α‐tubulin, and disturbed actin polymerization. Additionally, MC-LR exposure impaired cytoplasmic maturation by disturbing mitochondria distribution. Moreover, MC-LR also produced abnormal epigenetic modifications, and induced high levels of oxidative stress and DNA damage. The administration of VC provided partial protection from all of the defects observed in oocytes exposed to MC-LR. Conclusions: These results demonstrate that MC-LR has a toxic effect on oocyte meiosis through the generation of excessive ROS levels and DNA damage. Supplementation of VC was able to protect against MC-LR-induced oocyte damage and represents a potential therapeutic strategy to improve the quality of MC-LR-exposed oocytes.

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Exposure to Copper Compromises the Maturational Competency of Porcine Oocytes by Impairing Mitochondrial Function

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.678665 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jingyue Chen ◽

Zhaokang Cui ◽

Yawei Qiu ◽

Xingxing Zhang ◽

Fang Chen ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Dna Damage ◽

Reactive Oxygen ◽

Oocyte Quality ◽

Actin Dynamics ◽

Oxygen Species ◽

Cortical Granules ◽

Porcine Oocyte ◽

Mitochondrial Distribution ◽

And Function

Copper (Cu) is an essential trace element for animals, and also an important nutritional component for the normal physiology and metabolism of animal reproductive systems. An excess or lack of Cu will directly or indirectly affect animal reproductive activities. However, the effect of Cu, in particular excessive Cu, on the reproductive performance of sows has not been studied. Here, we report that excessive Cu had negative effects on oocyte maturation and organelle functions. We showed that Cu exposure perturbed porcine oocyte meiotic maturation and impaired spindle/chromosome structure, resulting in a defective spindle assembly, as well as the abnormal distribution of actin dynamics and cortical granules. In addition, single-cell transcriptome analysis identified the target effectors of Cu actions in porcine oocytes, further demonstrating that Cu exposure affects the mitochondrial distribution and function, leading to the high levels of reactive oxygen species, DNA damage, and early apoptosis of porcine oocytes. These findings demonstrate that Cu exposure causes abnormalities in the mitochondrial distribution and function, resulting in the increased oxidative stress and levels of reactive oxygen species, DNA damage, and apoptosis, ultimately leading to a decreased porcine oocyte quality.

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132 Influence of oocyte retrieval methods and maturation media on invitro development of polar body extrusion in pig oocytes

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab132 ◽

2021 ◽

Vol 33 (2) ◽

pp. 174

Author(s):

K. M. Honneysett ◽

M. L. Mphaphathi ◽

A. M. Maqhashu ◽

E. C. Webb

Keyword(s):

Follicular Fluid ◽

Cell Layer ◽

Polar Body ◽

Cumulus Cell ◽

Oocyte Retrieval ◽

Cumulus Cells ◽

Reproductive Technology ◽

Maturation Stage ◽

Significant Difference ◽

Polar Body Extrusion

Oocyte recovery is a reproductive technology that can be done by using two techniques, aspiration and slicing. Invitro maturation (IVM) is an additional reproductive technology used to advance an oocyte to a maturation stage; thereafter, it may be used during IVF. The objectives of the present study were (1) to compare two different oocyte retrieval methods (aspiration and slicing) from pig ovaries on oocyte quality and quantity, and (2) to compare three different IVM media [NCSU 37, TCM-199, and modified porcine follicular fluid (mpFF=porcine follicular fluid+FSH+LH] on oocytes’ polar body extrusion. During aspiration, an 18G needle was attached to a 10-mL syringe and all visible follicles were aspirated. During slicing, a surgical blade was used to slice the ovaries held in mDPBS. Follicular fluid collected from both methods was assessed for the presence of oocytes with the aid of a microscope. The collected oocytes were then categorized as Grade A, B, or C: Grade A=oocytes with compacted, multilayered cumulus cells and a homogeneous ooplasm; Grade B=oocytes with a compact cumulus cell layer with homogeneous ooplasm; Grade C=oocytes with a less compact cumulus cell layer with irregular ooplasm containing dark granules. The IVM media were placed in a four-well multidish; thereafter Grades A and B oocytes were allocated per treatment groups and matured for 44h. The treatment means were compared using the Fisher’s protected t-test least significant difference. The results showed significant differences between the grades of oocytes (P<0.05) with Grade A and B oocytes accounting for 50.8% of total oocytes (193.8) for aspiration and 58.7% of total oocytes (488.6) for slicing. The oocytes polar body extrusion was recorded as 25.3, 84.2, and 73.8% for NCSU 37 (P<0.05) and TCM-199 and mpFF respectively (P>0.05). In conclusion, the slicing method proved to be better than aspiration with regards to the retrieval of Grades A and B oocytes as well as the total number of oocytes retrieved. The TCM-199 and mpFF media had a higher percentage of oocytes with polar body extrusion than NCSU 37.

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