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Haoya Chang Huanhuan Chen Lei Zhang Yile Wang Xiaogang Xie Yong Zhang Fusheng Quan 《Molecular reproduction and development》2019,86(11):1603-1614
As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation. 相似文献
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Oogenesis is one of the first processes to fail during aging. In women, most oocytes cannot successfully complete meiotic divisions already during the fourth decade of life. Studies of the nematode Caenorhabditis elegans have uncovered conserved genetic pathways that control lifespan, but our knowledge regarding reproductive aging in worms and humans is limited. Specifically, little is known about germline internal signals that dictate the oogonial biological clock. Here, we report a thorough characterization of the changes in the worm germline during aging. We found that shortly after ovulation halts, germline proliferation declines, while apoptosis continues, leading to a gradual reduction in germ cell numbers. In late aging stages, we observed that meiotic progression is disturbed and crossover designation and DNA double‐strand break repair decrease. In addition, we detected a decline in the quality of mature oocytes during aging, as reflected by decreasing size and elongation of interhomolog distance, a phenotype also observed in human oocytes. Many of these altered processes were previously attributed to MAPK signaling variations in young worms. In support of this, we observed changes in activation dynamics of MPK‐1 during aging. We therefore tested the hypothesis that MAPK controls oocyte quality in aged worms using both genetic and pharmacological tools. We found that in mutants with high levels of activated MPK‐1, oocyte quality deteriorates more rapidly than in wild‐type worms, whereas reduction of MPK‐1 levels enhances quality. Thus, our data suggest that MAPK signaling controls germline aging and could be used to attenuate the rate of oogenesis quality decline. 相似文献
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Vitale AM Calvert ME Mallavarapu M Yurttas P Perlin J Herr J Coonrod S 《Molecular reproduction and development》2007,74(5):608-616
In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage. 相似文献
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Qi Zhang JianXiu Hao BoWen Liu YingChun Ouyang JiaNi Guo MingZhe Dong ZhenBo Wang Fei Gao YuanQing Yao 《Cell proliferation》2023,56(3)
Maternal ageing is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age‐related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aged oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. We cultivated endometrial mesenchymal stem cells (EnMSCs) from aged mice and extracted mitochondria from EnMSCs. To improve the quality of oocytes, GV oocytes were supplemented with mitochondria via microinjection. And MII oocytes from aged mice were fertilized by intracytoplasmic sperm injection (ICSI), combining EnMSCs'' mitochondrial microinjection. In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aged oocytes. Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aged mice after ICSI. We also found that the birth rate of mitochondria‐injected ageing oocytes was significantly increased after embryo transplantation. Our study demonstrates that supplementation with EnMSC‐derived mitochondria can improve the quality of oocytes and promote embryo development in ageing mice, which might provide a prospective strategy for clinical treatment.In this study, we chose endometrial mesenchymal stem cells (EnMSCs) as the sources of mitochondria. We isolated the EnMSCs from 10‐month‐old mice and then extracted the mitochondria of EnMSCs. Then, the GV oocytes and MII oocytes from aged mice were injected with mitochondria. We found that mitochondria derived from EnMSCs could significantly improve the quality of oocytes, promote the embryonic development and improve the birth rates of aged mice. 相似文献
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Chikara Kubota Xiangzhong Yang Andras Dinnyes Junichi Todoroki Hiroshi Yamakuchi Kazunori Mizoshita Satoru Inohae Norio Tabara 《Molecular reproduction and development》1998,51(3):281-286
Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc. 相似文献
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Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed. 相似文献
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排卵后老化卵母细胞的染色体形态变化 总被引:1,自引:0,他引:1
小鼠排卵后的卵母细胞停滞在MⅡ期, 如果此时的卵母细胞未能及时受精, 随着在输卵管中停留时间的延长, 卵母细胞会逐渐发生老化。这种卵母细胞的老化会导致包括人在内的哺乳动物的胚胎发育异常, 所以很有必要研究排卵后卵母细胞的老化机理。本实验主要研究小鼠排卵后的卵母细胞在体内老化过程中的染色体形态变化, 发现随着老化时间的延长, 有更高比例(65%, hCG后34 h)的卵母细胞的染色体呈不对称和松散的状态, 进一步研究表明, 这种染色体的变化可能与H3K14、H4K16的乙酰化升高, 及H3K9的甲基化降低有关。 相似文献
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Pierre G. Adenot Maria S. Szllsi Patrick Chesn Sylvie Chastant Jean-Paul Renard 《Molecular reproduction and development》1997,46(3):325-336
The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22–26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and micro-tubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro-tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997. © 1997 Wiley-Liss, Inc. 相似文献
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Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes
as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental
potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically
fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin
with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct
fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes
were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell
embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation.
This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of
camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes. 相似文献
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Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts. 相似文献
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Anelise dos Santos Mendon?a Ana Luíza Silva Guimar?es Naiara Milagres Augusto da Silva Alexandre Rodrigues Caetano Margot Alves Nunes Dode Maurício Machaim Franco 《PloS one》2015,10(10)
DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP), specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5%) than MII oocytes (34.6%) (p = 0.039); spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001). Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results may contribute to our understanding of the reprogramming of imprinted genes during bovine oogenesis. 相似文献
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Qian‐Nan Li Jun‐Yu Ma Wen‐Bo Liu Tie‐Gang Meng Feng Wang Yi Hou Heide Schatten Qing‐Yuan Sun Xiang‐Hong Ou 《Molecular reproduction and development》2020,87(7):800-807
The genome methylation is globally erased in early fetal germ cells, and it is gradually re‐established during gametogenesis. The expression of some imprinted genes is regulated by the methylation status of CpG islands, while the exact time of DNA methylation establishment near maternal imprinted genes during oocyte growth is not well known. Here, growing oocytes were divided into three groups based on follicle diameters including the S‐group (60–100 μm), M‐group (100–140 μm), and L‐group (140–180 μm). The fully grown germinal vesicle (GV)‐stage and metaphase II (M2)‐stage mature oocytes were also collected. These oocytes were used for single‐cell bisulfite sequencing to detect the methylation status of CpG islands near imprinted genes on chromosome 7. The results showed that the CpG islands near Ndn, Magel2, Mkrn3, Peg12, and Igf2 were completely unmethylated, but those of Peg3, Snrpn, and Kcnq1ot1 were hypermethylated in MII‐stage oocytes. The methylation of CpG islands near different maternal imprinted genes occurred asynchronously, being completed in later‐stage growing oocytes, fully grown GV oocytes, and mature MII‐stage oocytes, respectively. These results show that CpG islands near some maternally imprinted genes are not necessarily methylated, and that the establishment of methylation of other maternally imprinted genes is completed at different stages of oocyte growth, providing a novel understanding of the establishment of maternally imprinted genes in oocytes. 相似文献
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Ahmed Gad Lucie Nemcova Matej Murin Jiri Kanka Jozef Laurincik Michal Benc Lazo Pendovski Radek Prochazka 《Molecular reproduction and development》2019,86(4):426-439
Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte‐specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing. Oocytes were aspirated from large (LO; 3–6 mm) or small (SO; 1.5–1.9 mm) follicles and tested for developmental competence and chromatin configurations. Small RNA libraries were constructed from both groups and then sequenced in an Illumina NextSeq. 500. Oocytes from the LO group exhibited higher developmental competence and different chromatin configuration compared with oocytes from the SO group. In total, 167 and 162 known miRNAs were detected in the LO and SO groups, respectively. MiR‐205, miR‐16, miR‐148a‐3p, and miR‐125b were among the top 10 highly expressed miRNAs in both groups. Eight miRNAs were differentially expressed (DE) between both groups. Target gene prediction and pathway analysis revealed 46 pathways that were enriched with miRNA‐target genes. The oocyte meiosis pathway and signaling pathways including FoxO, PI3K‐Akt, and cAMP were predictably targeted by DE miRNAs. These results give more insights into the potential role of miRNAs in regulating the oocyte development. 相似文献