MPF与MAPK在生殖细胞减数分裂中相互作用研究进展

许多研究显示生殖细胞减数分裂是由MPF调控的,MAPK对细胞增殖、细胞分裂和细胞周期调控同样起到关键性作用.以前都是将MPF与MAPK作为两种行使不同功能的物质,分别进行研究,但经过近年的研究发现,MPF与MAPK在卵母细胞成熟中有一定的关系,不能将两者绝对割裂开.文章就生殖细胞减数分裂调节机制研究中有关MPF与MAPK的相互关系进行了综述.     

哺乳动物卵母细胞孤雌激活研究进展

孤雌激活是指处于第2次减数分裂中期卵母细胞不经过雄性配子的作用,而在某些理化因素的刺激下恢复并完成减数分裂,进行有丝分裂发育到胚胎的过程.卵母细胞的激活是以Ca2+为第二信使,成熟促进因子(MPF)、细胞分裂素活化蛋白激酶(MAPK)、细胞静止因子(CSF)等细胞因子综合作用的结果.卵母细胞激活是核移植的关键技术,也是研究哺乳动物受精机制及发育机理的有效方法.现介绍卵母细胞孤雌激活的机制以及卵母细胞孤雌生殖激活方法.     

锌对哺乳动物卵母细胞质量的影响及其作用途径

卵母细胞质量直接影响受精率、早期胚胎存活,甚至成年后的疾病。锌是动物必需微量元素之一,是迄今为止发现的动物必需微量元素中功能最多的一种,近年来科学家们发现其在卵母细胞质量调控上发挥了重要作用。研究表明,锌可通过影响成熟促进因子(MPF)活性和丝裂原活化蛋白激酶(MAPK)信号通路影响卵母细胞减数分裂,通过谷胱甘肽影响卵母细胞抗氧化功能,并能通过影响组蛋白和DNA甲基化水平等途径影响动物卵母细胞质量。本文就锌对哺乳动物卵母细胞质量的影响及其作用途径作一综述。     

蛋白激酶C在哺乳动物卵母细胞第1次减数分裂成熟过程中的调节作用

蛋白激酶C（PKC）是一个广泛分布在真核细胞中的丝氨酸/苏氨酸蛋白激酶家族。它在卵母细胞的生发泡破裂（GVBD）、染色体凝集、MⅠ期纺锤体组装和第一极体排放等过程中起着重要的调节作用。PKC的活性变化调节着GVBD的发生，GVBD标志着第1次减数分裂的启动。PKC活性在卵母细胞成熟过程中逐渐升高，在第1次减数分裂中/后期转变时活性下降，使卵母细胞得以释放出第一极体，至此卵母细胞完成第1次减数分裂进入第2次减数分裂。作者就PKC在卵母细胞第1次减数分裂成熟过程中的作用综述如下。     

哺乳动物卵母细胞发育机理的研究进展

卵母细胞的成熟过程是一个复杂的减数分裂过程，许多分子参与了这个过程的调控，本文就卵母细胞成熟抑制因子（OMI）、成熟促进因子（MPF）、生长分化因子（GDF）、微管辅助蛋白（MAP）、激素、Ca^2 及钙调素等对卵母细胞发育调控机理作一综述。     

甘氨酸对猪卵母细胞体外成熟质量及卵丘细胞的影响

本实验旨在研究甘氨酸对卵丘细胞及卵母细胞体外成熟质量的影响,优化猪卵母细胞体外成熟(IVM)培养体系。在猪卵母细胞体外成熟培养液中添加6 mmol/L甘氨酸,体外培养至44 h后,通过倒置显微镜测量卵丘细胞扩散直径,结合流式细胞术与qPCR检测卵丘细胞凋亡情况;进一步检测成熟卵母细胞中谷胱甘肽(GSH)、细胞成熟促进因子(MPF)和丝裂原活化蛋白激酶(MAPK)水平及细胞成熟相关基因表达。结果表明:甘氨酸能显著提高卵丘细胞扩散直径,减少卵丘细胞的总凋亡率,并能显著提高卵丘细胞中Bcl-2的mRNA表达;甘氨酸能显著增加成熟卵母细胞中的GSH、MPF和MAPK水平以及成熟相关基因BMP15、GDF9、CyclinB1、CDK1、C-MOS的mRNA表达。综上可知,甘氨酸可提高猪卵母细胞体外成熟质量,降低卵丘细胞凋亡率。     

猪卵母细胞体外成熟过程中p90rsk未被MAPK激活

从屠宰场获得的初情期母猪卵巢中的卵母细胞 ,在修改后的 TCM199中进行体外成熟培养。在卵母细胞发育的一定时期取样 ,利用 SDS- PAGE蛋白质电泳和免疫印迹技术 ,研究了 MAPK(丝裂原激活蛋白激酶 ,又称 ERK)和p90 rsk (ribosomal S6 kinase)对猪卵母细胞成熟的调节。结果表明 ,修改后的 TCM199可以满足猪卵母细胞成熟的需要 ,但猪卵母细胞在体外成熟过程中 ,p90 rsk未随 MAPK激活而激活 ,说明维持猪卵母细胞的 M 阻滞与 MAPK有关 ,而与 p90 rsk无关。     

谷胱甘肽和活性氧对哺乳动物配子作用的研究进展

文章综述了近年来关于谷胱甘肽对哺乳动物精子、卵母细胞和胚胎早期发育的作用及活性氧对精子功能的影响。在雄性和雌性配子中,谷胱甘肽起着保护精子和卵母细胞免受氧化损害的作用。在卵母细胞减数分裂中谷胱甘肽具有维持纺锤体形态的作用,受精后,巯基在精核的形成中起着积极的作用,并促进早期胚胎发育到囊胚阶段。在卵母细胞成熟过程中,谷胱甘肽的浓度发生着变化。它的合成受促性腺激素的调节,在精子的发生过程中随着精子的成熟,谷胱甘肽的浓度逐渐降低,但活性氧对精子功能也有多方面的作用。文章对卵丘细胞中小分子巯基化合物在谷胱甘肽合成中的重要作用也进行了综述。     

微丝调控卵母细胞减数分裂成熟的分子机制研究进展

卵母细胞在与精子融合形成合子前会经历2轮减数分裂。与有丝分裂不同的是,2次减数分裂都是不对称的,最终会产生1个大体积的具有全能性单倍体卵母细胞和2个小体积的注定退化的极体。肌动蛋白丝作为卵母细胞中的细胞骨架,与分裂过程中的囊泡转运、细胞核定位、纺锤体迁移与锚定、极体排出和染色体分离等生物学事件存在重要联系。本文以哺乳动物为模型,总结了肌动蛋白在卵母细胞减数分裂成熟过程中的重要调节机制与信号通路,以期为进一步研究卵母细胞成熟过程的调控机制提供参考。     

C型钠肽和生长分化因子9预处理对绵羊体外成熟卵母细胞质量和发育能力的影响

为探讨生长分化因子9(growth differentiation factor 9,GDF9)和骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)在协同C型钠肽(C-type natriuretic peptide, CNP)调节卵母细胞减数分裂进程中的作用，提高卵母细胞体外成熟的质量，试验基于CNP预处理的“两步法”体外成熟(in vitro maturation, IVM)体系，研究了预处理阶段添加GDF9和BMP15在调节绵羊卵母细胞减数分裂进程和胞质成熟事件中的作用，并探讨CNP和GDF9联合预处理后对卵母细胞体外成熟质量及发育能力的影响。结果显示，GDF9可显著提高卵丘细胞中CNP受体NPR2基因的表达，从而增加CNP阻滞绵羊卵母细胞减数分裂的效率，而BMP15无此作用。预处理阶段联合添加CNP和GDF9能改善卵丘细胞功能，并且在CNP的作用基础上进一步增加卵母细胞线粒体的活性和数量、GSH含量，并降低ROS水平，从而提高卵母细胞体外成熟后的质量以及体外受精后的发育效率，卵裂率和囊胚率得到显著增加。研究结果不仅表明GDF9能强化CN...     

Inhibition of mitogen activated protein kinase activity induces parthenogenetic activation and increases cyclin B accumulation during porcine oocyte maturation

The inhibition of mitogen activated protein kinase (MAPK) activation during porcine oocyte maturation leads to decreased maturation promoting factor (MPF) activity and to the induction of parthenogenetic activation. In the present study, in order to analyze the mechanism underlying the suppression of MPF activity in MAPK-inhibited porcine oocytes, we injected mRNA of SASA-MEK, a dominant negative MAPK kinase, or antisense RNA of c-mos, a MAPK kinase kinase, into immature porcine oocyte cytoplasm. The injection of SASA-MEK mRNA or c-mos antisense RNA inhibited the MAPK activity partially or completely, respectively, decreased the MPF activity slightly or significantly, respectively, and induced parthenogenetic activation in 17.1% or 96.6% of mature oocytes, respectively, although no parthenogenetic activation was observed in the control oocytes. Immunoblotting experiments revealed that cyclin B accumulation in these MAPK-suppressed porcine oocytes was increased significantly after 50 h of culture and that a considerable amount of MPF was converted into inactive pre-MPF by hyperphosphorylation. These results indicate that the inhibition of MAPK activity in porcine oocytes did not promote cyclin B degradation but rather suppressed it; also the decrease in MPF activity in MAPK-suppressed porcine oocytes correlated with the conversion of active MPF into inactive pre-MPF.     

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of 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.     

PI3-kinase and mitogen-activated protein kinase in cumulus cells mediate EGF-induced meiotic resumption of porcine oocyte

Previous studies have shown that epidermal growth factor (EGF) has the ability to promote in vitro cultured porcine oocyte maturation. However, little is known about the detailed downstream events in EGF-induced meiotic resumption. We designed this study to determine the relationship of EGF, EGFR, phosphatidylinositol 3-kinase (PI3-kinase), MAPK, and germinal vesicle breakdown (GVBD) during oocyte maturation. Our results showed that GVBD in cumulus-enclosed oocytes (CEOs) but not in denuded oocytes (DOs) was induced by EGF in a dose-dependent manner, which indicated that cumulus cells but not oocyte itself were the main target for EGF-induced meiotic resumption. Furthermore, we found that MAPK in cumulus cells rather than in oocyte was activated immediately after EGF administration. To explore whether EGF exerts its functions through MAPK pathway, the activities of EGF receptor (EGFR) and MAPK were inhibited by employing AG1478 and U0126, respectively. Inhibition of MAPK blocked EGF-induced GVBD, whereas inhibition of EGFR prevented MAPK activation. Both AG1478 and U0126 could lead to the failure of EGF-induced GVBD singly. Notably, we found that LY294002, a specific inhibitor of PI3-kinase, effectively inhibited EGF-induced MAPK activation as well as subsequent oocyte meiotic resumption and this inhibition could not be reversed by adding additional EGF. Thus, PI3-kinase-induced MAPK activation in cumulus cells mediated EGF-induced meiotic resumption in porcine CEOs. Together, this study provides evidences demonstrating a linear relationship of EGF/EGFR, PI3-kinase, MAPK and GVBD and presents a relatively definitive mechanism of EGF-induced meiotic resumption of porcine oocyte.     

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus‐oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.     

Effect of EGF on expression and localization of maturation‐promoting factor,mitogen‐activated protein kinase,p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes

This study aimed to investigate the localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus–oocyte complexes. Cumulus–oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34^cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.     

The localization and function of p38α mitogen‐activated protein kinase in rat oocytes

P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.     

Adiponectin enhances in vitro development of swine embryos

Recent studies have shown that factors from adipose tissue influence and regulate the reproductive system. Hormones such as leptin and resistin are now known to regulate several reproductive processes. Adiponectin is the most abundant protein secreted by adipose tissue, and its circulating concentration is inversely related to adiposity and body mass index. Little is known about the involvement of adiponectin in reproduction. In the present study, the effect of recombinant adiponectin on the meiotic maturation and early embryo development in vitro was investigated, using porcine oocytes. Adiponectin receptors, AdipoR1 and AdipoR2, were found to be expressed in porcine oocytes and cumulus cells of both small and large follicles. Both AdipoR1 and AdipoR2 were immunolocalized to cumulus-oocyte complexes (COCs), oocytes, and early developing embryos. When included in oocyte maturation medium for 46 h, adiponectin significantly decreased the frequency of meiotic immature oocytes derived from large follicles (3-6 mm) but not from small follicles (<3mm). From studies of oocytes matured in the presence of adiponectin and mitogen-activated protein kinase (MAPK) pathway inhibitors MEK1 (PD98059), MEK1/2 (U0126), and p38MAPK (SB203580) it was concluded that adiponectin enhances oocyte maturation thought the p38MAPK pathway. Finally, a superior rate of embryo development to the blastocyst stage was achieved by embryos cultured in the presence of adiponectin. These results indicate that adiponectin has a positive effect on the meiotic maturation and in vitro embryo development of porcine oocytes and suggests a physiological role for this adipokine in early development in mammals.