哺乳动物卵母细胞凋亡机制研究进展

细胞凋亡是细胞程序性死亡过程,在生殖细胞的发生过程中具有重要作用。哺乳动物卵母细胞在生长发育过程中,除排卵受精的成熟卵母细胞外,其他卵母细胞在卵泡发育的不同阶段凋亡。作者综述了卵母细胞的发育与细胞凋亡、细胞凋亡的主要参与分子及凋亡途径等研究进展,旨在为进一步研究哺乳动物卵母细胞的生长、发育和排卵机制提供帮助。     

Role of cell-death ligand-receptor system of granulosa cells in selective follicular atresia in porcine ovary

In the mammalian ovary, more than 99% of follicles degenerate without ovulation and few oocytes ovulate and succeed to the next generation. Granulosa cell apoptosis plays a critical role in this process, follicular atresia. However, the molecular mechanisms responsible for the regulation of granulosa cell apoptosis have not been clarified. Death ligand and receptor systems are major apoptosis-inducing factors. This review describes the granulosa cell apoptosis via death ligand and receptor systems during follicular atresia in the porcine ovary.     

miRNA在哺乳动物配子发生中的作用研究进展

哺乳动物配子发生的基因表达调控包括编码基因的阶段特异性表达调控及非编码基因的转录和转录后水平调控。微小RNA(microRNA, miRNA)作为一类小的非编码RNA, 通过识别靶基因非翻译区的结合位点, 导致mRNA降解或者蛋白质翻译抑制, 从而在转录后水平发挥调控作用。近年来, miRNA在哺乳动物生殖活动中的作用逐渐被揭示, 越来越多的研究表明, miRNA在哺乳动物精子发生、精子成熟、卵母细胞成熟、卵泡发育及早期胚胎发育等过程中都发挥着重要的调节作用, 其可通过调节支持细胞的增殖和凋亡或精原细胞、精母细胞及精细胞的细胞周期进程, 在精子发生的不同阶段发挥间接或直接调控作用, 也可通过调节卵母细胞、卵丘细胞以及颗粒细胞的增殖、凋亡、激素合成和细胞间作用, 对卵母细胞的发育和成熟过程进行调控。作者主要介绍了在哺乳动物配子发生过程中, miRNA的细胞和阶段特异性表达及其对靶基因的调节作用, 以期为深入研究哺乳动物配子发生的调控机制提供参考。     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.     

生长激素对卵巢功能的调节

人们一般认为生长激素 ( GH)仅对动物生长发育起重要的促进作用 ,而最近的研究发现 GH也参与了雌性动物生殖的诸多过程 ,是重要的繁殖调节因子之一。GH可促进早期卵泡的发生 ,阻止卵泡的闭锁 ,加快卵母细胞细胞核和质的成熟 ,同时增强卵泡对促性腺激素的敏感性和减少卵泡细胞的凋亡利于排卵。此外 ,GH通过 IGF-1依赖和不依赖两种机制影响类固醇激素的合成。尽管生长激素对卵巢功能调节的研究取得了一些进展 ,但是生长因子和代谢类激素对动物繁殖的影响有待进一步研究     

The role of forskolin as a lipolytic stimulator during in vitro oocyte maturation and the in vitro embryo production of livestock

Cryopreservation is a modern technique which assists in the preservation of genetic material from oocytes and embryos for a long time. However, elevated vulnerability to cryopreservation due to the large accumulation of intracellular lipids within oocytes or embryos avoids success of this method. These lipids remain the main crucial factor limiting survival rates of oocytes and embryos after thawing. Lipid ingathering in the oocyte cytoplasm augments lipid peroxidation (LPO) and oxidative stress increases the apoptosis process, declines the viability after thawing, declines cytoskeleton actin filament injuries, lowers the blastocyst rates and reduces cryotolerance in the early stages of embryo development. There have been several attempts to reduce the ingathering of intracellular lipids in oocytes or embryos during the cryopreservation process, in that way enhancing the competence of cryopreserved oocytes or embryos and increasing their viability. One of the most applied agents for chemical delipidation is forskolin. Forskolin exhibited a possible part in improving the oocytes cryopreservation through stimulating cyclic adenosine monophosphate (cAMP) production. The main purpose of cAMP modulation is to provide energy to sustain the mammalian oocytes´ meiotic arrest. The purpose of the existing article is to assess and offer more evidence concerning the forskolin utilization as a modulator of cAMP during the cryopreservation of oocytes and its influence on meiosis completion and the reorganization of cytoplasm, which are prerequisites for the development of oocytes in addition to the contribution to fertilization and subsequently, the development of embryos.     

miRNA调节卵泡颗粒细胞凋亡及其机制的研究进展

微小核糖核酸(microRNA,miRNA)是一类内源性非编码RNA,具有广泛的基因表达调控作用,可以在转录后水平通过影响靶基因来调控相应蛋白质的表达,进而调节细胞的生命活动。miRNA在哺乳动物卵泡颗粒细胞中表达,并调控颗粒细胞的凋亡。颗粒细胞作为卵巢卵泡中数量最多的细胞群,在卵泡发育过程中起着至关重要的作用,不仅为卵母细胞提供营养物质,还调控其发育和成熟。颗粒细胞凋亡是导致卵泡闭锁的重要原因,影响卵泡的数量和质量从而影响雌性动物的繁殖性能。颗粒细胞凋亡过程受多种因素的调控。文章简述了miRNA对卵巢颗粒细胞凋亡的调控作用及其机制,其中包括miRNA通过调控激素分泌和细胞凋亡相关因子的表达进而调节颗粒细胞的凋亡,miRNA对颗粒细胞凋亡相关信号通路的影响,miRNA调控颗粒细胞凋亡导致的卵巢相关疾病,并总结了对颗粒细胞凋亡有调控作用的miRNA,以及miRNA在疾病诊断和治疗中的潜在作用,以期为后续相关卵巢疾病的发病机制和治疗方案研究,以及提高雌性哺乳动物生殖性能提供指导和参考。     

Bmp/Smad信号通路及其在哺乳动物卵巢发育中的作用

开展绵羊多羔性状基因的研究对于揭示其分子调控机制和提高绵羊繁殖力具有重要意义。骨形态发生蛋白(BMPs)为转化生长因子-β(TGF-β)超家族成员,与哺乳动物繁殖活动密切相关。研究表明,BMPs可促使原始卵泡向次级卵泡转化,对哺乳动物卵巢颗粒细胞增殖、生殖激素的合成和分泌以及卵母细胞成熟和排卵等方面起重要调节作用,而BMPs发挥功能主要依赖于经典的Bmp/Smad信号通路。本文就Bmp/Smad信号通路成员的表达、对早期胚胎发育的影响以及在哺乳动物卵巢发育中的作用等方面的研究进行总结,以期为进一步研究BMPs及其信号通路的调控机制提供参考。     

Relationship between oocyte maturation and fertilization on zygotic diversity in swine

Two experiments were conducted to examine how oocyte maturation and fertilization influence zygotic diversity in swine. In the first experiment, the distribution of oocyte maturation was compared to that of zygotic development. Oocytes were aspirated from follicles of 31 gilts and classified into stages of meiosis. Zygotes were flushed from oviducts of 19 additional gilts and classified into stages of meiosis and fertilization. The second experiment examined whether the time from ovulation to fertilization was constant among all oocytes. To test this premise, four to six oocytes from follicles of 10 mated gilts were aspirated just before or during ovulation, stained and transferred back into the oviducts of these same gilts. Zygotes were recovered 10 h later to determine whether the first oocytes ovulated were the more developed zygotes and, conversely, whether the last oocytes to be ovulated represented the lesser developed contemporaries. The skewed (P less than .05) distribution of oocyte maturation was similar to that of zygotic development. Regression of the frequency distribution describing early oocyte maturation resulted in a line with a slope (.59) that was similar to the slope (.58) of the regressed distribution of zygotic development. Likewise, the order of ovulation and order of subsequent stages of zygotic development were similar. These data suggest that variation in zygotic development in swine was due to variability in oogenesis; the time from ovulation to fertilization appeared to be constant.     

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.     

hCG促进马属动物排卵的研究进展

马属动物属季节性单胎动物,从母马或母驴出现发情表现到排卵,持续3~13 d不等,该生理特征极大地影响其繁殖效率。hCG作为LH类似物不会受性激素反馈调节机制的制约,广泛应用于马属动物卵泡发育和排卵。从性激素在哺乳动物卵泡发育过程中的作用、马属动物主卵泡波与LH诱导的优势卵泡排卵、外源性激素对马属动物卵泡发育的影响、hCG的结构与功能、hCG在延长母马黄体期的应用和hCG在马属动物超数排卵中的应用6个方面内容阐述hCG促进马属动物排卵的研究进展。     

β-神经生长因子在家兔诱导排卵中的研究进展

家兔是刺激性排卵动物,需要性交刺激释放促性腺激素释放激素（GnRH）,引发促黄体生成素（LH）增加,从而诱导成熟卵母细胞的排卵,但其排卵机制尚未完全明晰。在家兔的人工授精中,由于缺少性交的刺激,肌注或阴道内输入GnRH及其类似物被用来诱导排卵,然而这些化合物通常会对家兔繁殖产生不利影响,因此需要一种新的方法来取代激素刺激。β-神经生长因子（β-NGF）是新发现的在雌性生殖生理学中发挥作用的最有前途的物质之一,β-NGF及其受体广泛存在于家兔生殖系统中并具有重要的生理作用。同时β-NGF本身就存在于动物的精液中,且很有可能是兔的一种天然促排卵因子,使用β-NGF或许能够减少激素或其他药物的使用。作者简述了β-NGF促进家兔排卵作用及研究现状,主要从β-NGF及其受体在家兔生殖系统中的表达、β-NGF系统在诱导家兔排卵中的物种特异性,以及β-NGF诱导家兔排卵的作用机制等方面进行分析,旨在为研究家兔刺激排卵机理提供思路与理论基础,并促进β-NGF在家兔辅助生殖技术中的应用。     

The combination treatment of cholesterol-loaded methyl-β-cyclodextrin and methyl-β-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO

Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-β-cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl-β-cyclodextrin (MβCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38–39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MβCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-β-cyclodextrin and methyl-β-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.     

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

Acceleration of follicular development by administration of vascular endothelial growth factor in cycling female rats

To address the role of follicular angiogenesis in the determination of ovulatory follicles and the effects of different vascular endothelial growth factor (VEGF) isoforms on follicular angiogenesis and development, mature female rats were treated with an angiogenic inhibitor (TNP-470), and also with VEGF 120 or 164 at different dosages (0.4, 0.8, 4.0 or 8.0 microg/kg body weight) for 3 days during the estrous cycle. Ovarian follicular angiogenesis, the population of large follicles and ovulation were examined. VEGF 120 (0.8 microg/kg) and 164 (8.0 microg/kg) treatments stimulated follicular angiogenesis in the theca interna layer, while TNP-470 treatment showed severe depression of follicular angiogenesis, and completely inhibited ovulation. After administration of VEGF 120 or 164, the number of healthy preovulatory follicles and ovulated oocytes increased significantly, concomitantly with a decrease in the number of atretic preovulatory follicles. The oocytes ovulated had normal fertilizability and developed to term with the same litter size as in the control rats. Our findings suggest that follicular angiogenesis may be a determinant of follicular development during the periovulatory phase, and that VEGF isoforms may play different important roles in regulating follicular angiogenesis.     

哺乳动物卵母细胞孤雌激活的研究

研究哺乳动物卵母细胞的激活及孤雌胚发育,对了解哺乳动物早期胚胎发育、深入细致地研究核移植技术和动物克隆技术、建立生产孤雌哺乳动物品系及加速家畜育种进程等均具有重要意义。本文主要从卵母细胞孤雌激活的机制及影响因素、不同动物卵母细胞的激活、提高卵母细胞孤雌激活效果的技术途径以及对孤雌激活的研究意义和发展前景等方面作了详尽论述,为同类研究提供参考。     

小鼠输卵管卵卵龄对体外受精影响初探

本文用昆白小鼠不同卵龄的卵子作体外受精试验，结果表明，新排出的卵细胞尚未成熟，排卵后１－２小时基本成熟，３－４小时充分成熟，５小时后虽有较高受精率，但受精卵继续培养１０小时，大部分退化。     

排卵不同阶段氯胺酮麻醉对昆明小鼠卵母细胞和早期胚胎的影响

主要研究排卵不同阶段氯胺酮III期麻醉深度对昆明小鼠卵母细胞和早期胚胎的影响。结果表明:排卵前麻醉(150mg/kg体重)造成卵母细胞和早期胚胎数量的极显著下降(P<0.01),而排卵期间麻醉及排卵后麻醉对排卵及早期胚胎数量影响不显著(P>0.05),排卵不同阶段麻醉均造成一定数量异常卵母细胞和非正常胚胎出现。因此,排卵期间麻醉小鼠相比排卵前及排卵后麻醉小鼠对卵母细胞和早期胚胎的影响最小,但仍有可能造成卵母细胞异常、胚胎畸形的发生。