卵泡分化排卵和黄体发生的分子基础

本文综述了近二十年来在雌性生殖研究领域中五个方面的研究进展。（1）原始卵泡的启动生长和分化：原始卵泡生长启动可能受卵巢内在因子,像生长因子的调控,而不是人所想象的FSH;启动生长的卵泡受生长因子,FSH,激活素,孤儿受体和其他卵巢旁分泌因子调控,并发展成“优势卵泡”;（2）优势卵泡和闭锁卵泡：FSH,激活素／抑制素,卵泡抑素和雌激素调节卵泡分化的趋向,它们的相互作用和平衡决定“优热卵泡”和“闭锁卵泡”的形成,其中雌激素在此起关键性作用;它一方面促进FSH对LH受体的诱导作用,增加GC上的LH受体数目;另一方面可促进GC芳香水酶的产生,使此卵泡进入良性循环,发展为优势卵泡;（3）两种细胞两种促性尕激素学说：在FSH和LH作用下,GC产生的孕酮可被膜细胞利用转化为雄激素;因此GC和TC相互作用是卵巢雌激素产生的前提;（4）排卵:GC主要产生tPA,而TC主要表达tPA的抑制因子PAI－1,在激素作用下,GC的tPA和TC的PAI－1相互作用,形成一个局部蛋白水解窗口,它对卵泡破裂起决定性作用;（5）黄体形成和萎缩：uPA和MMP／TIMP系统对黄体发生起重要作用;而tPA对黄体萎缩起关键作用。     

促性腺激素对小鼠腔前卵泡及其卵母细胞体外发育的影响研究

通过使用改良的TCM199无血清培养系统,在96孔细胞培养板中,每孔放入一个腔前卵泡等条件下,探索促性腺激素(FSH、LH)对小鼠腔前卵泡卵母细胞体外生长发育的影响,旨在摸索一套适合小鼠腔前卵泡体外生长发育的培养体系.结果表明:随着浓度的增加,卵泡和卵母细胞的直径都不同程度的有所增长,在卵泡培养的同期相比,卵泡增长值最大组为FSH400+LH200组,而卵母细胞增长值最大组却是FSH400组.从添加物上看,各个实验组与对照组之间,卵泡直径和卵母细胞直径增长值都存在显著性差异(P0.05).随着培养时间的延长,FSH400+LH200组卵泡存活率、成腔率表现为最高,分别为68.6%,47%.从卵泡发生生发泡破裂率和第一极体排出率来看,FSH400+LH200组发生生发泡破裂率高于其他组,为17.6%,但第一极体排出率却低于FSH400组.因此,改良的TCM199无血清基础培养液中联合添加400mIU/mL FSH和200mIU/ML LH效果优于单独添加组的效果.     

Germline stem cells and follicular renewal in the postnatal mammalian ovary

A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.     

抗氧化剂对小鼠腔前卵泡及其卵母细胞体外发育的影响研究

通过使用改良的TCM199无血清培养系统,在96孔细胞培养板中,每孔放入一个腔前卵泡等条件下,探索添加物抗氧化剂维生素C对小鼠腔前卵泡卵母细胞体外生长发育的影响,旨在摸索一套适合小鼠腔前卵泡体外生长发育的培养体系.结果表明:随着浓度的增加,卵泡和卵母细胞的直径都不同程度地有所增长,其中50μg/m L浓度组在卵泡培养相同时间下,卵泡和卵母细胞的增长值相对于其他浓度组略大些,但各浓度组之间无论是卵泡直径增长值还是卵母细胞直径增长值都不存在显著性差异(P0.05).培养到第6天时,50和100μg/m L浓度组卵泡存活率最高,为59.5%,且50μg/m L浓度组第一极体排出率最高,为2.38%,但发生生发泡破裂率次于150μg/m L浓度组.可得结论:在改良的TCM199无血清培养系统中,添加50μg/m L维生素C有利于腔前卵泡卵母细胞的生长,而添加150μg/m L维生素C有利于生发泡破裂.     

Localization of orphan receptor TR3 mRNA in early developmental follicles in rat

Byin situ hybridization, the localization of orphan receptor TR3 mRNA has been observed in early developmental follicles. TR3 mRNA is first expressed in the ovarian interstitial cells on day 2 after birth, and then in granulosa cells (GC) in primary follicles on day 4. The expression level of TR3 mRNA in GC increases following the follicular development. Its higher expression can be observed in the outer layer of GC and inner layer of theca cells (TC) on day 6, where the cells present active proliferation and differentiation. The expression of TR3 is in an increasing manner until the large antral follicles on day 30. The mRNA is only expressed in the healthy, but not atretic follicles in adult rat ovaries. Injection of epidermal growth factor (EGF) has dramatically enhanced its expression in the early stage of developmental follicles. It is therefore suggested that TR3 may play a role in regulating growth and differentiation of ovarian somatic cells in the early stage, and its expression is regulated by EGF.     

白棘三列海胆生殖系统的组织学研究

从组织学方面研究了白棘三列海胆生殖系统的构造及其性腺发育分期．结果表明,白棘三列海胆的生殖腺为5列,由系膜悬挂于各生殖腔内．卵巢为滤泡型,在滤泡的边缘为卵原细胞和卵黄形成前期卵母细胞,而在滤泡的内部为卵黄形成初期卵母细胞、卵黄形成后期卵母细胞和成熟卵细胞,当卵母细胞内的卵黄完成积累后,经两次成熟分裂,分裂为成熟卵子．精巢也为滤泡型,精原细胞由滤泡壁原始的生殖细胞产生,进而依次发育为初级精母细胞、次级精母细胞、精细胞和精子．白棘三列海胆生殖腺发育周期可分为：恢复期、生长期、成熟前期和排放后期,通过解剖观察及组织切片显示,全年中除5月份之外,其余各月份均可进行白棘三列海胆的人工繁殖．     

虎纹捕鸟蛛卵巢发育的组织学

根据卵巢的组织学和形态特征分析,虎纹捕鸟蛛的卵巢为若干由生殖上皮向卵巢腔内突起的卵巢小体构成.在卵细胞发育过程中,一直有滋养细胞形成的托柄向其供应养分.卵巢发育可分为5个时期：形成期、生长期、成熟期、排卵期和恢复期.在生殖季节里可多次产卵,为多次产卵类型.卵巢的大小重量与蛛体重量成正相关,而成熟的蜘蛛性腺指数仅随卵细胞的发育而变化,不随蛛体大小改变.     

不同发育时期小鼠卵巢腔前卵泡的分布及形态学研究

通过石蜡切片-HE染色技术,系统研究小鼠生后不同发育时期即3,5,7,10,15,20,30,45 d卵巢的卵泡分布、形态结构及发育规律.研究结果表明,随着小鼠卵巢的发育,由最初的无皮髓质之分到皮髓明显可见,卵泡从只见聚集呈条索状到发育成原始卵泡、初级卵泡、次级卵泡,直至有腔的形成.从数量上看,原始卵泡逐渐减少,初次级卵泡逐渐增多,有腔卵泡数量也从无到有从少到多.卵巢由最初的无卵泡发育到有许多大小不等、处于不同发育时期的卵泡、大部分有腔样结构.在此发育过程中会伴有卵泡的闭锁.小鼠卵巢中各级腔前卵泡的分布具有区域性.     

中华绒螯蟹卵子发生中卵母细胞表面和滤泡细胞的超微结构

利用透射电镜技术观察了中华绒螯蟹卵子发生过程中卵母细胞表面和滤泡细胞超微结构的变化.结果显示:从卵原细胞期到卵黄大量合成时期,滤泡细胞逐渐向卵母细胞迁移,细胞膜向外伸出许多突起,胞质中的细胞器逐渐增多,它们与卵黄蛋白物质一同移入卵周隙.到卵黄合成晚期,滤泡细胞逐渐解体.与此同时卵母细胞表面在卵黄发生初期可见微胞饮小泡;卵黄大量发生期出现大量微绒毛;卵黄合成晚期,卵黄膜形成,卵黄膜上出现多条通道,这些结构为卵母细胞吸收来自滤泡细胞的卵黄蛋白物质提供了保障.     

短额负蝗卵子发生及卵母细胞凋亡的组织学观察

用组织学方法对短额负蝗卵子发生和卵母细胞凋亡进行了显微观察.根据卵母细胞的大小、形态、胞核的变化、卵黄物质的形成以及滤泡细胞的形态变化等特征,将短额负蝗卵子发生分为3个时期8个阶段,并描述了各个阶段卵母细胞和滤泡细胞的形态特征.卵子发生过程中卵母细胞凋亡现象主要发生在卵母细胞生长期,特征主要表现为滤泡细胞向卵母细胞质内分裂增殖,卵母细胞的胞质不均匀,出现许多空泡状物质.     

Role of plasminogen activators and inhibitors in reproduction

The recent progress in the studies on the role of local and directed fibrinolysis controlled by plasminogen activators (PAs) and regulated by their inhibitors (PAIs) in reproduction is summarized. Hormone-induced coordinated expression of tissue type PA (tPA) and PAI type-1 (PAI-1) in the ovary is involved in the processes of ovulation and luteal regression; increases in urokinase type PA (uPA) and PAI-1 activity in the early stage of luteinized follicles may be responsible for ovarian tissue remodeling and angiogenesis; the PA system has been found to play an important role in spermatogenesis in testis and modulation of sperm maturation in epididymis. PA and matrix matalloproteanase (MMP) and their respective inhibitors have also been identified in trophoblast and uterus. The targeted proteolytic activity generated by the two systems may play an essential role in the processes of the cyclic uterine angiogenesis, implantation and placentation as well as parturition.     

P53蛋白在三种爬行动物卵泡内的免疫组织化学定位

应用免疫组织化学ABC法对无蹼壁虎、赤链蛇、中华鳖的卵泡进行P53定位。结果发现：无蹼壁虎卵泡内少数颗粒细胞胞浆有阳性表达，而赤链蛇卵泡颗粒细胞胞浆均呈P53免疫反应强阳性，中华鳖的早期卵泡细胞呈P53阳性。由此推测P53蛋白在无蹼壁虎、赤链蛇卵泡中表达可能与颗粒细胞凋亡有关，在中华鳖卵泡高表达对早期卵泡的生长分化和原始卵泡的启动可能有重要的调控作用。     

大熊猫（Ailuropoda Melanoleuca）卵巢的年龄变化

对比观察了5只不同年龄大熊猫(包括青年、中年和老年)的卵巢皮质石蜡切片。可见皮质结构有较大的年龄变化。主要表现在:性成熟后,卵巢皮质的厚度随年龄的增长有减薄的趋势。成年的各级正常卵泡数量较少,初级和次级的更少;闭锁卵饱和间质腺较多,并呈现出随年龄增长而增多的动向。老年卵巢结构变化明显,皮质纤维化,卵泡全部消失,仅有较多囊泡,小动脉管壁增厚,管腔变小。老年卵巢的外形、体积和重量等方面与成年之间未见明显年龄变化。     

三疣梭子蟹第一次卵巢发育过程中卵母细胞和卵泡细胞超微结构的观察

通过透射电镜技术研究了东海三疣梭子蟹初产卵巢发育过程中卵巢的超微结构的变化.1)三疣梭子蟹初产卵巢发育过程中卵黄发生期可分为初期和后期;2)卵黄发生初期在7~9月间,此阶段卵黄生成以内源性合成为主,卵母细胞核物质外输明显,胞浆内大量的粗面内质网正在进行卵黄物质的内源性合成;3)卵黄发生后期在10月至第二年4月间,卵泡细胞与卵母细胞的结合阶段,卵母细胞借助卵泡细胞进行外源性卵黄物质合成,卵母细胞与卵泡细胞间的界线逐渐消失,部分卵泡细胞由于胞质大量输出仅见胞核,此时内源性卵黄合成仍然存在;4)排卵后,由于大量成熟卵细胞的排出,卵巢组织存在大量的基膜,残留成熟卵母细胞中的卵黄物质已经解体,卵黄颗粒自中央向边缘逐渐松散.     

Regulation of ovarian function by the matrix metalloproteinase system

In most organs of mammals, cyclic remodelling of tissues after morphogenesis is minimal; however, repro-ductive tissues of female animals including endometrium, mammary gland, ovarian follicle and corpus luteum un-dergo growth, maturation and involution at various stages in the reproductive cycle or lifespan of the animal. Recon-struction of the extracellular matrix (ECM) is required for the dynamic tissue reorganization characteristic of these tissues. The ECM consists of proteinaceous and nonpro-teinaceous molecules that provide the tissue-specific, ex-tracellular architecture to which cells attach. Furthermore, interaction of cellular receptors with proteins of the ECM can regulate cellular structure, second messenger genera-tion and gene expression. Maintenance of ECM homeo-stasis depends largely on coordinated action of matrix metalloproteinases (MMPs) and tissue inhibitors of met-alloproteinases (TIMPs)-- an important proteinase sys-tem responsible for degradating and remodelling of ECM[1]. MMPs/TIMPs have been recognized as the cru-cial role players in regulating follicular and luteal function for their extensive involvements in the cyclic changes of dynamic ovarian tissues. In recent years, literature that MMP system has important roles in ovary is accumulating. The focus of this review is on the effects of MMPs and their inhibitors, TIMPs on follicular growth, atresia, ovu-lation, luteal development, and luteolysis. Emphasis has been given to the recent progress in the new field when-ever possible.     

北京鸭腔上囊的研究Ⅸ.胚胎后期及胚后腔上囊的发育

对北京鸭26日胚、胚后3周龄、7周龄、10周龄、12周龄和16周龄的腔上囊进行了比较观察,结果表明北京鸭26日胚、胚后3周龄和12周龄是腔上囊及其淋巴上皮滤泡发育的3个代表性阶段。26日胚腔上囊组织结构已基本发育成熟。胚后3周龄腔上囊进入发育高峰。胚后12周龄腔上囊出现退化。综合分析光镜和电镜的观察结果,以及不同分化阶段淋巴细胞的数量统计,表明胚胎后期及胚后腔上囊发育过程中,分化早期的前淋巴母细胞和淋巴母细胞在细胞群中的优势逐渐被分化晚期的中淋巴细胞和分化成熟的小淋巴细胞的优势所代替。     

Mitochondrial functions on oocytes and preimplantation embryos

Oocyte quality has long been considered as a main limiting factor for in vitro fertilization (IVF). In the past decade, extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA (mtDNA) damages, chromosomal aneuploidies, the incidence of apoptosis, and changes in mitochondrial gene expression. All these dysfunctions may cause a high level of developmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species (ROS) that is closely associated with the oxidative energy production or calcium overload, which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.     

Sources of cumulus expansion enabling factor (CEEF) in porcine follicles

It was shown that expansion of porcine cumulus did not depend on oocyte-secreted factor(s), and it is therefore presumed that porcine CEEF may not be produced exclusively by the oocyte. In this experiment, we used mouse oocytectomized complexes (OOX), which were incapable of CEEF production, to assess the secretion of CEEF by evacuated zona, oocytes of different quality and somatic cells in the porcine follicles. The results showed that: (ⅰ) Evacuated zonae from both porcine and mouse oocytes did not produce CEEF. (ⅱ) Porcine oocytes of A, B and C types from 3—6 mm follicles were not significantly different in both production and activity of CEEF. (ⅲ) Both porcine OOX from 3—6 mm follicles and granulose cells from < 1 mm follicles secreted CEEF in a large quantity, independent of gonadotropins; mural granulose cells from 3—6 mm follicles, however, produced neglectable amount of CEEF. (ⅳ) The follicular fluid from 3—6 mm porcine follicles contained CEEF activity that was concentration-dependent, and thus it enabled cumulus expansion in 60% mouse OOX when used at 10% of concentration, but the expansion rate of mouse OOX decreased to 9% when the concentration was increased to 50%. (ⅴ) Mouse OOX cultured in porcine CEEF-containing M199 expanded only in the presence of gonadotropins, suggesting that the activity of porcine CEEF is hormone-dependent.     

热应激对 ICR 雌性小鼠超数排卵及卵泡发育的影响

摘要:目的 探讨短期热应激对小鼠超数排卵和卵泡发育的影响。 方法 取超数排卵处理后体质量近似的 7 ~ 8 周龄 ICR 雌性小鼠 20 只,随机分为对照组和热应激组,每组 10 只,饲养于屏障环境内。 热应激组小鼠放入 38 ℃ 的生化培养箱中,热应激处理 10 h,期间自由采食和饮水。 热应激结束后,将小鼠放回屏障设施内饲养,持续 6 d,6 d后,进行超数排卵处理并处死小鼠,获取小鼠输卵管内卵子以及卵巢。 结果 热应激组小鼠超排卵数高于对照组( P<0. 05) 。 对卵巢切片进行苏木素-伊红( HE)染色后,观察发现热应激组小鼠的总卵泡数和黄体数均明显高于对照组( P<0. 01) 。 热应激组小鼠的卵泡闭锁率高于对照组,但无明显差异( P> 0. 05) 。 结论 热应激严重影响小鼠的繁殖机能,尤其是卵泡的发育过程。