mTOR与卵泡发育

原始卵泡由单层扁平前颗粒细胞包围卵母细胞形成,是女性的基本生殖单位,其数目在胎儿期就已决定。随着年龄的增长,原始卵泡数量不断减少,其数目一定程度上代表了女性生育年限的长短。而哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)与细胞生长发育密切相关。在静止期卵母细胞与前颗粒细胞中,mTOR表达异常可导致细胞内蛋白合成增加,卵母细胞发育及颗粒细胞分裂、分化,使原始卵泡被激活,引发原始卵泡库过早耗竭,从而导致女性卵巢储备能力降低、生育年限缩短。近年来,mTOR及其介导的信号通路作为女性生育力保护的潜在靶点已成为生殖医学领域的研究热点。其对卵泡发育的调节作用,将为延缓女性卵巢衰老提供新思路。现从哺乳动物原始卵泡激活及其与mTOR信号网络的相关性等方面进行综述,并探讨其与女性不孕的关系。     

mTOR与卵泡发育

原始卵泡由单层扁平前颗粒细胞包围卵母细胞形成，是女性的基本生殖单位，其数目在胎儿期就已决定。随着年龄的增长，原始卵泡数量不断减少，其数目一定程度上代表了女性生育年限的长短。而哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）与细胞生长发育密切相关。在静止期卵母细胞与前颗粒细胞中，mTOR表达异常可导致细胞内蛋白合成增加，卵母细胞发育及颗粒细胞分裂、分化，使原始卵泡被激活，引发原始卵泡库过早耗竭，从而导致女性卵巢储备能力降低、生育年限缩短。近年来，mTOR及其介导的信号通路作为女性生育力保护的潜在靶点已成为生殖医学领域的研究热点。其对卵泡发育的调节作用，将为延缓女性卵巢衰老提供新思路。现从哺乳动物原始卵泡激活及其与mTOR信号网络的相关性等方面进行综述，并探讨其与女性不孕的关系。     

卵泡发育早期细胞因子对卵母细胞发育的调控

在卵泡内,卵母细胞及其周围颗粒细胞间的旁分泌作用对卵母细胞的发育和功能有重要作用.研究证实了源于颗粒细胞的酪氨酸激酶受体配体在卵母细胞生长过程中起重要作用.酪氨酸激酶受体配体和源于卵母细胞的生长分化因子-9以及骨生成蛋白-15间的相互作用表明卵母细胞和颗粒细胞在分子水平上存在复杂的作用.卵泡刺激素调控这些旁分泌作用并调节卵泡正常发育.促进卵母细胞发育的分子机制可应用于人的体外生长系统并改善生育.     

生长分化因子9在卵泡发育中的调控作用

生长分化因子9(GDF-9)是转化生长因子β(TGF-β)超家族成员之一,主要表达于卵母细胞,调节卵泡形成。GDF-9除了促进卵母细胞生长与分化外,还作为旁分泌因子促进颗粒细胞增生和卵丘膨胀,抑制颗粒细胞和卵泡膜细胞分化。综述了GDF-9的表达时间和定位,与垂体卵巢激素相互调节影响卵泡生成的生理作用,及在卵泡发育障碍性疾病发生中的可能机制。     

生长分化因子9在卵泡发育中的调控作用

生长分化因子9(GDF-9)是转化生长因子β(TGF-β)超家族成员之一,主要表达于卵母细胞,调节卵泡形成.GDF-9除了促进卵母细胞生长与分化外,还作为旁分泌因子促进颗粒细胞增生和卵丘膨胀,抑制颗粒细胞和卵泡膜细胞分化.综述了GDF-9的表达时间和定位,与垂体卵巢激素相互调节影响卵泡生成的生理作用,及在卵泡发育障碍性疾病发生中的可能机制.     

颗粒细胞在卵母细胞发育成熟中的作用

卵泡的发育是涉及多种细胞、多个阶段的复杂过程,其中颗粒细胞和卵母细胞是构成卵泡的重要细胞,卵泡的形成需要颗粒细胞与卵母细胞间密切的相互作用。在促性腺激素等多种激素和信号分子作用下,颗粒细胞增殖、分化,并通过间隙连接与周围的颗粒细胞、卵母细胞、膜细胞进行物质交换和信号传递,卵母细胞也可分泌多种因子反作用于颗粒细胞。颗粒细胞与卵母细胞间的相互作用涉及多条信号通路,这些信号通路间同样存在相互影响,决定卵泡的成熟或闭锁。当这些信号通路被过度激活或抑制时,可影响细胞因子的生成或传递,从而引起颗粒细胞或卵泡的凋亡,具体机制尚未明确。本文就卵泡的形成,颗粒细胞与卵母细胞、膜细胞在发育过程中的相互作用以及涉及的重要信号通路进行综述。     

人卵泡液蛋白质组学的研究进展

卵泡液是由卵母细胞、颗粒细胞和膜细胞等共同分泌的一种含多种蛋白质的生物液,其作为卵母细胞生长的微环境或培养基,直接影响到卵泡的发育和卵母细胞的成熟。过去十几年,对卵泡液蛋白质组学的研究主要集中于新蛋白筛选及疾病相关蛋白等的识别上。本文从卵泡液新蛋白的筛选、年龄相关生物学标志鉴定、确定卵泡成熟标志、疾病相关标志的筛选和体外受精（IVF）成功的标志等5方面介绍近年卵泡液蛋白质组学的相关研究。筛选和鉴定某些生殖和发育相关的关键蛋白,并可能成为卵泡液中的生物标志,可用于预测卵母细胞的质量、受精及胚胎发育潜能。     

人卵泡液蛋白质组学的研究进展

卵泡液是由卵母细胞、颗粒细胞和膜细胞等共同分泌的一种含多种蛋白质的生物液,其作为卵母细胞生长的微环境或培养基,直接影响到卵泡的发育和卵母细胞的成熟。过去十几年,对卵泡液蛋白质组学的研究主要集中于新蛋白筛选及疾病相关蛋白等的识别上。本文从卵泡液新蛋白的筛选、年龄相关生物学标志鉴定、确定卵泡成熟标志、疾病相关标志的筛选和体外受精(IVF)成功的标志等5方面介绍近年卵泡液蛋白质组学的相关研究。筛选和鉴定某些生殖和发育相关的关键蛋白,并可能成为卵泡液中的生物标志,可用于预测卵母细胞的质量、受精及胚胎发育潜能。     

缝隙连接和卵巢卵泡发育的关系研究进展

卵巢中的卵泡处于无血管的微环境中,因而缝隙连接介导卵母细胞和其周围体细胞(颗粒细胞和卵泡膜细胞)的代谢交换,传递内分泌和旁分泌的生长因子。卵巢卵泡的整个发育过程离不开细胞间的缝隙连接。连接蛋白Cx32、Cx37、Cx43、Cx45和Cx57参与卵泡的发育,其中Cx43和Cx37对卵巢卵泡的发育起了至关重要的作用。现就卵巢卵泡的发育和缝隙连接的关系进行综述。     

PCOS患者糖脂代谢特点及其相关细胞因子参与卵泡发育的研究现状北大核心CSCD

多囊卵巢综合征(polycystic ovary syndrome,PCOS)是常见的女性内分泌、代谢紊乱性疾病,其糖脂代谢异常与生殖功能障碍密切相关。卵巢是能量代谢活跃的器官,颗粒细胞的增殖,卵母细胞的发育成熟及排卵,都是耗能的过程,需要足够的能量供应。糖、脂类在颗粒细胞中被分解,通过缝隙连接为卵母细胞发育提供能量。PCOS患者糖脂代谢异常表现在卵巢颗粒细胞和卵泡液中信号分子及细胞因子、卵泡液中代谢产物的变化,影响卵泡发育及卵母细胞质量。本文就PCOS患者糖脂代谢特点及与糖脂代谢相关的细胞因子对卵泡发育和卵细胞质量的影响进行综述,为临床提供参考。     

Ultrastructural localisation of calcium deposits in the mouse ovary

Follicle-enclosed mouse oocytes contain numerous calcium deposits. The ultrastructural distribution of calcium deposits in the nuclei, mitochondria and cytoplasm of mouse oocytes and granulosa cells of primary, secondary and antral follicles was examined using the combined oxalate-pyroantimonate method. The mitochondria of oocytes from all types of follicles had the highest levels of calcium deposits of all oocyte compartments, with the exception of primary follicles, in which oocyte nuclei contained the same level of calcium deposits as the mitochondria. Calcium deposits in the cytoplasm of oocytes from primary follicles were significantly lower than those in the cytoplasm of oocytes from secondary and antral follicles. Calcium deposits in the cytoplasm of granulosa cells were significantly lower than calcium deposits in the mitochondria of granulosa cells and this difference persisted throughout all categories of follicles. Calcium deposits in the nuclei of granulosa cells did not differ from levels in the mitochondria in primary and secondary follicles. In contrast, the nuclei of granulosa cells from antral follicles had lower levels of calcium deposits than the mitochondria. The differences observed in calcium deposits in various cellular compartments in oocytes and granulosa cells in the follicles of ovaries of adult mice can be attributed to their acquisition of meiotic competence and follicular development.     

Expression of mRNAs encoding oestrogen receptor (ER) alpha and ERbeta, androgen receptor and progesterone receptor during gonadal and follicular development in the marsupial brushtail possum (Trichosurus vulpecula)

The objective of the present study was to determine which ovarian cells express mRNAs for oestrogen (ERalpha and ERbeta), androgen (AR) and progesterone (PR) receptors during ovarian and follicular development in the brushtail possum. Expression of ERalpha and/or ERbeta mRNA was observed from birth, initially in cells of the blastema, then in the medullary cords from Day 20. ERalpha was expressed in the oocytes and granulosa cells of secondary and antral follicles. Preovulatory follicles did not express ERalpha mRNA, although their oocytes were not examined for any gene. ERbeta mRNA was observed in oocytes at all follicular stages examined, but was not consistently observed in granulosa or theca cells. Expression of AR mRNA before Day 40 was very faint; thereafter, expression was observed in the medullary cords, peaking between Days 60 and 120. Oocytes, granulosa cells and theca of secondary and antral, but not preovulatory, follicles expressed AR mRNA. PR mRNA was expressed throughout the gonad by Day 20. Granulosa cells of some secondary and antral follicles and theca of antral follicles expressed PR mRNA. Thus, the expression of mRNAs encoding steroidogenic receptors in a time- and cell-specific manner supports a role for steroids in the process of ovarian follicular formation and growth.     

小鼠腔前卵泡体外培养方法的建立

目的建立可供毒理学研究的小鼠腔前卵泡体外培养方法。方法采用机械方法分离出生后12～14天(C57B1/6JxCBA/Ca)小鼠卵巢内直径为100～130μm的腔前卵泡,96孔板单个卵泡连续培养12天后诱导排卵16h,观察卵泡发育、激素分泌和卵子形成,并将体外卵母细胞的成熟情况(IVG)与体内卵母细胞的生长情况(IVM)进行比较,以判断体外培养方法是否成功。结果分离完整卵泡率为89.74%(376/419);培养12天卵泡存活率为96.81%(364/376),有腔形成率为48.94%(184/376);卵泡和卵母细胞直径显著增加。诱导排卵后卵丘细胞-卵母细胞复合体排出率为56.38%(212/376);培养卵泡经历从腔前卵泡到卵母细胞成熟和极体排出的形态学变化,也有部分卵泡不发生腔样结构改变。体外培养卵泡分泌E2和P激素与体内分泌特征一致。IVG组4.61%(10/217)卵母细胞不能恢复成熟[停止在生发泡期(GV期)],76.50%(166/217)停止减数分裂在生发泡破裂期(GVBD期),18.89%(41/217)卵母细胞停止在第2次减数分裂(MII期)(PB);而IVM组1.24%(3/241)的卵母细胞不能恢复成熟(停止在GV期),45.23%(109/241)停止减数分裂在GVBD,53.53%(41/241)卵母细胞停止在MII期(PB);卵母细胞染色体核型完整,无染色体数目和结构异常。结论本研究建立的小鼠腔前卵泡体外培养方法基本成功,可作为卵泡发育生理研究和毒理学研究的模型。     

自然周期黄体生成激素峰诱发卵子成熟的分子机制

在自然月经周期中,月经中期的黄体生成激素（LH）峰启动复杂的排卵过程,引起卵子完成减数分裂进入MⅡ期、卵丘复合物脱离卵泡壁排出、剩余颗粒细胞的黄素化。在辅助生殖技术的临床促排卵方案中,最常使用人绒毛膜促性腺激素（hCG）代替LH峰的作用,因两者作用于同一受体。而LH受体被激活后引发的排卵分子机制至今尚未研究清楚。随着卵泡的生长,颗粒细胞LH受体含量逐渐增加,在成熟前达到峰值,为应答LH峰做好准备;LH峰作用于壁颗粒细胞受体,通过刺激型G蛋白/腺苷酸环化酶/环磷酸腺苷/环磷酸腺苷依赖的蛋白激酶（Gs/AC/cAMP/PKA）途径,使细胞内cAMP浓度上升,激活转录因子,包括cAMP反应元件结合蛋白1（CREB1）与cAMP反应元件调节蛋白（CREM）,引起卵泡内细胞卵子成熟及排卵相关重要基因表达的激活与抑制,最终导致排卵的发生及黄体的形成。     

Relationship between follicle size and oocyte developmental competence in prepubertal and adult pigs

The present study compared the distribution and steroid composition of 3-, 4- and 5-8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5-8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5-8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5-8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5-8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.     

Immunohistochemical localization of steroidogenic enzymes and comparison with hormone production during follicle development in the pig

The steroidogenic enzymes, P450 aromatase (P450arom) and P450 17alpha-hydroxylase (P45017alpha), were precisely located within the healthy porcine follicle by immunohistochemistry. Enzyme distribution was examined throughout follicular development during natural oestrous cycles (n = 14 gilts) and was compared with steroid production by healthy whole and theca-only follicles. All follicles 2 mm or more in diameter were either fixed for immunohistochemistry (n = 380 of which 197 were assessed as healthy) or incubated as whole (n = 110) or theca-only (n = 110) follicles to measure steroidogenesis. P45017alpha was confined to the theca layer. The number of positive cells and staining intensity increased with follicle size. P450-arom was consistently detected in the granulosa layer of follicles measuring 6 mm or more in diameter and those cells furthest from the antrum were most strongly stained. P450arom was also detected in the theca layer of these large follicles. Whole and theca-only follicles produced oestradiol and androstenedione, and the levels of both hormones increased with follicle size (P<0.001). Whole follicles produced more oestradiol (P<0.001), but less androstenedione (P = 0.01) than theca-only follicles of the same size. Although granulosa cells contained P450arom and synthesized oestradiol, only theca cells contained P45017alpha. Theca cells therefore provided granulosa cells with androgen substrate. In addition, theca cells possessed P450arom, making them capable of independent oestradiol production, which may be required to trigger the LH surge. This study confirms the vital role of theca cells in follicular steroidogenesis in the pig.     

Development of the membrana granulosa of bovine antral follicles: structure, location of mitosis and pyknosis, and immunolocalization of involucrin and vimentin

The membrana granulosa of the ovarian follicle is termed the 'follicular epithelium', yet there have been no studies considering its epithelial nature and how it changes during follicular development. Therefore, these issues were investigated using histology (n = 45 ovaries), considering its structure and the location of proliferating and dying cells, and drawing analogies with other epithelia. Additionally, differences between the layers of granulosa cells were demonstrated by immunohistochemistry (n = 7 ovaries). The structure of the membrana granulosa differed between follicles. Six arbitrary classifications were designed based on these structures, 80 follicles were allocated (n = 13 ovaries) to these classes and the follicular diameters were then measured. For the first time, differences in membrana granulosa structure were shown to correspond to follicle size. Follicles in classes 1-3, where basal granulosa cells were columnar with nuclei positioned basally in the cell, were all < or = 3 mm in diameter. All follicles larger than 3 mm had either columnar basal cells with nuclei positioned centrally (class 4), or had rounded basal cells (class 5), and all follicles > 5 mm had only rounded basal cells. In all these classes, cells in the middle zone were rounded; cells aligning the antrum were often flattened. Irrespective of follicle class, cell proliferation and cell death were shown to be predominantly in the middle portions, rather than the most antral or most basal portions, of the membrana granulosa of healthy and atretic follicles. Involucrin, a marker of keratinocyte differentiation, was localized to the suprabasal region of the membrana granulosa of healthy follicles, particularly in the second and third cellular layers in from the follicular basal lamina. Conversely, the staining intensity for the intermediate filament protein vimentin was lowest in this region, and greatest in the more antral and basal regions. In atretic follicles, there was widespread staining for involucrin and vimentin throughout the membrana granulosa. In conclusion, the membrana granulosa is highly structured, and alters with follicular development. Layers in the membrana granulosa can differ in terms of cell shape, and differ in proliferation and gene expression. In the light of the current work, and an associated study, it is proposed that proliferation occurs in the middle layers, and that granulosa cells, then progress basally or antrally, the latter undergoing terminal differentiation.     

骨形态发生蛋白15调控卵泡发育及与生殖内分泌疾病的关系

骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）作为卵母细胞分泌因子,在调控卵泡发育过程中起着重要的作用。BMP15通过协同双调蛋白、上调卵丘扩张基因的表达,促进卵母细胞发育成熟;BMP15能增加颗粒细胞对卵泡刺激素（follicle-stimulating hormone,FSH）的敏感性,刺激颗粒细胞中Kit配体的表达从而促进颗粒细胞的增殖分化;BMP15通过调节卵泡凋亡相关因子的表达抑制卵泡闭锁;BMP15还可通过调节FSH受体及下游Smad信号通路介导对类固醇激素的调节。BMP15表达降低或基因突变,可能通过诱导颗粒细胞凋亡,阻碍早期卵泡发育及卵子发生过程,介导多囊卵巢综合征、早发性卵巢功能不全等多种疾病的发生。综述BMP15在卵泡发生过程中的作用及与相关生殖内分泌疾病的最新研究进展。