冻融小鼠卵巢移植后细胞凋亡及血管内皮生长因子表达的变化及意义研究

目的:探讨冻融小鼠卵巢同种异体移植后细胞凋亡及血管内皮生长因子表达的变化及意义。方法:收集C57BL/6j雌鼠和BALB/c雄鼠杂交后F1代4周龄小鼠卵巢,慢冻速融后移植至杂交后F1代8～12周雄鼠的肾被膜下,分别于移植后1d(24h)、2d(48h)和7d回收移植物,将冻融以及移植后不同时间段的卵巢组织进行HE染色、全卵巢卵泡计数、电镜观察、免疫组织化学分析细胞凋亡及RT-PCR检测VEGF基因表达。结果:冻融小鼠卵巢移植后随着时间的推移、各级卵泡数和卵泡存活率逐渐下降;移植后48h内细胞凋亡指数最高;电镜观察发现小鼠卵巢组织移植后损伤主要发生在移植后48h内;移植后VEGF的表达有上升的趋势,至第7d仍维持较高水平;移植后48hVEGF120mRNA和VEGF188mRNA水平明显升高(P0.05),至7d下降恢复至移植前水平,而VEGF164mRNA水平移植后无明显变化(P0.05)。结论:小鼠卵巢组织移植后48h内细胞凋亡最为严重,移植后引起大量卵泡的丢失;在移植后血管化的过程中VEGFmRNA表达量增加,VEGF120mRNA和VEGF188mRNA可能参与卵巢移植后早期血管化过程。     

血管紧张素Ⅱ在小鼠卵母细胞中的免疫组化定位

本文研究了血管紧张素Ⅱ在小鼠卵母细胞中的免疫组织化学定位,结果表明血管紧张素Ⅱ不仅分布在卵巢内的黄体细胞,卵泡的膜细胞,基质和血管,在恢复减数分裂过程中,处于生发泡破裂和第一极体排放期的卵母细胞内也检测到血管紧张素Ⅱ的免疫阳性物,因此,血管紧张素Ⅱ有可能在卵泡的生长发育和卵母细胞的成熟过程中起着重要作用。     

卵母细胞在哺乳动物卵巢排卵过程中的决定性作用

哺乳动物卵巢排卵是一个复杂的调控过程。卵泡成熟破裂后,卵母细胞从卵巢中排出。卵泡细胞感受排卵刺激,并诱导卵母细胞减数分裂的恢复及其随后的释放。卵母细胞及其周围颗粒细胞的旁分泌在对此起关键性作用,其中卵母细胞对其释放具有决定性作用。作者先前已经阐述过颗粒细胞在哺乳动物卵巢排卵过程中的调控作用,该文将从卵母细胞的发育及其调控角度重点阐明其在排卵过程中的决定作用,旨在进一步理解哺乳动物卵巢的排卵过程,同时为不孕不育等卵巢疾病的治疗提供重要的研究方向和理论基础。     

血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞顶体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和/或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和／或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

MAPK和p90rsk在大鼠卵泡发育中的表达与活性

利用免疫组织化学方法研究丝裂原激活蛋白激酶(mitogen-activated protein kinases, MAPK)及其底物之一p90rsk在大鼠卵泡发育过程中的表达与活性.结果表明,非活性形式的MAPK存在于大鼠各生长期卵泡的卵母细胞和颗粒细胞中,但磷酸化活性形式的MAPK只存在于部分具有分裂增殖活性的颗粒细胞中.MAPK的作用底物p90rsk只在各期卵泡的卵母细胞中表达,在颗粒细胞中无着色,说明MAPK信号级联在卵母细胞和颗粒细胞中具有不同的作用方式.另外,胎鼠卵巢的免疫组化染色结果显示,MAPK在卵原细胞增殖过程中具有活性,表明MAPK信号级联在这一过程中起作用.     

MAPK和p90 rsk 在大鼠卵泡发育中的表达与活性

利用免疫组织化学方法研究丝裂原激活蛋白激酶（mitogen-activated protein kinases,MAPK)及其底物之一p90^rsk在大鼠卵泡发育过程中的表达与活性。结果表明,非活性形式的MAPK存在于大鼠各生长期卵泡的卵母细胞和颗粒细胞中,但磷酸化活性形式的MAPK只存在于部分具有分裂增殖活性的颗粒细胞中。MAPK的作用底物p90^rsk只在各期卵泡的卵母细胞中表达,在颗粒细胞中无着色,说明MAPK信号级联在卵母细胞和颗粒细胞中具有不同的作用方式。另外,胎鼠卵巢的免疫组化染色结果显示,MAPK在卵原细胞增殖过程中具有活性,表明MAPK信号级联在这一过程中起作用。     

血管紧张素II在小鼠卵母细胞中的免疫组化定位(英文)

本文研究了血管紧张素II在小鼠卵母细胞中的免疫组织化学定位。结果表明血管紧张素II不仅分布在卵巢内的黄体细胞、卵泡的膜细胞、基质和血管,在卵母细胞的细胞质和细胞膜上也见有阳性分布。颗粒细胞和卵丘细胞上未见着色。在恢复减数分裂过程中,处于生发泡破裂和第一极体排放期的卵母细胞内也检测到血管紧张素II[(\265\304\303\342\322\337\321\364\320\324\316\357\241\243)238.1(\322\362\264\313)],血管紧张素II有可能在卵泡的生长发育和卵母细胞的成熟过程中起着重要作用。     

扬子鳄卵巢性类固醇激素受体的免疫细胞化学研究

为探讨扬子鳄卵巢内不同性类固醇激素受体在卵泡发育中的调控作用,研究采用组织学和免疫细胞化学方法,运用激光共聚焦显微镜,对扬子鳄不同发育时期卵泡中的雌激素受体、雄激素受体和孕激素受体进行了检测。结果发现,3种类固醇激素受体在卵巢各期滤泡细胞中均有表达,在4月Ⅱ-Ⅳ期卵泡的滤泡细胞中阳性反应最强;9月卵巢的滤泡细胞中阳性反应最弱;ER和AR不仅在各期滤泡细胞中存在阳性位点,在6月卵泡的卵母细胞胞质中也有表达。结果说明,在扬子鳄卵母细胞生长发育和成熟过程中,3种激素受体通过与其对应的激素结合对滤泡细胞的发育、卵黄的合成与积累以及排卵起着重要的调控作用。       

斯钙素与哺乳动物生殖的研究进展

斯钙素(stanniocalcin,STC)是一类首先在硬骨鱼类发现的糖蛋白激素,其生理作用在于调节钙磷稳态。近年来在入和哺乳动物组织中也发现存在有STC,并且以旁分泌方式参与机体的多种生理功能。本文综述了近年来哺乳动物胚胎发育、妊娠及哺乳期间STC基因在卵巢内的表达,早期胚胎植入过程中STC基因在子宫内膜的表达,以及人绒毛膜促性腺激素(hCG)对STC分泌的调节作用及其作用机制。研究表明,新生个体的STC基因表达首先出现在卵巢的膜细胞内,随后出现于卵巢基质的间质细胞和内膜细胞;但STC基因的表达产物STC却聚积于卵母细胞和黄体细胞内,提示STC参与卵母细胞的成熟过程。胚胎植入、妊娠、分娩与哺乳期间卵巢内STC基因表达量的动态变化行为,提示STC在哺乳动物生殖过程的各个环节均发挥一定的作用。     

Up-regulation of alpha-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis

We recently demonstrated that the number of primordial follicles was significantly reduced in the ovaries of near-term baboon fetuses deprived of estrogen in utero and restored to normal in animals administered estradiol. Although the baboon fetal ovary expressed estrogen receptors alpha and beta, the mechanism(s) of estrogen action remains to be determined. It is well established that inhibin and activins function as autocrine/paracrine factors that impact adult ovarian function. However, our understanding of the expression of these factors in the primate fetal ovary is incomplete. Therefore, we determined the expression of alpha-inhibin, activin beta(A), activin beta(B), and activin receptors in fetal ovaries obtained at mid and late gestation from untreated baboons and at late gestation from animals in which fetal estrogen levels were reduced by >95% by maternal administration of the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 and estradiol benzoate. Immunocytochemical expression of alpha-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, alpha-inhibin was abundantly expressed in pregranulosa cells of interfollicular nests and granulosa cells of primordial follicles. Thus, the number (mean +/- SEM) per 0.08 mm2 of fetal ovarian cells expressing alpha-inhibin, determined by image analysis, was similar at mid and late gestation and increased approximately 8-fold (P < 0.01) near term in baboons treated with CGS 20267 and was restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol. Activin beta(A) was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin beta(B) was also expressed in pregranulosa cells and granulosa cells at mid and late gestation, respectively, but was not detected in oocytes. Neither the pattern nor the apparent level of expression of activin beta(A) or beta(B) were altered in fetal ovaries of baboons treated with CGS 20267 or CGS 20267 and estrogen. Activin receptors IA, IB, IIA, and IIB were detected by Western blot analysis in fetal ovaries at mid and late gestation, and expression was not altered by treatment with CGS 20267 or CGS 20267 and estrogen. Activin receptors IB and IIA were localized to oocytes and pregranulosa cells at midgestation and to granulosa cells and oocytes of primordial follicles at late gestation. Thus, the decrease in the number of follicles in the primate fetal ovary of baboons deprived of estrogen in utero was associated with increased expression of alpha-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling alpha-inhibin expression and, thus, the intraovarian inhibin:activin ratio.     

Localization and developmental expression of the activin signal transduction proteins Smads 2, 3, and 4 in the baboon fetal ovary

We recently demonstrated that the reduction in the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was associated with increased expression of alpha-inhibin, but not activin betaA and betaB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.     

Expression of Fas,p53 and AFP in development of human fetal germ cells in vitro

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.     

Relationship between Numerous Mast Cells and Early Follicular Development in Neonatal MRL/MpJ Mouse Ovaries

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

秀丽白虾卵母细胞不同发育阶段滤泡细胞的超微结构

用透射电镜技术观察了秀丽白虾(Exopalaemon modestus)不同发育阶段卵巢滤泡细胞的超微结构及其与卵母细胞的联系。随着卵母细胞的发育进程,滤泡细胞经历了发育和退化过程。在卵黄大量发生期,卵母细胞被多层滤泡细胞包绕,血窦伸入层间;滤泡细胞内含有丰富的内质网、高尔基体、线粒体、核糖体及原始卵黄颗粒。在卵子成熟期,滤泡细胞由内向外依次解体,血窦萎缩。这些形态变化支持滤泡细胞具有吸收血液营养、合成并向卵母细胞输送原始卵黄物质的功能的观点。与锯缘青蟹、长毛对虾和中华绒螯蟹的滤泡细胞的作用方式稍有不同。