磷酸二酯酶5在小鼠卵巢内的表达

目的探讨cGMP特异性结合的磷酸二酯酶5（PDE5）在小鼠卵巢内的定位和表达情况。方法应用免疫组织化学对小鼠卵巢切片进行染色,检测PDE5在卵巢内不同部位的表达情况。利用Western blot检测PDE5在小鼠不同组织和细胞内的表达情况。结果PDE5在小鼠卵巢的黄体细胞（CL）和卵泡膜细胞（TC）上有强表达,卵母细胞（Oos）上以及大有腔卵泡的卵丘细胞（CCs）也有表达,而在小卵泡的颗粒细胞上没有表达。结论揭示了PDE5在小鼠卵巢内的表达情况,为进一步研究PDE5对卵巢功能的调节提供了生理学基础。     

比格犬卵巢子宫内雌激素受体ERα、ERβ的免疫组织化学定位

目的研究雌激素受体α,β在比格犬卵巢及子宫内的定位。方法采用免疫组化SP法DAB显色结合BCIP/NBT及AEC显色检测ERα、ERβ在比格犬子宫及卵巢内的表达。结果比格犬ERα主要表达于卵泡颗粒细胞、卵巢间质腺腺上皮细胞及子宫内膜腺体腺上皮细胞胞核内,胞质内仅有少量表达,而在卵泡膜内膜的间质细胞,腺体周围的基质细胞及小动脉血管内皮细胞和平滑肌细胞、小静脉内皮细胞的胞核内有少量表达。而ERβ则以相同的组织特异性主要表达于上述组织细胞的胞质内,在胞核内有微弱表达。ERα表达于膜黄体细胞的胞核内,而在黄体颗粒细胞胞核与胞质内均有表达。而ERβ则仍特异表达于不同生理阶段黄体细胞的胞质内。BCIP/NBT与AEC双染未见ERα、ERβ在子宫内有明显的共表达现象。结论比格犬ERα、ERβ在子宫与卵巢组织内定位不同,ERα主要定位于胞核,在胞质内有微弱表达,而ERβ主要定位于胞质,在胞核内有零星表达。     

EGF对大鼠卵巢颗粒细胞增殖与分化影响的研究

许多研究发现,表皮生长因子(EGF)对生殖功能有重要的调节作用。本文用体外细胞培养的方法,研究了EGF对大鼠卵巢颗粒细胞增殖与分化的影响及其作用方式。结果如下:EGF可以明显抑制颗粒细胞DNA的合成,但促进孕酮的生成,后者是因为EGF能显著提高细胞内3β-羟甾脱氨酶(3β-HSD)的活性。放射受体分析表明,颗粒细胞上存在EGF的特异性受体,其K_d为1.83±0.3×10~(-8)mol/L,B_(max)为1.75±0.29×10~4个位点/细胞。卵巢免疫组化结果未发现颗粒细胞有EGF样免疫染色,而卵泡膜、黄体及间质内等均有阳性染色。以上结果提示,EGF可能通过旁分泌机制作用于颗粒细胞的EGF受体,从而调节细胞的生长和性激素的分泌,这对于颗粒细胞的成熟及卵泡的发育有着重要意义。     

莱芜黑山羊卵巢FSHr、LHr的免疫组化定位研究

为从形态学角度理解内分泌调节过程,揭示促性腺激素(GTH)对雌性哺乳动物卵巢调节作用机制,对处于非繁殖季节的莱芜黑山羊卵巢中FSHr、LHr分布进行免疫组化SABC方法定位.分别选取相邻的6张连续阳性切片,光镜观察、图像分析.结果显示:FSHr、LHr阳性细胞主要分布于卵泡颗粒细胞、膜细胞、卵母细胞、血管周围间质,尤其以在卵泡颗粒细胞、膜细胞的胞质中分布最多.原始卵泡卵母细胞中便有两种受体阳性物质分布,在各级卵泡中两种受体阳性细胞数量、染色强度随卵泡发育水平呈正向增加趋势.     

成体雌性小熊猫卵巢组织学观察

2000 年至2009 年,12 只固定于10% 福尔马林中非生殖系统疾病死亡的小熊猫卵巢组织,按常规组织学技术制作组织切片,HE 染色,光学显微镜观察。结果:(1)不同发情时期卵巢均有原始卵泡、初级卵泡和次级卵泡分布。发情期的卵巢未观察到典型的成熟卵泡和卵母细胞; (2)原始卵泡数量较少,初级卵泡数量较多,多数初级卵泡和大多数的次级卵泡都处在闭锁状态;(3)卵泡腔出现之前,卵母细胞的直径和卵泡直径同时增长;卵泡腔出现之后,卵母细胞直径增长较慢,卵泡直径增长较快; (4)不同发情时期的小熊猫卵巢均存在大量的间质腺细胞;(5)妊娠小熊猫和发情间期无妊娠小熊猫的卵巢均有发育正常的黄体;(6)卵泡细胞发育呈低柱状至柱状时出现透明带。结论:(1) 卵泡闭锁主要发生在初级卵泡阶段,仅少数卵泡能发育至次级卵泡;(2)卵母细胞和卵泡生长呈双相生长的趋势; (3) 不同发情时期的小熊猫卵巢间质腺都发达; (4)发情排卵后,非妊娠黄体与妊娠黄体维持的时间相似,证实了小熊猫存在假孕现象。     

猪分离卵泡体外培养过程中Fas/FasL对颗粒细胞凋亡的作用

从猪卵巢分离完整有腔卵泡,按质量分为3类：健康卵泡、早期闭锁卵泡和晚期闭锁卵泡。猪分离卵泡经眼观检查后再行石蜡切片和HE染色,形态学研究表明,眼观检查对于健康卵泡的判定准确率为92％。取健康卵泡按直径大小分为3组：直径＞5 mm大卵泡组、3～5 mm中卵泡组和≤3 mm小卵泡组。卵泡培养8、16和24 h,以Annexin-V FITC/PI双染流式细胞仪检测壁层颗粒细胞凋亡情况,结果发现培养卵泡颗粒细胞的总凋亡率（早期凋亡＋晚期凋亡）在8 h时就已达到70%以上,至24 h则为81.1%～94.6%。收集无血清培养0、8、16、24、48和72 h的卵泡颗粒细胞,用real time PCR SYBRgreen法检测各组卵泡颗粒细胞FasL和Fas mRNA相对表达量。各级卵泡颗粒细胞中FasL mRNA水平随培养时间显著增加,培养至24 h达最大值（P＜0.05）;小卵泡颗粒细胞FasL mRNA水平均高于大、中卵泡组。各级卵泡颗粒细胞Fas mRNA相对表达量在培养前（0 h）差异不显著,8 h时显著增加,48 h达最大值。该实验表明,所用无血清卵泡培养体系可有效诱导卵泡颗粒细胞的凋亡,细胞凋亡是卵泡闭锁的主要诱因,但卵泡闭锁程度可因卵泡大小而异,小卵泡似乎更容易发生闭锁。     

甲状腺激素受体在扬子鳄卵巢中的免疫组织化学定位和不同繁殖时期的mRNA表达水平变化检测

目的研究扬子鳄甲状腺激素受体(thyroid hormone receptor,TR)基因的系统进化关系和在卵巢组织中的定位,分析成年扬子鳄在不同繁殖活动期卵巢组织中甲状腺激素受体的动态变化规律。方法通过Clustal W软件对扬子鳄和其它脊椎动物TRs氨基酸序列进行序列比对,使用MEGA 6.0软件构建NJ系统发育树;应用q RT-PCR方法检测TRα和TRβm RNA在不同繁殖时期卵巢组织中的表达水平差异;应用免疫组织化学法对扬子鳄卵巢卵泡中TR进行组织定位。结果系统发育分析显示扬子鳄与鸟类亲缘关系较近;扬子鳄卵巢中TR的m RNA表达水平在繁殖期最高,在繁殖前期和繁殖后期相对较低;免疫组织化学染色显示,TR阳性反应主要位于卵泡的颗粒层细胞、卵泡膜、卵母细胞质和卵母细胞核中,其中颗粒层细胞阳性反应最明显。结论 TR基因的进化时间较早,因此序列保守性较高;TRα和TRβ对扬子鳄卵泡发育成熟具有重要作用;颗粒层细胞是TRs的功能作用主要位点。     

乌梢蛇卵泡不同发育期颗粒细胞的显微结构变化

采用光学显微镜主要观察了乌梢蛇(Zaocys dhumnades)卵泡发育过程中颗粒细胞的显微结构变化.结果表明,乌梢蛇的滤泡前体细胞在形态、大小与嗜色性上均与生殖基的表面上皮相似;在原始卵泡期,卵母细胞周围的滤泡前体细胞围绕成一圈;在颗粒层细胞期的时期I至时期Ⅲ,颗粒细胞分化为由小细胞、梨形细胞与中间型细胞构成的异型颗粒细胞;在时期Ⅳ,这些异型颗粒细胞又转变成为只有小细胞的同型颗粒细胞.乌梢蛇卵泡颗粒细胞来源于生殖基的表面上皮,其发育特征是首先由同型发育成为异型,再由异型转变为同型颗粒细胞,具有同源异型的特征.     

闭锁卵泡中肾素—血管紧张素质系统的变化

目的：探讨卵巢局部的肾素－血管紧张素系统（RAS）在卵泡闭锁中的作用。方法：应用卵巢细胞双室培养,放射免疫测定（RIA）及免疫组化方法研究猪卵巢的健康卵泡和闭锁卵泡的颗粒细胞和卵泡内膜细胞与RAS的关系;观察了血管紧张素Ⅱ（AngⅡ)拮抗剂Saralasin及血管紧张素转换酶（ACE）抑制剂Captopril的作用。结果（1）与健康卵泡相比较,闭锁卵泡的卵泡液及卵泡内膜细胞培养液中的AngⅡ浓度明显升高,肾素浓度则明显降低;而颗粒细胞培养液中的AngⅡ和肾素浓度均无明显改变;（2）闭锁卵泡切片的AngⅡ,2型血管紧张素Ⅱ受体（AT2）染色明显强于健康卵泡;AngⅡ和AT2水平在双室培养的闭锁卵泡的卵泡内膜细胞中明显强于健康卵泡的卵泡内膜细胞;健康和闭锁卵泡的颗粒细胞中的AngⅡ,AT2水平变化不大;（3）双室培养中加入Saralasin或Captopril培养48h后,（a)与健康卵泡相比,闭锁卵泡的卵泡内膜细胞培养液中的AngⅡ浓度显降低,肾素浓度明显升高;（b)健康和闭锁卵泡的卵泡内膜细胞AngⅡ和AT2免疫组化染色均呈现下降,（c)RIA结果显示,健康和闭锁卵泡的颗粒细胞培养液中的AngⅡ和肾素浓度无明显变化;免疫组化结果显示,颗粒细胞的AngⅡ,AT2水平亦无显改变。结论：卵泡闭锁与卵巢RAS密切相关,AngⅡ和AT2是卵泡闭锁的重要相关因子;卵泡闭锁过程中卵巢局部的RAS变化主要发生在卵泡内膜细胞;Saralasin和Captopril可能通过调节卵巢RAS而具有抑制卵泡闭锁的作用。     

干扰素α应答基因的克隆及在兔早期胚胎发育中功能分析

干扰素(interferon,IFN)在哺乳动物早期胚胎发育过程中具有重要的生理功能,但是其作用机制尚不清楚.通过筛选卵巢cDNA文库和5′-RACE方法,克隆了兔卵巢干扰素α应答基因(interferonresponsivegene,IFRG)的全长cDNA(登录号:AJ584672).利用RT-PCR证明IFRG在兔卵母细胞和植入前胚胎中均有表达,这将为深入研究IFN在早期胚胎中的作用机制提供理论参考,卵巢原位杂交表明IFRG在成熟卵泡(类型5)的颗粒细胞中有表达,在初级和次级卵泡的膜鞘细胞和粒层细胞中有很高的表达,鉴于这些细胞与卵泡的发育密切相关,推测IFRG在卵泡的发育、成熟和排卵中发挥重要作用.     

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.     

A role for the androgen receptor in follicular atresia of estrogen receptor beta knockout mouse ovary.

Estrogen receptor beta (ERbeta) is highly expressed, but ERalpha is not detectable in granulosa cells in the mouse ovary. In ERbeta knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERbeta there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.     

Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Follicle-restricted compartmentalization of transforming growth factor beta superfamily ligands in the feline ovary

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Throughout reproductive life, follicle fate is balanced between growth and apoptosis. These opposing forces are controlled by numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor beta (TGFbeta) superfamily. TGFbeta, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor 9 (GDF-9) are present in the ovary of many animals; however, no comprehensive analysis of the localization of each ligand or its receptors and intracellular signaling molecules during folliculogenesis has been done. The domestic cat is an ideal model for studying ovarian follicle dynamics due to an abundance of all follicle populations, including primordial stage, and the amount of readily available tissue following routine animal spaying. Additionally, knowledge of the factors involved in feline follicular development could make an important impact on in vitro maturation/in vitro fertilization (IVM/IVF) success for endangered feline species. Thus, the presence and position of TGFbeta superfamily members within the feline ovary have been evaluated in all stages of follicular development by immunolocalization. The cat inhibin alpha subunit protein is present in all follicle stages but increases in intensity within the mural granulosa cells in large antral follicles. The inhibin betaA and betaB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are produced in primordial and primary follicle granulosa cells. Additionally, inhibin betaA subunit is detected in the theca cells from secondary through large antral follicle size classes. GDF-9 is restricted to the oocyte of preantral and antral follicles, whereas the type II BMP receptor (BMP-RII) protein is predominantly localized to primordial- and primary-stage follicles. TGFbeta1, 2, and 3 ligand immunoreactivity is observed in both small and large follicles, whereas the TGFbeta type II receptor (TGFbeta RII) is detected in the oocyte and granulosa cells of antral follicles. The intracellular signaling proteins Smad2 and Smad4 are present in the granulosa cell cytoplasm of all follicle size classes. Smad3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicle size classes. These data suggest that the complete activin signal transduction pathway is present in small follicles and that large follicles primarily produce the inhibins. Our data also suggest that TGFbeta ligands and receptors are colocalized to large antral follicles. Taken together, the ligands, receptors, and signaling proteins for the TGFbeta superfamily are present at distinct points throughout feline folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation.     

Effect of macrophages on proliferation of granulosa cells in the ovary in rats.

The effect of macrophages on proliferation of granulosa cells was examined in gonadotrophin-primed immature female rats. The mouse anti-rat macrophage monoclonal antibodies TRPM-2 and TRPM-3 were used and macrophages were observed in the granulosa layer and antrum of follicles and in corpora lutea and stroma around follicles. There was no difference in distribution between TRPM-2-positive cells and TRPM-3-positive cells. Macrophages with some cytoplasmic vacuoles of various sizes were also demonstrated in growing follicles. The average ratios of macrophages to granulosa cells in preantral, antral and mature follicles were 0.008, 0.007 and 0.002, respectively. Labelling with [3H]thymidine of granulosa cells cultured with peritoneal macrophages was significantly greater and the labelling index peaked to 25.0% when the ratio of macrophages to granulosa cells was 0.01, compared with the value of 14.2% when the granulosa cells were cultured alone. This ratio of macrophages to granulosa cells was similar to that in the preantral and antral follicles in vivo. These results suggest that macrophages participate in promoting proliferation of granulosa cells as local mediators in growing follicles.     

An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects

An enzymatic method was developed to collect intact follicles at different stages of development from cyclic hamsters to study ovarian folliculogenesis under various circumstances. Ovaries from 6 adult hamsters on each day of the cycle (Day 1 = ovulation) were collected, corpora lutea and large preantral and antral follicles were dissected, and follicles saved. Minced ovaries were then incubated with a mixture of collagenase, DNAse and pronase at 37 degrees C for 20 min to disperse intact follicles. Histological studies with 2191 isolated follicles revealed 10 different stages of follicular development (depending on the number of granulosa cell layers surrounding the oocyte and development of the antrum). Of the total follicular population, 14% showed signs of atresia, with 50% of those having 1-3 layers of granulosa cells (Stages 1-3); a second peak of 18% was observed in antral follicles (Stages 8-10). No signs of thecal cells were evident until the follicles reached Stage 6 (7-8 layers of granulosa cells), which possibly accounts for reduced atresia in this class and beyond. Ultrastructural study revealed that there were no signs of morphological damage to the basement membrane or to other subcellular organelles in the small preantral follicles. The presence of subnuclear lipid droplets in follicles with 3 layers of granulosa cells provided evidence for potential steroidogenesis by small follicles. The number of Stage 1-10 follicles was remarkably constant throughout the estrous cycle (460 +/- 34 per animal on Day 1 vs. 492 +/- 66 on Day 4). The usefulness of this method in analyzing follicular kinetics is illustrated in experiments involving hypophysectomy and the effects of unilateral ovariectomy. This procedure offers an improved method to study the factors responsible for the growth and the differentiation of small preantral follicles in the mammalian ovary.