Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

Expression of follicle stimulating hormone and luteinizing hormone receptors during follicular growth in the domestic cat ovary

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 microm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 microm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue.     

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles

The purpose of this study was to establish a culture model for isolated intact porcine antral follicles and investigate the relationship between granulosa cell apoptosis and follicular atresia. Small (<3 mm), medium (3–5 mm) and large (>5 mm) healthy porcine follicles were isolated and cultured in serum‐free TCM199 with or without follicular stimulating hormone (FSH). Microscopic identification of healthy follicles was confirmed by histology. A spontaneous onset of apoptotic cell death in granulosa cells was observed from cultured antral follicles. The apoptotic rate of granulosa cells from small follicles cultured for 24 hr was higher than those of large and medium follicles, accompanied with high FasL mRNA abundance in granulosa cells. Supplementation with 3 or 5 IU/ml FSH significantly inhibited the percentage of granulosa cells that became apoptotic. FSH did not significantly alter estradiol secretion from cultured follicles. Progesterone secretion significantly decreased after culture for 48 hr, coinciding with the morphological changes observed. FasL and Fas mRNA were expressed in the healthy, early atretic, and progressed atretic porcine follicles regardless of follicular size. However, FasL but not Fas mRNA levels increased during follicular atresia. Addition of FSH significantly decreased FasL rather than Fas mRNA levels in granulosa cells and could attenuate apoptosis. Small follicles seemed to be more susceptible to atresia as compared to medium and large follicles. Mol. Reprod. Dev. 77: 670–678, 2010. © 2010 Wiley‐Liss, Inc.     

Size-frequency analysis of atresia in cycling rats

The purpose of this study was to delineate when, during follicular growth, the alternative developmental pathways leading to ovulation or atresia diverge. By using computerized image analysis techniques, random samples of healthy and atretic follicles in ovaries of cycling rats were subjected to size-frequency analysis. The vast preponderance of atretic follicles were of the early antral size class (approximately 300-350 micron diameter, 800-1000 granulosa cells in the largest cross-section); atretic small follicles (less than 250 granulosa cells in the largest cross-section) were rare. Follicles in early stages of atresia were uncommon in ovaries of animals killed at estrus, but were found with great frequency in ovaries of animals killed the following day (metestrus). These results suggest that, under normal cyclic conditions, there may be only one major branching point during follicular development when growing follicles become susceptible to atresia. The alternative developmental pathways leading to ovulation and atresia may not diverge until the penultimate stage of growth, immediately preceding the final transformation into a preovulatory follicle.     

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.     

Expression of tPA, LH receptor and inhibin α, β_A subunits during follicular atresia in rats

By using DNA 3'-end labeling, immunocytochemistry and mRNA in situ hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (ⅰ) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ⅱ) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (ⅲ) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in     

Expression of tPA, LH receptor and inhibin α, βA subunits during follicular atresia in rats

By using DNA 3′-end labeling, immunocytochemistry and mRNA insitu hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte.     

Follicular atresia in relation to oocyte morphology in non-pregnant and pregnant women

Antral follicles from normally menstruating women and women in the third trimester of pregnancy were classified as healthy or atretic by flow cytometric DNA measurements on aspirated granulosa cells and by the concentration of steroids in the follicular fluid. The oocytes contained in these follicles were characterized as healthy or degenerating by their morphology at the light microscopic level. In 98% of the cases (61/62) morphologically healthy or degenerating oocytes were found in follicles which were classified as healthy or atretic respectively. In the normally menstruating women degenerative changes in the oocyte and the remainder of the follicle appeared to occur in synchrony. In pregnant women asynchrony was noted between the oocyte and the remainder of the follicle as follicular atresia progressed. The present study demonstrated the usefulness of flow cytometric DNA measurement in characterizing antral follicles of all sizes as healthy or more or less atretic.     

Immunocytochemical study of cell proliferation in the cystic ovarian follicles in cows

We examined the frequency of proliferating cells in cystic, atretic and healthy antral follicles to determine whether a disorder of cell proliferation was responsible for the occurrence of bovine cystic follicles. Paraffin sections of healthy follicles and various stages of atretic and cystic follicles were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). The PCNA-positive cells were counted in 4 different regions of a follicle from the apical to the basal side. In the granulosa layer, a significantly higher frequency of PCNA-positive cells was observed in the healthy follicle in the basal region as compared with the apical region. A similar pattern of PCNA-positive cells population was observed in the granulosa layer of atretic follicles, although the frequency in the basal region was significantly lower in the atretic than the healthy follicle. The rate of cell proliferation in the granulosa layer of cystic follicles was markedly lower at the basal region than that of atretic follicles. In the theca interna, the frequency of PCNA-positive cells in atretic follicles at the early stages was higher than that in cystic follicles at the early stages. These results suggest that in the healthy follicle the proliferative activity of granulosa cells is higher in the basal than the apical region, and that the cell proliferation activity in the granulosa and theca interna may decrease in association with the induction of a follicular cyst.     

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.     

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.     

Expression of tPA, LH receptor and inhibin a,βA subunits during follicular atresia in rats

By using DNA 3′-end labeling, immunocytochemistry and mRNA insitu hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte. Project supported by the National Natural Science Foundation of China (Grant Nos. 39770099 and 39770290), Chinese Academy of Sciences and Rockefeller Foundation/WHO HPR research project.     

Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Histological study of follicular atresia in the ovary of the domestic hen (Gallus domesticus)

Semi-serial (1 in 20) sections of ovaries were studied and only two types of atresia were identified--non-bursting and bursting. Smaller, non-yolky follicles (less than 1 mm diameter) showed non-bursting atresia. Atresia in follicles greater than 1 mm diameter was invariably of the bursting type which involved the rupture of the follicular wall, and the extrusion of yolk and cellular debris through the rupture site into the stroma. However, this rupture site was small and consequently was not visible in every section but it could always be seen when the follicle was followed in semi-serial sections. The mitotic index of granulosa cells in bursting atretic follicles was much lower than that for normal follicles. The most common criteria for distinguishing non-bursting atretic follicles were the extremely shrunken, irregularly shaped oocytes and the separation of the granulosa from the theca. In bursting atretic follicles, reliable indications were the presence in the ooplasm of some cells or cellular debris, and disorganization of the yolk and granulosa tissue. The presence of pycnotic nuclei in the granulosa cells was not a consistent feature of all atretic follicles of the hen.