Localization of apoptotic cells in the cystic ovarian follicles of cows: a DNA-end labeling histochemical study

We examined the frequency of apoptosis in cystic follicular cells to investigate the cause of the delay in regression of cystic follicles. Paraffin sections of healthy antral follicles, early and late atretic ones, and early and late cystic ones were stained using the terminal deoxynucleotidyl transferase (Tdt)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labeling (TUNEL) method to detect apoptotic cells. In the granulosa layer of early cystic and atretic follicles, TUNEL-positive cells were evident. In the theca interna of both early and late atresia, high frequencies of TUNEL-positive cells were observed. In the theca interna, a high frequency of TUNEL-positive cells was noted in the early cystic follicles, whereas their frequency decreased in late cystic follicles. These results suggest that apoptosis occurs in the granulosa and theca interna cells of cystic as well as atretic follicles, but the frequency of apoptosis in theca interna cells decreases in late cystic follicles, which may be responsible for the delay of follicular regression.     

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.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.     

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.     

Steroidogenic activity of atretic follicles in the cycling hamster ovary and relation to ultrastructural observations

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17-hydroxylase/C17–C20 lyase (17-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles. Even in the early stage of atresia, the granulosa cells showed remarkable morphological change characteristic of apoptosis, as visualized by scanning and transmission electron microscopy. Cessation of granulosa cell proliferation may occur earlier than apoptotic change and the degeneration of the granulosa cells becomes rapid once atresia starts.     

Aspects of cellular proliferation during follicular atresia in the dog ovary

Summary Autoradiography after pulse labelling with [³H] thymidine was applied to investigate the proliferation processes in the granulosa and theca related to follicular atresia of the dog ovary during metestrus.The number of proliferating cells depends on the follicle type and its atretic stage. There is less proliferation in smaller secondary follicles than either in larger ones or tertiary follicles. While in early atresia tertiary follicles show the highest labelling indices, in advanced atresia the larger secondary follicles are those with the highest values. For each follicle type a decline in the labelling indices can be observed from early to terminal atresia. Tertiary follicles show a precipitous decrease in the labelling index between early and advanced atresia. There is a continuous gradient of proliferation from the center of the follicle over the peripheral granulosa to the theca. In tertiary follicles, an inverse correlation between labelling and necrosis of granulosa cells can be observed.     

Follicle growth in the ovary of the rabbit after ovulation-inducing application of human chorionic gonadotropin

Summary The growth of tertiary follicles, i.e., the proliferation of cells in the stratum granulosum and in the capillary network of the theca interna, after injection of ovulation-inducing human chorionic gonadotropin (HCG), was investigated in the rabbit by means of autoradiographic and morphometric methods.Based on the frequency distribution of follicles with different sizes and on the labeling index (LI) of granulosa cells as a function of follicle size and of time prior to and after HCG stimulation, two groups of tertiary tollicles can be distinguished: growing (250–900 m in diameter) and mature (>900 m in diameter) elements. The growth of both groups is influenced by the release of gonadotropins.After HCG stimulation, follicles belonging to the first group grow rapidly. During, and a short time after ovulation, almost all non-ruptured follicles larger than 600 m in diameter become atretic. Within 35–50 h the ruptured and atretic mature follicles (>900 m in diameter) are replaced by follicles out of the group of growing follicles.From these results the following concept for regulation of follicle growth is derived: In principle, all growing follicles possess the potential to develop into mature follicles. When a sufficient number of mature follicles is generated, these mature follicles determine the number of succeeding growing follicles. Follicles that are not required for providing mature follicles become atretic as soon as they reach a diameter of 700 m. When the majority of mature follicles is lost during ovulation (by rupture or atresia), this inhibition regulated by mature follicles is abolished, and all of the growing follicles again are capable to develop into mature follicles.The relative amount of capillaries in the theca interna of growing and mature follicles remains constant with increasing follicle size. This means that the capillary network grows parallel to the increasing size of follicles. No differences are found between intact and atretic follicles; advanced atretic follicles were excluded from this study.The labeling index (LI) of granulosa cells in the stratum granulosum and of endothelial cells in the theca interna, as a function of follicle size and of time after HCG stimulation, are closely correlated. A change in the LI of granulosa cells is usually followed with a certain delay by a similar alteration of the LI of endothelial cells in the theca interna. This suggests that granulosa cells have a certain regulatory function on capillary growth.     

Localization of relaxin in the pig follicle during preovulatory development

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.     

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was performed in 55 cases of morphologically normal human ovaries by using a specific polyclonal antibody against purified human placental 3 beta-HSD. In small developing follicles, immunoreactivity was observed only in the theca interna but also became recognizable in the membrana granulosa with development of the follicle. At a late stage of folliculogenesis, the intensity of the 3 beta-HSD activity in the membrana granulosa was nearly equal to that of theca interna in 2 or 3 large follicles examined. One to several layers of theca interna cells just beneath membrana granulosa did not demonstrate any immunoreactivity of 3 beta-HSD or that of cytochrome P-450 17 alpha-hydroxylase. These unstained theca interna cells did not appear to be directly involved in ovarian steroidogenesis and might be designated as 'enzymically inactive theca interna cells.' Marked immunoreactivity was observed in luteinized theca and granulosa cells of the corpus luteum.     

Luteinization in the atresia of maturing ovarian follicles in mouse

Maturing ovarian follicles undergoing atresia were ultrastructurally analyzed. Using the method of Okr?s (1968) the process of luteinization was traced, taking place in the basal layer of cells of granulous membrane and in the cells of inner layer of theca interna during the atresia. In the atresia process, numerous electron dense granules arranged in packets and surrounded by tubular mitochondria are observed in the basal cell layer of granulous membrane. A considerable accumulation of the grains of reaction product and the presence of tubular mitochondria are also found in the cells of theca interna. Such a behaviour of those cells can be associated with an enhanced production of steroid hormones (mainly estrogens) in atretic follicles.     

Ultrastructure of the theca interna of ovarian follicles in sheep

Summary The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy.In follicles <3.0mm in diameter, the theca interna consisted of about 8–12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure.In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages.It was concluded that: (1) the theca interna of 2.0–2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca interna to the membrana granulosa.     

Functional morphology of the theca interna of the vesicular follicle in human ovary]

A study of histological serial sections of human ovaries and histochemical reactions established that several cell types differentiate in the theca interna during the formation and development of tertiary follicles (2nd growth period). This leads, in turn, to triple-layering of the theca interna in follicles of the 2nd resting period. The inner thecal layer consists of fusiform cells from the basement membrane, which are apposed to the membrana granulosa and show a strong positive reaction to alkaline phosphatases. A tropic function for the follicular epithelium must be assigned to this layer. The middle layer consists of epitheloid thecal cells, which show a strong positive reaction to 3beta-ol-steroid hydrogenase. These represent the estrogen glands of the follicle. The outer layer of the theca interna, whose transition to the theca externa is indistinct, also has fusiform cells, which are available as reserve material for the differentiation of further epitheloid thecal cells to pre-ovulatory follicles in later periods of development.     

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.     

Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles 相似文献   

Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular atresia in the infant human ovary.

The pattern of follicular atresia was studied in nine ovaries from children between the ages 3 months and 8 years. Atretic follicles were found among follicles at all stages of development. The percentage of follicles with signs of atresia became larger as the size of the follicles increased. Only 2% of small follicles (Type 3b) showed signs of atresia, while all follicles greater than 1 mm in diameter (Type 8) were atretic. In follicles of Type 5 and larger, four stages of atresia, which represent consecutive stages of a single atretic process, were defined. The beginning of atresia was characterized by the presence of pyknotic granulosa cells. As atresia progressed, the granulosa layer disappeared, the oocyte became necrotic, the follicle collapsed and the theca cells became hypertrophied. The oocyte can degenerate in several ways: it can be penetrated by cells, the nucleus can become pyknotic or it may complete meiotic prophase. It is suggested that the last event is only possible after the oocyte has reached its full size and has completed RNA synthesis.     

Evolution of prothymosin alpha and proliferating cell nuclear antigen (PCNA) immunoreactivity through the development of rat ovarian follicles

Summary The cellular distribution of prothymosin alpha (ProT) was studied in ovarian follicles of adult cycling rats. We found positive granulosa and theca cells throughout follicular maturation. When both ProT and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied, we observed that both proteins were expressed in the same granulosa and theca cells, although sometimes ProT immunoreactivity was weak or absent in the mitotic (M) phase. Moreover, both peptides share the nuclear distribution, but ProT immunoreactivity was never seen in nucleoli. Therefore, we conclude that in mitotic cells ProT is expressed only in actively proliferating cells, since all ProT-positive cells were also positive for PCNA. ProT and PCNA immunoreactivities during the meiotic division were studied in oocytes. The presence of PCNA was, unlike ProT, constant throughout follicle development (except atretic oocytes). Oocytes expressed ProT from primordial follicles to the eighth generation, but more developed oocytes and atretic oocytes were not immunoreactive. In hypophysectomized rats, all oocytes were immunoreactive. Interestingly, in hypophysectomized rats treated with follicle stimulating hormone (FSH) that promoted follicle development, the more developed oocytes did not show ProT immunoreactivity. Since hypophysectomized rats were not treated with luteinizing hormone we conclude that ProT expression is not required to complete meiotic division I.     

Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.