Androgen receptor in early apoptotic follicles in the porcine ovary at pregnancy

Localization of androgen receptor (AR) was investigated in ovarian follicles developing and undergoing atresia during pregnancy in the pig. Immunohistochemical staining was conducted on ovarian antral follicles isolated on different days of gestation: 10, 18, 32, 50, 70, and 90. Paraffin sections were also subjected to in situ DNA labeling. TUNEL staining revealed the presence of positive follicles on all days of pregnancy but the amount of atretic follicles increased with time. However, even on day 90 of gestation many follicles were normal, with no signs of atresia. In atretic follicles, apoptotic cells were localized predominantly in the granulosa while theca was much less affected. Atretic follicles with many apoptotic cells were negative for AR. Nuclear immunostaining for AR was positive in follicles with limited amount of apoptotic cells. The same relationship was observed in ovarian follicles isolated at various days of pregnancy.     

Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells

The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small ( 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/ml in serum-free media at 39 degrees C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in pr     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.     

Immunocytochemical Localization of Aromatase in The Ovary of Superovulated Cattle,Pigs and Sheep

The localization of aromatase, an enzyme converting androgens to estrogen, in the ovaries of superovulated cattle, pigs and sheep was studied immunocytochemically in the preovulatory and postovulatory period using anti-human placental aromatase cytochrome P-450 antiserum. Immunostaining for aromatase was detected in the granulosa cells of preovulatory follicles of all species studied. Theca interna cells were stained in preovulatory follicles in the pig but not in cattle and sheep. Interstitial gland cells, cumulus cells and oocytes were unstained in all species. In cattle and pig the corpora lutea were unstained whereas they displayed staining in the sheep. Preantral and small antral follicles were unstained during both the preovulatory and postovulatory period in all species. It is concluded that granulosa cells of preovulatory follicles are the main residence for aromatase activity in superovulated cattle, pig and sheep, whereas the activity of theca interna and corpora lutea is species specific.     

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.     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

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.     

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.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Ovarian follicle apoptosis at the onset of standing estrus in virgin and repeat-breeder dairy heifers

There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.     

Expression of prostasin serine protease and protease nexin-1 (PN-1) in rhesus monkey ovary during menstrual cycle and early pregnancy.

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.     

Quantitative analysis of in-vitro incorporation of [3H]thymidine into hamster follicles during the oestrous cycle

Follicles were isolated from hamster ovaries at 09:00 h and 15:00 h on each of the 4 days of the oestrous cycle (Day 1 = oestrus; Day 4 = pro-oestrus) by microdissection and by a mixture of enzymes and classified into 10 stages with pre-calibrated pipettes (stage 1 = preantral follicles with 1 layer of granulosa cells; stage 10 = preovulatory antral follicles). The follicles at each stage were incubated for 4 h with [3H]thymidine with incorporation expressed per microgram follicular DNA or per follicle. A significant increase in thymidine per follicle occurred at 15:00 h on Days 1 and 3 of the cycle from stage 2 (bilaminar follicle) to stage 6 (7-8 layers granulosa cells plus theca). When expressed as thymidine per follicle or microgram DNA, there was a significant increase in incorporation for stages 1-4 (4 layers granulosa cells) on Day 4 at 15:00 h compared to 09:00 h, presumably as a consequence of the preovulatory increase in gonadotrophins. Follicles in stages 5 to 8 (preantral follicles with 5 or more layers of granulosa cells to small antral follicles), from which the next set of ovulatory follicles will be selected, did not show a significant peak in incorporation per microgram DNA until Day 1 at 09:00 and 15:00 h when the second increase in FSH is in progress. DNA synthesis was similarly sustained throughout Day 1 for stage 1-4 follicles. These results suggest that periovulatory changes in FSH and LH, directly or indirectly, are not only responsible for ovulation and the recruitment of the next set of follicles destined to ovulate but also stimulate DNA replication in smaller follicles which develop over the course of several cycles before they ovulate or become atretic.     

Developmental competence of bovine oocytes is not related to apoptosis incidence in oocytes, cumulus cells and blastocysts

The number of follicles undergoing atresia in an ovary is very high, and isolation of cumulus-oocyte complexes (COCs) from such atretic follicles may impair subsequent embryo development in vitro. Our aim was to study if stringent selection by morphological assessment of COCs can improve embryo development, and to evaluate whether oocyte diameter is related with apoptotic ratio in oocytes and blastocysts. COCs from slaughtered cattle were recovered by follicle aspiration and classified depending on oocyte diameter: (A) <110 microm; (B) 110-120 microm; (C) >120 microm. COCs were matured, fertilized and cultured in vitro. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively, in denuded oocytes, COCs and blastocysts. Immature oocytes from Group A showed higher apoptotic ratio assessed by TUNEL assay, and the COCs corresponding to this group also showed a higher proportion of apoptotic cumulus cells. After maturation, no differences were present in the incidence of apoptosis among oocytes from different groups, but COCs corresponding to the largest diameter showed less apoptotic cumulus cells. In addition, the percentage of apoptotic oocytes decreased during in vitro maturation in all groups. Apoptotic cell ratio (ACR) in blastocysts was not related to oocyte diameter. In conclusion, oocyte selection and oocyte morphological evaluation prior to maturation was not sufficient to select non-atretic oocytes. When oocyte diameter was used as an additional selection the embryonic developmental potential increased together with oocyte diameter, but this improvement was not related to a lower incidence of apoptosis in the largest oocytes.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Structural changes occurring during atresia in sheep ovarian follicles

Summary The structural changes that characterize primary, secondary and tertiary atresia in sheep Graafian follicles have been studied by means of histological, histochemical and ultrastructural techniques.In primary atresia vacuoles representing swollen endoplasmic reticulum are prominent along the antral border together with disorganized granulosa cells containing pyknotic nuclei. Phagocytic cells, which increase in number as atresia progresses, were seen within the membrana granulosa and are considered to be transformed granulosa cells. Even in follicles classified as nonatretic, a few antral vacuoles and occasional pyknotic nuclei are present.During secondary atresia there is a large increase in the number of cells with pyknotic nuclei; many of these nuclei had been extruded and had fused to form the characteristic Feulgen-positive atretic bodies found along the edge of the antral cavity. These bodies usually have a diameter of up to 15 m but occasionally reached as much as 400 m. A second area of degeneration is frequently present in the membrana granulosa, two or three cell layers from the basal lamina, and it is at this level that exfoliation of granulosa cells occurs in tertiary atresia. In contrast to the membrana granulosa, there are during secondary atresia, only slight indications of degeneration in the cumulus.In tertiary atresia the membrana granulosa is highly disorganized; the atretic bodies are often fewer in number than at earlier stages. The basal lamina remains essentially intact. It is at this stage that the first clear signs of degeneration occur in the theca interna. Despite some disintegration of the cumulus, the integrity of the oocyte is maintained and its nucleus remains vesicular.Changes in the thecal microcirculation may play a key role in atresia: adjacent to the basal lamina of non-atretic follicles, there is a well-developed capillary network which is significantly reduced as atresia progresses.The authors are greatly indebted to Dr. H.M. Dott and Mr. G.C. Foster for carrying out the analysis with the Quantimet image analysing computer. The skilled technical assistance of Mrs. Linda Collins is also gratefully acknowledged     

Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy

Alterations in the basal lamina (BL) of developing follicles were studied by immunofluorescent microscopy using antibodies against type IV collagen, laminin, and fibronectin, and by electron microscopy. Ovarian development was induced in immature rats by sequential administration of estradiol, follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). A continuous BL was observed in healthy follicles treated with estradiol and FSH. As determined by immunofluorescence, laminin, type IV collagen, and fibronectin were restricted to the BL and the theca but not to the granulosa. When follicles were allowed to undergo atresia or induced to ovulate with hCG, the BL became fragmented. This was confirmed by electron microscopy of healthy, atretic, and luteinizing follicles which showed that in healthy follicles the BL was continuous, whereas in both atretic and luteinizing follicles, it was fragmented. Atresia was also associated with the penetration of thecal cells into the follicles. These observations indicate that the intact BL present in healthy follicles undergo extensive changes during atresia and ovulation.