Expression and localisation of ephrin-B1 and EphB4 in steroidogenic cells in the naturally cycling mouse ovary

Ephrin receptors and ligands are membrane-bound molecules that modulate diverse cellular functions such as cell adhesion, epithelial–mesenchymal transition, motility, differentiation and proliferation. We recently reported the co-expression of ephrin-B1 and EphB4 in adult and foetal Leydig cells of the mouse testis, and thus speculated that their co-expression is a common property in gonadal steroidogenic cells. Therefore, in this study we examined the expression and localisation of ephrin-B1 and EphB4 in the naturally cycling mouse ovary, as their expression patterns in the ovary are virtually unknown. We found that ephrin-B1 and EphB4 were co-expressed in steroidogenic cells of all kinds, i.e. granulosa cells and CYP17A1-positive steroidogenic theca cells as well as in 3β-HSD-positive luteal cells and the interstitial glands; their co-expression potentially serves as a good marker to identify sex steroid-producing cells even in extra-gonadal organs/tissues. We also found that ephrin-B1 and EphB4 expression in granulosa cells was faint and strong, respectively; ephrin-B1 expression in luteal cells was weak in developing and temporally mature corpora lutea (those of the current cycle) and likely strong in regressing corpora lutea (those of the previous cycle) and EphB4 expression in luteal cells was weak in corpora lutea of the current cycle and likely faint/negative in the corpora lutea of the previous cycle. These findings suggest that a luteinising hormone surge triggers the upregulation of ephrin-B1 and downregulation of EphB4, as this expression fluctuation occurs after the surge. Overall, ephrin-B1 and EphB4 expression patterns may represent benchmarks for steroidogenic cells in the ovary.     

OBSERVATIONS ON THE FINE STRUCTURE OF LUTEIN CELLS : II. The Effects of Hypophysectomy and Mammotrophic Hormone in the Rat

Corpus luteum formation was induced in 26-day-old rats which were subsequently hypophysectomized and injected with mammotrophic hormone (MH, LTH). Sections of corpora lutea from these animals were examined with the electron microscope and compared with similarly prepared (Caulfield's fixed, Araldite embedded) corpora from normal pregnancy and from controls, the latter consisting of corpora prior to hypophysectomy and corpora from uninjected rats 7 to 14 days after hypophysectomy. Lutein cells from corpora lutea of injected animals and of normal pregnancy are characterized by abundant, tortuous, tubular agranular endoplasmic reticulum and by mitochondria, many of which are disc-shaped with dense matrices and both villiform and lamelliform cristae. The endoplasmic reticulum is most abundant in lutein cells from pregnant animals, in which cells it is in the form of thin, highly tortuous tubules. The form of the lipid droplets seen in cells of stimulated animals varies greatly. Marginal foldings of the lutein cells on the perivascular space were found in all instances. Lutein cells from hypophysectomized animals have a less highly developed agranular endoplasmic reticulum. The mitochondria have irregular outlines and a relatively lucid matrix. The lipid droplets in these cells show less tendency to be extracted, but are not so large or abundant as in the cells of onset controls. Granules believed to contain lipid pigments are common in the lutein cells of these control animals. It is suggested that lutein cells from corpora lutea which are actively secreting progesterone may be readily distinguished from lutein cells from non-active corpora by means of the multiple characteristics enumerated. It is further suggested that mammotrophic hormone has a general effect on the metabolism of lutein cells rather than solely affecting a specific organelle, the abundance or composition of which may be the limiting factor in the production of progesterone.     

Histochemical changes in acid and alkaline phosphatase activities in the growing follicles and corpora lutea of the rat ovary

Changes in acid and alkaline phosphatase activities are studied histochemically in different components of the growing follicles and corpora lutea of different generations. Theca cells of the growing follicles showed strong acid phosphatase activity as compared to that observed in the oocyte and granulosa cells. Alkaline phosphatase activity is localized only in the theca interna cells of growing follicles. However, after ovulation with the formation of corpora lutea, the granulosa lutein cells also showed this activity. Changes in the histochemical distribution of the acid and alkaline phosphatases in the follicles and corpora lutea are discussed in relation to folliculogenesis and corpus luteum and regression.     

Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.     

Immunohistochemical localization of taurine in the rat ovary, oviduct, and uterus.

The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:1133-1142, 2001)     

Ultrastructural changes in granulosa cells and plasma steroid levels after administration of luteinizing hormone-releasing hormone in the Western painted turtle, Chrysemys picta

In this study we investigated the effects of treatment by luteinizing hormone-releasing hormone (LHRH) on the morphology and steroid release of ovarian tissues in the Western painted turtle, (Chrysemys picta). In Experiment I, four adult female turtles were injected with synthetic mammalian LHRH (i.p., 500 pg/g bodyweight) and four with saline 2-3 weeks prior to ovulation. Granulosa cells from LHRH-treated turtles vs controls contained both preovulatory follicles (16-20 mm in diameter) and small follicles (0.5-1.00mm in diameter) with increased RER, free ribosomes and mitochondria with swollen cristae. An increase in the amount of cytoskeletal material (microfilaments) was observed in granulosa cells of the experimental turtles compared to the controls. Cytoplasmic extensions of the oocyte and granulosa cells were longer in the small follicles of treated animals, accounting for the observed increase in the thickness of the zona pellucida (ZP) over the controls. In Experiment II, administration of LHRH (i.p.) to 10 turtles during the same period triggered a substantial increase in plasma progesterone and estradiol-17beta levels over the 10 saline-injected controls. This supports the idea that in this species, as in mammals, steroidogenic activity in the ovarian follicles are under the control of the hypothalamic-pituitary axis. The ultrastructure and hormonal levels of the experimental animals were typical of untreated turtles just prior to ovulation. In this species the development of follicles and steroidogenesis can be stimulated prematurely by a releasing hormone from a nonreptilian origin.     

中国大鲵闭锁卵泡的显微和超微结构观察

用光镜和电镜观察了中国大鲵卵泡闭锁过程和闭锁小体的显微和超微结构。结果显示 ,大鲵闭锁小体是卵泡细胞侵噬卵母细胞并增殖形成细胞团 ,膜细胞未参与。在大部分卵泡处于缓慢生长期时 ,未发现卵泡闭锁现象 ;在 5、 6月份 ,卵巢内大部分卵母细胞进入卵黄形成前期 ,部分卵泡闭锁 ,但闭锁小体细胞的类固醇激素分泌结构特征不明显 ;在 7、 8月份 ,大多数卵母细胞处于卵黄形成期 ,闭锁小体细胞具有管泡状嵴线粒体、丰富的滑面内质网和脂滴、发达的高尔基体等。这些细胞学特征表明闭锁小体可分泌类固醇激素 ,以调节正常卵子的成熟。在大鲵中观察到的闭锁小体属于排卵前黄体     

Localization of some steroidogenic enzymes in the ovary of the rabbit

The distribution of delta 5-3 beta-HSD, peroxidase and cytochrome oxidase in immature, sexually mature and pregnant rabbit ovary has been studied histochemically. Corpora lutea are found only in pregnant rabbits. delta 5-3 beta-HSD is present in the theca interna of mature follicles, corpora lutea and interstitial gland cells but is absent in the granulosa cells of both developing and mature follicles. The granulosa cells of mature and developing follicles, hypertrophied theca interna and the luteal cells all show intense cytochrome oxidase activity. Peroxidase is present in the corpora lutea only. It is suggested that delta 5-3 beta-HSD in the theca interna and interstitial gland cells is the enzyme responsible for steroid synthesis in the ovaries of immature as well as sexually mature rabbits, while peroxidase and delta 5-3 beta-HSD present in the corpora lutea together regulate luteal steroidogenesis during pregnancy. The intense cytochrome oxidase activity together with peroxidase and delta 5-3 beta-HSD confirms the observations that this tissue is a site of intense oxidative activity.     

Luteal regression in the primate: different forms of cell death during naturaland gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis

Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated by light and electron microscopy. Functional corpora lutea were studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora lutea induced to regress by administration of GnRH antagonist or prostaglandin F(2alpha) analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate.     

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.     

Postnatal androgenization induces premature aging of rat ovaries

In the present paper, we report that ovaries of adult rats treated with testosterone propionate (TP) on a critical postnatal Day 5 exhibit histologic and immunohistochemical findings which resemble those of the anovulatory ovaries in middle-aged female rats. The sterile rat model has been long known whereas ovarian failure seems to be a reason for anovulation with normal hypothalamo-pituitary-gonadotropin background. Appropriate function of ovarian steroidogenic cells is also regulated by mesenchymal cells. To characterize the ovarian failure, we studied the histology, luteinizing hormone receptor (LHR) expression, and characterized changes of vascular pericytes, T cells, and dendritic cells in ovarian steroidogenic compartments consisting of interstitial cells (ISC) of ovarian interstitial glands, and granulosa and theca interna cells of ovarian follicles. Normal adult ovaries contained 63% of mature interstitial glands. The mature ISC exhibited moderate cytoplasmic and strong surface LHR expression and fine (<5 micrometer) cytoplasmic vacuoles (ISC of 'luteal type'). They originated from young ISC of 'thecal type,' which exhibited strong cytoplasmic LHR expression. Remaining 37% were aged interstitial glands, which consisted of aged ISC (increased cytoplasmic vacuolization, nuclear pyknosis, and reduced surface LHR expression) and regressing ISC (weak cytoplasmic and no surface LHR expression). However, no mature ISC of 'luteal type' were detected in anovulatory ovaries of adult rats (45- and 60-day-old) injected with TP (100 or 500 microgram) on postnatal Day 5 (TP rats). Their ovaries contained 96% of aged interstitial glands with aged and regressing ISC. Remaining 4% were abnormal interstitial glands with direct transition of young ISC of 'thecal type' into aged ISC (young/aged glands). Lack of mature ISC, and similar amount of aged (96%) and young/aged interstitial glands (4%) was also detected in anovulatory ovaries of untreated persistently estrous middle-aged (10-month-old) females (aging PE rats). The aging process in TP and aging PE rats was accompanied by regression of vascular pericytes, T cells, and dendritic cells within the interstitial glands. In addition, anovulatory ovaries of TP rats and aging PE females contained mature follicles exhibiting LHR overexpression by granulosa cells, and aged (cystic) follicles with reduced layers of granulosa cells lacking LHR expression. In contrast, when the rats were injected with 500 microgram of TP later, on postnatal Day 10, the adult females exhibited estrous cycles and normal ovaries with corpora lutea. These results show that injection of TP during the critical postnatal period causes a lack of mature and preponderance of aged ISC in adult ovaries, accompanied by degeneration of mesenchymal cells. We suggest that mesenchymal cells regulate qualitative aspects of tissue-specific cells, and this function of mesenchymal cells is programmed during the critical period of development.     

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.     

Absence of specific follicle stimulating hormone receptors in porcine corpora lutea

Crude cell membrane preparations of corpora lutea from 13 pigs in different phases of the estrous cycle or pregnancy were assayed for the presence of specific follicle stimulating hormone (FSH) receptors using highly purified ovine FSH. Testicular tissue from boars and granulosa cells from porcine follicles served as positive controls. Scatchard analysis was used to determine binding affinity of FSH to target tissues as well to study human chorionic gonadotropin (hCG) bindability to corpora lutea membranes. In contrast to testicular tissue and granulosa cells, no specific FSH binding was detected in luteal tissue during the estrous cycle or pregnancy in pigs.     

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: Tissue culture approach

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P₄) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.     

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.     

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.     

Persistent vaginal cornification in mice treated with estrogen prenatally.

Twelve of 14 female mice of the ICR strain which had received a single injection of 50 mug estradiol-17beta on day 17 of fetal life exhibited irreversible cornification or stratification of the vaginal epithelium which persisted after ovariectomy until sacrifice performed 42-48 days later. Eight of the 12 mice had corpora lutea in their ovaries removed at 3-5 months of age. A similar injection of estradiol on day 15 of fetal life induced irreversible cornification or stratification of the vaginal epithelium in 6 of 12 females and only one of the 6 had corpora lutea in its ovaries when removed at 3-5 months. Mice given the same dose of estradiol on the day of birth or at 3 days of postnatal age invariably had ovaries bearing follicles of varying sizes and hypertrophied interstitial tissue but no corpora lutea. Changes in the vaginal epithelium in these animals were less remarkable as compared to that in prenatally treated mice.     

Effects of gonadotrophin on the ovary of Apodemus flavicollis in winter anoestrus

Wild-caught female Apodemus flavicollis were given daily subcutaneous injections of PMSG + HCG on two consecutive days (2xPMSG + HCG), HCG on two days (2 X HCG), PMSG + HCG until their vagina became perforate (Perforate), and PMSG + HCG until they became perforate and were left undisturbed for another five days (Perforate + 5). Untreated females served as controls. The ovaries and uteri increased in weight as a result of the treatment. The number of healthy follicles increased in "perforate" females and decreased in "2 X HCG". Luteinization of the follicles and possibly of the interstitial tissue was seen in "2 X HCG". In this group, the mean diameter of the corpora lutea increased, and it decreased in "perforate" and "perforate + 5". The mean number of corpora lutea was unaffected by the treatment. The activity of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) was strong in the corpora lutea of the untreated females and remained so throughout treatment. The interstitial tissue of the untreated females showed only weak 3 beta HSD activity, but the activity was strong in all the experimental groups. One female ovulated, but it is not clear if it was in response to treatment.     

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.