Regulation of follicular development by diethylstilboestrol in ovaries of immature rats

Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (less than 200 microns, 200-400 microns and greater than 400 microns) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes. Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36-48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries. These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully.     

In vitro effects of dihydrotestosterone on granulosa cell production of estrogen and progesterone

To investigate the direct effects of androgens on follicle development, intact, immature female rats were given 8 IU PMSG (0 hour) and four injections of either vehicle or dihydrotestosterone (DHT), 1 mg/kg body weight, at 0, 12, 24, and 36 hours after PMSG. Granulosa cells from small (less than 200 microns), medium (200 to 400 microns), and large (greater than 400 microns) follicles were isolated and cultured in the presence or absence of 0.5 microM DHT in vitro for 48 hours, and the medium was assayed for progesterone and estrogen. Results show that DHT caused an increase in progesterone accumulation in all granulosa cells, regardless of follicle size. However, DHT inhibited estrogen accumulation in granulosa cells from different-size follicles and the inhibition varied depending on the duration of androgen exposure in vivo. The inhibition of estrogen accumulation was seen in granulosa cells from small follicles without prior exposure to DHT in vivo, while an inhibition of estrogen accumulation was seen in granulosa cells from medium and large follicles exposed to DHT treatment in vivo. Taken together, the results of experiments with in vivo and/or in vitro DHT treatment show that the androgen increases granulosa cell progesterone synthesis regardless of follicle size. However, the estrogen accumulation by granulosa cell is dependent on follicle size and duration of DHT exposure.     

Direct ovarian effects of a GnRH agonist in hypophysectomized immature rats: inhibition of theca-interstitial luteinizing hormone receptor mRNA expression and induction of interstitial cell differentiation

The effect of a gonadotropin-releasing hormone (GnRH) agonist on luteinizing hormone (LH) receptor mRNA expression was examined histologically in the ovaries of immature hypophysectomized (HPX) rats by in situ hybridization. In the ovaries of HPX rats treated with diethylstilbestrol (DES) and pregnant mare serum gonadotropin (PMSG), LH receptor mRNA was expressed in the granulosa cells of mature follicles as well as the theca-interstitial cells. In DES-primed ovaries of rats treated with both GnRH agonist plus PMSG, many follicles were luteinized without ovulation, and the signal of LH receptor mRNA disappeared completely in the theca-interstitial cells as well as the luteinized cells, but remained in the granulosa cells of unaffected mature follicles. The complete suppression of the theca-interstitial LH receptor expression by GnRH agonist was also observed in HPX rats that received no other treatment. On the other hand, the coadministration of a GnRH antagonist with PMSG resulted in the hyperstimulation of follicular growth, accompanied by very strong expression of LH receptor mRNA in the granulosa cells as well as the thecainterstitial cells. In addition, morphological changes in the ovarian interstitial cells were also induced by the administration of GnRH agonist in HPX rats: loose connective tissue decreased and the interstitial cell mass markedly increased. The increase of the interstitial cells became more prominent when rats were treated with GnRH agonist and testosterone simultaneously. These results suggest that GnRH may be an important factor for modulating the interstitial cell function and differentiation in the rat ovary.     

Inhibition by diethylstilboestrol of proliferative potential of follicles of different sizes in immature rat ovaries

Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter less than 200 microns (small), 200-400 microns (medium) and greater than 400 microns (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells. Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentrations are highest.     

Role of ovarian theca and granulosa cell interaction in hormone productionand cell growth during the bovine follicular maturation process

We have investigated the possible role of theca and granulosa cell interaction in the control of the hormone-producing activity and growth of granulosa and theca cells during bovine ovarian follicular development, using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. When follicular cells were isolated from small follicles (3-5 mm), theca cells reduced estradiol, progesterone, and inhibin production by granulosa cells to 14 +/- 5%, 64 +/- 6%, and 27 +/- 4%, respectively, of the production by granulosa cells cultured alone. On the other hand, when the cells were isolated from large follicles (15-18 mm), theca cells increased these levels to 253 +/- 34%, 156 +/- 24%, and 287 +/- 45%, respectively. Theca cells did not affect the growth of granulosa cells. Androstenedione production by theca cells was augmented by granulosa cells to 861 +/- 190% (in small follicles) and 1298 +/- 414% (in large follicles), respectively. The growth of theca cells was also augmented by granulosa cells (small follicle, 210 +/- 43%, and large follicle, 194 +/- 24%, respectively). These results indicate that theca cells secrete factor(s) inhibiting the differentiation of immature while promoting that of matured granulosa cells; they also suggest that granulosa cells secrete factor(s) promoting both the differentiation and growth of theca cells throughout the follicular maturation process.     

Effect of dihydrotestosterone on the growth and function of ovarian follicles in intact immature female rats primed with PMSG

Intact, immature female rats were primed with PMSG and treated with 4 injections of DHT. DHT given at 0, 12, 24 and 36 h caused a significant decrease in the ovulation rate 72 h after the PMSG treatment. Concurrent treatment with oestrogen reversed the inhibitory effects of the androgen. The androgen effect was apparently exerted directly on the ovary since DHT did not alter the surge of LH and FSH which occurred at 58 h after PMSG treatment. The DHT inhibition of ovulation was observed in the treatment cycle as well as in subsequent cycles which followed a second PMSG injection. This finding suggests that intermediate size follicles were also adversely affected by the androgen. To confirm that androgen affects follicles of all size ranges, follicles less than 200 microns, 200-400 microns and greater than 400 microns in diameter were isolated from the ovaries of rats treated with PMSG and DHT or the vehicle. The follicles were isolated by density gradient separation of follicles followed by filtration with pre-calibrated Teflon sieves. In some experiments, granulosa cells were also harvested from isolated follicles. DHT treatment did not affect the numbers of follicles of any size but did reduce the oestrogen content of follicles of all sizes. Follicles from DHT-treated animals contained fewer granulosa cells and the cells from treated animals had lower aromatase activity than did cells from control rats. Taken together, these findings suggest that DHT reduces the ovulation rate by decreasing the number of granulosa cells/follicle and by altering the oestrogen synthetic abilities of the cells. All follicles, regardless of size, were sensitive to androgen treatment.     

Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-I and follicle-stimulating hormone in vitro.

Granulosa cells of antral follicles both proliferate and undergo differentiation. The aim of the present work was to study the mechanisms controlling the balance between proliferation and differentiation in granulosa cells during the development of antral follicles in the ewe. For this purpose, the responses of both activities to insulin-like growth factor-I (IGF-I) and to FSH in vitro were studied comparatively in granulosa cells from small antral follicles (1-3 mm in diameter) and large antral follicles (5-7 mm in diameter). In granulosa cells from large follicles, IGF-I enhanced both basal and FSH-induced progesterone secretion after a 24-h delay period; this effect was lower and further delayed in cells from small follicles. Reciprocally, FSH increased IGF-I-stimulated progesterone secretion in cells from large follicles. IGF-I increased the thymidine labeling index of granulosa cells from small follicles within 24 h and enhanced cell multiplication. In cells from large follicles, this effect was lower and delayed, but IGF-I also enhanced cell survival. Culture at high density of plating inhibited the proliferative response of both types of cells to IGF-I. FSH was without effect on granulosa cell multiplication. These results suggest that the cytodifferentiative and the growth-promoting effects of IGF-I are clearly distinct. We propose that they would be exerted on two distinct granulosa cell subpopulations, nonproliferating and proliferating cells, respectively. Differences in the responsiveness of cells from small and large follicles could be related to differences in the proportion of these two cellular subtypes.     

Effect of pregnant mares' serum gonadotrophin on the micromorphology of antral follicles in heifers, with special reference to atypical granulosa cells.

Five Dutch-Friesian heifers were injected i.m. with 3000 iu pregnant mares' serum gonadotrophin (PMSG) on day 10 of the oestrous cycle, to study the effects on the number and micromorphological quality of antral follicles (> or = 0.3 mm in diameter). The ovaries were collected 48 h after PMSG injection. As well as the presence of mitotic figures and the absence of pyknotic nuclei in the granulosa, atypical granulosa cells were found in nonatretic follicles. These cells had an oblong nucleus and stained with toluidine blue. They were characterized by their dark cell matrix, and the presence of numerous free ribosomes and intermediate filaments of varying quantity. Atypical granulosa cells were micromorphologically similar to fibroblast-like cells in the theca. Their presence coincided with the occurrence of degenerative changes in the cytoplasm of nearby granulosa cells and they were more frequent in atretic follicles. The presence of atypical granulosa cells in follicles hitherto called nonatretic is therefore probably associated with the onset of follicular atresia. In the PMSG-treated heifers, the mean number of large (> or = 6.0 mm in diameter) antral follicles was greater than in the control group (18.4 +/- 4.0 versus 3.0 +/- 1.0), because of an increase in the number of large nonatretic follicles (11.8 +/- 4.4 versus 0.4 +/- 0.2). After hormone treatment, the mean number of medium-sized (3.0-5.9 mm) nonatretic follicles also increased (6.4 +/- 1.3 versus 1.8 +/- 1.0). PMSG did not change the mean number of nonatretic follicles < 3.0 mm or that of atretic follicles in the different size categories. However, when follicles hitherto called nonatretic, with atypical granulosa cells, were taken together with the group of atretic follicles, PMSG appeared to increase the mean number of large atretic follicles (13.6 +/- 2.4 versus 3.0 +/- 1.0). The mean number of medium-sized and large nonatretic follicles without atypical granulosa cells was markedly increased (3.8 +/- 1.0 versus 0.2 +/- 0.2 and 4.6 +/- 1.9 versus 0.0, respectively). The data demonstrate that PMSG stimulates the formation not only of nonatretic follicles > or = 3.0 mm, but also of atretic follicles > or = 6.0 mm.     

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.     

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     

Increases in ovarian GnRH receptors by following GnRH treatment

Using a gonadotropin-releasing hormone (GnRH) analog, [D-Ser (tBu6)] des-Gly10-GnRH-N-ethlamide (GnRHa) as a ligand the binding capacity of the rat ovary to GnRH during sexual maturation and the mechanism regulating GnRH binding capacity were examined. Specific high affinity binding sites for GnRH were observed in the ovary and the Kd values for the granulosa cells and the residual tissue were similar to those of whole ovary. During sexual maturation, the GnRH binding capacity of the ovary rose from 7 days of age to a peak of 28 days and declined during the prepubertal period. The treatment with PMSG decreased GnRH binding capacity in the residual tissue as well as in the whole ovary but did not change the binding capacity in the granulosa cells in diethylstilbestrol (DES) primed hypophysectomized rats. Repeated injections of GnRH caused a significant increase in the number of GnRH receptors of the ovary in PMSG treated DES primed hypophysectomized rats but not in the saline treated rat. The granulosa cells exhibited increases in GnRH binding capacity following repeated administrations of GnRH more than the residual tissue did. In GnRH treated DES primed hypophysectomized rats, increasing doses of PMSG increased the binding capacity in the granulosa cells but decreased the binding capacity in the residual tissue. From these findings, GnRH in combination with PMSG seems to have stimulatory effects on GnRH binding capacity and to increase the sensitivity to GnRH in the granulosa cells.     

Sequential changes associated with the degeneration of preovulatory rat follicles.

In 26-day-old rats, follicles capable of ovulation were present 48 h after PMSG injection and they degenerated if not exposed to an ovulating dose of HCG. In such follicles 125I-labelled LH bound to the thecal and granulosa cells. By 60 h after PMSG, LH binding to the granulosa cells was reduced by 46% although these follicles retained their ability to ovulate. LH binding to the granulosa cells was lost in most follicles by 72 h and ovulation could not be induced. The thecal cells still possessed LH binding sites at 72 h after PMSG. HCG stimulation of these follicles resulted in disruption of the granulosa and the invasion of blood cells into the antrum.     

Action of PMSG on follicular populations in the heifer

The short-term action of PMSG on the population of growing follicles in cattle was studied using histological methods. On Day 7 of a synchronized oestrous cycle 10 Friesian heifers were unilaterally ovariectomized. The remaining ovary was immediately stimulated by an injection of PMSG (2000 i.u.) and was removed 48 h after the preovulatory discharge of LH. Control animals did not receive any injection of PMSG. In all ovaries, follicles greater than 70 micron diameter were counted, measured and checked for atresia. The mitotic index in granulosa cells of follicles of different sizes was estimated in both ovaries of all the PMSG-injected animals. Unilateral ovariectomy alone had no significant effect on follicular populations. In the interval between PMSG injection and removal of the second ovary (148 +/- 22.7 h), PMSG significantly increased the number of normal preantral follicles but did not change the number of normal antral follicles. The mitotic index doubled in preantral and early antral follicles but remained unchanged in large antral follicles. PMSG stimulated slightly the growth of the antrum in large antral follicles but did not stimulate its formation in preantral follicles. The incidence of atresia among antral follicles, particularly the largest ones (diam. greater than 1.7 mm), was significantly reduced after PMSG, suggesting some 'rescue' of follicles from atresia.     

Comparison of granulosa cell proliferation in small follicles of hypophysectomized, prepubertal, and mature rats

The factors that control the rate of granulosa cell proliferation during follicular development are unknown. The object of this study was to test the hypothesis that growth rates of small and medium follicles may be modulated by cyclic alterations in endogenous hormone concentrations. Follicular growth in adult cycling rats was compared with hypophysectomized rats, untreated prepubertal rats, and prepubertal rats treated with exogenous gonadotropins. Cell kinetics was studied using a metaphase arrest technique and by long-term infusion of [3H]thymidine. Many follicles of hypophysectomized rats showed evidence of continued cell proliferation despite the absence of gonadotropins. In hypophysectomized rats, follicular growth was able to proceed to the size of the largest healthy non-preovulatory follicles in the proestrous rat ovary. Follicular growth in prepubertal rats progressed little beyond this same size range. Granulosa cell proliferation rates differed in immature rats and cycling rats. Granulosa cells in small follicles (80-180 cells in the largest cross-section) of cycling rats grew slowly. However, granulosa cells in small follicles of immature rats were among the fastest growing in the ovary. These results suggest that, although gonadotropins are not absolutely required to maintain granulosa cell proliferation in small follicles, the rate at which these follicles grow varies under different hormonal conditions.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Alterations in the cell cycle characteristics of granulosa cells during the periovulatory period: evidence of ovarian and oviductal influences

Granulosa cells at different stages of differentiation were collected from ovarian follicles and oviducts during the periovulatory period, and their nuclear DNA content was monitored by flow cytometry to establish their cell cycle characteristics (G0 + G1, S, G2 + M). The proportion of cells in the three phases of the cell cycle varied in characteristics patterns depending upon the time they were collected, before or following ovulation. Granulosa (cumulus) cells recovered from ovulated oocytes were mitotically inactive as shown by the large proportion of cells with a 2C amount of DNA and the absence of cells in S phase. The proportion of granulosa cells in G2 + M decreased when recovery from the oviducts was delayed. In contrast, granulosa (cumulus and/or mural) cells recovered from preovulatory follicles prior to luteinizing hormone (LH) exposure contained a considerable population of cells undergoing DNA synthesis, and a decreased proportion of cells with a 2C DNA content. Our findings indicate that granulosa cells undergo dynamic and characteristics changes in all cell cycle phases during the periovulatory period, within follicular and oviductal environments. Intrafollicular events appear to play a major role in controlling DNA synthesis, proliferation, and related cell cycle events in the granulosa cells. Flow cytometric techniques provide objective and detailed information on the cell cycle characteristics of granulosa cell populations at different stages of differentiation. Elucidation of the mechanisms regulating cell cycle parameters of granulosa cells and their physiological significance thus seems feasible.     

Periovulatory changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in hormonally induced immature female rats

Immature female rats were treated with PMSG and human CG to induce ovulation. Sequential treatment with these hormones allowed us to investigate variations in the production of inhibin subunits shortly before ovulation and during the induced luteal phase. Using this model, we found that expression patterns for the alpha-, beta A-, and beta B-subunits were similar to those observed in mature cycling animals: administration of PMSG (to mimic the gonadotropin surge) led to a sharp increase in the expression of all three subunits in large preovulatory follicles whereas injection with human CG (to induce ovulation) caused a decrease in the levels of the respective mRNAs. In contrast to mature females, shortly before ovulation, levels of inhibin alpha-subunit mRNA were low in small antral follicles (approximately 350 microns). In addition, at that time, inhibin beta A- and beta B-subunits mRNAs were present in several large follicles (greater than 500 microns). More than 2 days after ovulation, inhibin beta A- and beta B-subunit mRNAs could not be detected in small antral size follicles (approximately 350 microns) of hormonally induced females. On the other hand, hybridization signals for the inhibin alpha-subunit were observed in some small antral and preantral size follicles, while signals were very low or undetectable in a large number of atretic follicles. Using this synchronized ovulation model, hybridization patterns for inhibin beta A-subunit mRNA was observed in interstitial cells, 8-10 h after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular development in the sheep after priming with PMSG

The administration of PMSG to sheep early in the oestrous cycle (Days 2 or 3) results in the formation of follicular cysts of varied morphology by Days 8 or 9. These may persist for up to 14 days after injection. If PMSG is given on Day 5 or later approximately 50% of such cysts ovulate. However, when PMSG is given at the beginning of the cycle (Day 2 or 3), the membrana granulosa is lost from the majority of the cysts and the theca interna luteinizes. The major hormone secreted by such luteinized follicles is progesterone. The structure of the steroid-secreting cells of the follicles is similar to that of large luteal cells of granulosa cell origin in cyclic corpora lutea. It is suggested that under suitable luteinizing conditions thecal cells may acquire many of the characteristics of granulosa cells.     

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.     

Formation of the hamster zona pellucida in relation to ovarian differentiation and follicular growth

Using an immunofluorescence technique on ovarian sections, zona-immunoreactive components were detected in the cytoplasm of the oocyte from the beginning of its growth, when it is surrounded by only a thin squamous follicular cell layer, up to the end of its growth. In parallel with oocyte growth, the staining intensity decreased in the ooplasm. No staining was observed in the cytoplasm of the granulosa cells during normal follicular development in adult cyclic females. However, staining of the granulosa cells was observed at some stages of follicular development in immature females. This staining was especially evident in the ovaries of immature females (22 or 26 days old) stimulated with PMSG. In addition, the staining of the granulosa cells was consistently observed in ovaries showing an abnormal histology. Increased staining of the zona at its outer and inner regions could be distinguished in normal follicles, but when staining occurred on the granulosa cells no such pattern was observed over the zona matrix. These studies indicate that the oocyte itself but not the granulosa cells elaborates the native immunogenic material of the zona pellucida. The administration of PMSG at particular stages of ovarian differentiation interferes with follicular development leading to an abnormal extracellular assembly of the zona and its degradation (phagocytosis) by the surrounding granulosa cells.