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.     

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.     

Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.     

The androgen receptor does not mediate progestin regulation of progesterone biosynthesis in cultured rat granulosa cells

In the present investigation the influence of androgens and progestins on the FSH modulation of progesterone biosynthesis was studied in cultured rat granulosa cells. Cells obtained from the ovaries of immature estrogen treated rats were cultured for three days in serum free medium or in medium supplemented with FSH or CPA, with or without reduced androgen DHT or the synthetic progestin R5020 alone or in combination with the anti-androgen CPA. Treatment with FSH increased pregnenolone, progesterone and 20 alpha-OHP accumulation in the culture medium 20-, 14- and 7-fold, respectively. Furthermore FSH increased the activity of the enzyme 3 beta-HSD. Concurrent treatment with DHT or R5020 augmented the FSH stimulated steroidogenesis of cultured cells. The androgen enhancement of FSH stimulated steroidogenesis of cultured granulosa cells was blocked by concomitant treatment with CPA, whereas treatment of cultures with anti-androgen did not affect the stimulatory effect of the synthetic progestin R5020.     

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.     

Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.     

Modulation of 17 alpha-hydroxylase/C17,20-lyase activity in porcine theca cells

The major source of ovarian androgen is the theca cells. Androgens are produced by the conversion of progestins by the 17 alpha-hydroxylase/C17,20 lyase enzymatic system (lyase). The 3 beta-hydroxysteroid dehydrogenase and aromatase enzymes in the theca cells are modulated by gonadotropins as well as by steroids produced locally. Therefore, the combined effects of hCG plus progesterone, estradiol, or dihydrotestosterone (DHT) on microsomal lyase activity in theca cells from large and medium-sized follicles were determined. Theca cells (3 x 10(6) cells/6 ml/well) were cultured in Medium 199 (M199) containing only insulin (10 micrograms/ml) and transferrin (5 micrograms/ml). At 24 h, theca cells were treated with M199, hCG (15 ng/ml), progesterone, estradiol, or DHT (100 ng/ml) or a combination of hCG + one of the three steroids. Media were removed at various times of culture (27-72 h) and levels of androgen determined by RIA. Microsomes were incubated with 1 microCi [3H]progesterone +0.5 mM NADPH and radioactive conversion products were measured after purification by thin layer chromatography. Administration of progesterone, estradiol, or DHT alone had little effect on lyase activity in theca cells from medium-sized follicles whereas the addition of hCG alone significantly increased lyase activity in these cells. However, concomitant addition of any steroid with hCG inhibited the increase in lyase activity after the addition of hCG alone. Theca cells from large porcine follicles had a higher basal level of lyase activity compared to theca cells from the smaller follicles. Lyase activity in theca cells from large follicles was enhanced by progesterone; estradiol was inhibitory. DHT initially stimulated lyase activity in theca cells from large follicles, but was inhibitory later in culture. In contrast to its marked effect on theca cells from medium follicles, hCG had only a small effect on lyase activity in theca cells from large follicles. Thus, thecal lyase activity increased as the follicle matured, providing more androgen substrate for the production of estrogen. Lyase activity in theca cells of medium follicles appears to be regulated predominantly by gonadotropin from the pituitary while intraovarian regulation of lyase activity by steroids may be more important in larger follicles.     

Flow cytometric analysis of granulosa cells from developing rat follicles.

Immature rats were treated with diethylstilboestrol (DES) or pregnant mares' serum gonadotropin (PMSG) and forward angle light-scatter (FALS) and 90 degrees light-scatter (90 degrees LS) signals were used to measure the size and the granularity (internal organization) of the granulosa cells, respectively. The results confirmed the presence of two major populations of granulosa cells in the ovaries of both groups of rats, with the same percentage of larger cells in both treatments (52.3% in DES, 49.5% in PMSG). Since DES treatment brings about granulosa cell growth while PMSG treatment causes growth and differentiation, it is evident that there is heterogeneity in granulosa cell sizes during different states of growth and differentiation. There was also heterogeneity in sizes of granulosa cells harvested from follicles of small (less than 210 microns), medium (210-420 microns) and large (greater than 420 microns) diameter. Quadrant analysis of granulosa cells in various fractions collected from Percoll gradients suggested an increase in granularity in the small and large granulosa cell populations. Cell cycle analysis of small and large granulosa cell populations collected from large follicles of rats treated with PMSG indicated that each population was distributed in G0/G1, S and G2/M phases. These results demonstrate that populations of small and large granulosa cells exist in rat ovarian follicles during various stages of growth and differentiation.     

Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles

The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles. Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM). Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration. Northern blot analysis revealed a single band approximately 2.1 kb in size. Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels. Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH. Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar. In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles. These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion. This last observation indicates that control at sites other than SCC mRNA can affect progesterone production.     

Role of estrogen and androgen in maintaining the preovulatory follicle

Summary The effects of Nitromifene citrate (CI 628), an antiestrogen, and Flutamide, an antiandrogen, on the ultrastructure and viability of the preovulatory follicle and granulosa cells were examined both in vivo and in vitro. In vivo administration of either antihormone induced degeneration within the granulosa cells. In some of the affected granulosa cells, the nuclear material was condensed while the cytoplasm and associated organelles were unaltered. In others, the density of the cytoplasm was reduced, the smooth endoplasmic reticulum was dilated but the nucleus remained unaltered. In vitro, either antihormone reduced granulosa-cell viability but the granulosa cells were twenty times more sensitive to CI 628 than to Flutamide. In addition, exposure to CI 628 induced nuclear condensation without affecting the cytoplasm, while Flutamide induced the deterioration of the cytoplasm without altering the nucleus. These observations suggest that: (1) both estrogen and androgens control the viability of the granulosa cells and thereby the follicle, (2) the action of estrogen and androgen is mediated through receptors within the granulosa cells since these antihormones prevent the nuclear uptake of their respective hormone, (3) the granulosa cells of preovulatory follicles appear to be more dependent on estrogen than on androgen, and (4) each steroid appears to have a specific role in maintaining the granulosa cell; estrogens control the integrity of the nucleus while androgens preserve the cytoplasmic organization of the granulosa cell.The authors are indebted to Dr. Neri of Schering AG for donating the Flutamide and to Dr. Westland of Warner-Lambert/Parke-Davis for providing CI-628     

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.     

Effect of activin-a on goat granulosa cell steroidogenesis

Growth factors are said to play a significant role in the development of ovarian follicles. We wished to measure the content of one growth factor, activin-A in goat ovarian follicles, and study its effect on goat granulosa cells steroidogenesis. The follicular fluid content of activin-A from small, medium and large antral follicles was determined by two-site enzyme immunoassay. The results showed that activin-A concentration in the follicular fluid increased as the size of the follicle increased and, thus, may act as a local regulator of follicle development. To examine this possibility, the effect of increasing concentration of activin-A (0, 1, 10, 100 ng/mL) on differentiated goat granulosa cells steroidogenesis was evaluated in vitro for 48 hours in a chemically defined medium. Activin-A treatment resulted in a significant inhibition of progesterone production concomitant with a significant stimulation of estradiol production. These results were confirmed by time-effect of 50 ng/mL activin-A on goat granulosa cells steroidogenesis for 24, 48 and 72 hours. Granulosa cells displayed differential steroidogenic responses to activin-A, estradiol production becoming enhanced and progesterone production suppressed. Based on these findings, it appears that activin-A is a local regulator of goat granulosa cell steroidogenesis, and may act to promote granulosa cell differentiation and inhibit its luteinization.     

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.     

Steroidogenesis in porcine atretic follicles: loss of aromatase activity in isolated granulosa and theca

To evaluate the mechanisms involved in the reduction of estrogen concentrations in porcine follicular fluid during atresia, nonatretic and atretic follicles ranging from 4 to 7 mm in diameter were selected. Follicular fluid estrogen concentrations were 7-16-fold less in the atretic follicles. Isolated granulosa cells from atretic follicles demonstrated a significant reduction in aromatase activity and in follicle-stimulating hormone (FSH)-induced progesterone production in vitro compared to granulosa cells from nonatretic follicles. Isolated theca from atretic follicles also demonstrated a reduction in estrogen production. However, androgen concentrations were equivalent in the follicular fluid of atretic and nonatretic follicles, and theca from atretic follicles maintained testosterone and androstenedione production in vitro. The loss of thecal aromatase activity with atresia is not secondary to a reduction in FSH responsiveness, since FSH did not increase thecal progesterone production in vitro. Cell degeneration also does not account for the reduction in thecal estrogen production, since both androgen output in vitro and follicular fluid androgen concentrations were maintained. These data thus demonstrate that a mechanism other than reduced FSH responsiveness must account for the selective loss of thecal aromatase activity in this stage of atresia.     

The source of follicular androgens in the hamster follicle

The comparative ability of granulosa cells and theca of the hamster preovulatory follicle to produce androgens in vitro from endogenous and exogenous substrates was assessed. The results indicate that theca are the major source of follicular androstenedione, but that the granulosa cells may be the major source of follicular testosterone. Theca and granulosa cells accumulate comparable amounts of dihydrotestosterone from exogenous androstenedione and testosterone and both may be a significant source of follicular DHT. LH stimulates the conversion of progesterone and 17 alpha-OH progesterone to androstenedione, testosterone and DHT in theca. LH does not stimulate the conversion of androstenedione to testosterone or DHT, and that of testosterone to DHT in either granulosa cells or theca. FSH, in granulosa cells but not in theca, stimulates the conversion of adrostenedione to testosterone but it has no effect in DHT accumulation from exogenous testosterone.     

Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

Effect of TNF-alpha on LH and IGF-I modulated chicken granulosa cell proliferation and progesterone production during follicular development

This study demonstrates the effects of recombinant human tumour necrosis factor a (rhTNF-alpha) and conditioned medium of the HD11-transformed chicken macrophage cell line on cultured chicken granulosa cells. Effects were studied on basal, IGF-I- and LH-stimulated progesterone production and cell proliferation. Recombinant human TNF-alpha stimulated basal progesterone production in a dose-dependent manner in the granulosa cells of the largest follicle but had no effect on cells from the third largest follicle. TNF-alpha stimulated and sometimes inhibited progesterone production stimulated by IGF-I and LH alone or in combination depending on the size of the follicle and the concentration of LH or IGF-I applied. However, the inhibitory effect of TNF-alpha was significantly more pronounced in cells from the third largest follicle when high concentrations of IGF-I, LH or a combination of both were applied. TNF-alpha had no effect on basal cell proliferation in both the largest and the third largest follicles, but regulated responses to IGF-I and a combination IGF-I and LH in the cells of the third largest follicle but not those of the largest follicle. The data indicate that the normal hierarchy of follicles is maintained in the chicken ovary through the regulation of the activity of IGF-I and its interaction with LH. Conditioned medium of LPS-activated HD11 macrophages mimicked the effects of TNF-alpha and its interaction with IGF-I and LH on progesterone production and cell proliferation. The observation that the HD11-conditioned medium contained TNF-alpha indicates that TNF-alpha produced by macrophages found in chicken follicles modulates granulosa cell growth and differentiation.     

Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary.

In the study reported here, we investigated the effect of androgens on recruitment of resting, primordial follicles into the actively growing pool. Healthy, random-cycling female rhesus monkeys were treated with testosterone, dihydrotestosterone (DHT), or vehicle for 3-10 days, after which ovaries were collected for histological analysis. The first stage of follicle growth is the formation of the primary follicle, consisting of an oocyte surrounded by a single layer of cuboidal granulosa cells. The number of primary follicles was significantly increased over time in testosterone-treated animals. In situ hybridization showed that androgen treatment resulted in an increase to 3-fold in insulin-like growth factor I (IGF-I) and to 5-fold in IGF-I receptor mRNA in primordial follicle oocytes. DHT effects were comparable to those of testosterone, showing that these are androgen receptor-mediated phenomena. These data show that androgens promote initiation of primordial follicle growth and implicate oocyte-derived IGF-I in this activation process.     

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.     

Distribution of delta-5 -3beta-hydroxysteroid dehydrogenase activity in the Graafian follicle of the sheep.

The localization of delta-5 -3beta-hydroxysteroid dehydrogenase (3 beta-HSD) has been examined in ovarian follicles in vivo and in vitro, and related to oestrogen and progesterone production. In vivo, during the oestrous cycle, enzyme activity was restricted to the theca interna of the one or two most advanced follicles in each animal, but was present only between Day 2 and 5 and between Day 13 and ovulation. High levels of oestrogen were found in the ovarian venous blood only when follicles containing 3 beta-HSD were present. When sheep were injected with PMSG, the theca interna in a number ofsmall follicles acquired 3 beta-HSD activity and began to secrete oestrogen within 12 hr of the injection. The enzyme was not detected in the membrana granulosa of any follicles before ovulation but within a few hours of ovulation, 3 beta-HSD activity was present in the granulosa lutein cells. In vitro, large activated follicles exhibited 3 beta-HSD activity in the theca interna and secreted high levels of oestrogen into the culture medium. When LH was added to the medium oestrogen secretion was inhibited; within 48 hr, the follicles were secreting high levels of progesterone, and 3 beta-HSD activity was present in both the membrana granulosa and the theca interna. Dibutyryl cyclic adenosine monophosphate mimicked the effect of LH in suppressing oestrogen secretiion, but did not induce production of progesterone; the distribution of 3 beta-HSD activity infollicles treated with this nucleotide was the same as in those cultured in control medium.