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.     

Influence of in vitro plating density on rat granulosa cell responsiveness to follicle-stimulating hormone

The effects of cell plating density on granulosa cell sensitivity to follicle-stimulating hormone (FSH) were investigated, using a serum-free culture of cells obtained from immature, estrogen-treated rats. The cells were incubated at densities of 0.25 to 5 X 10(5) cells/dish with increasing concentrations of FSH for 2 days, and medium estrogen and progestin accumulation were measured by radioimmunoassay. Per-cell estrogen and progestin production rose with increasing FSH concentration and cell density up to 2 X 10(5) cells/dish. At a higher density (5 X 10(5)/dish), per-cell estrogen production fell; progestin production remained constant, although the major progestin produced was no longer progesterone, but rather its metabolite, 20 alpha-hydroxy-progesterone. The effects of changing cell density could not be accounted for by medium steroids or cytotoxic substances. It is concluded that in vitro plating density can markedly affect granulosa cell sensitivity to FSH. In vivo, changing intrafollicular cell densities may thus affect the ability of the whole cell complement to respond to gonadotropin.     

FSH-induced aromatase activity in hamster granulosa cells: effect of estradiol-17β in vitro

Summary We have shown previously that estradiol-17 (E₂) reduces number of ovulations in cyclic rats, induces atresia of the dominant preovulatory follicle in monkeys, and that the initial effects of this treatment include reduced viability and estrogen accumulation in vitro by aspirated granulosa cells (GC) from monkeys and hamsters. The present experiment was designed to determine whether the reduction in estrogen accumulation can be ascribed to a direct action of E₂ on the aromatization of androgen to estrogen in vitro. Female hamsters were injected with 30 I.U. pregnant-mare serum gonadotropin i.p. and sacrificed 3 days later. GC were aspirated from the largest follicles and incubated for 48 h (initial incubation period) in the presence of human pituitary follicle-stimulating hormone (hFSH, 100 ng/ml). Following initial incubation, GC were further incubated for up to 24 h (secondary incubation period). During this subsequent incubation, medium was supplemented with 100nM ³H-1-androstenedione (³H-A₄). Initial incubation with E₂ at doses of 10 ng/ml, 100 ng/ml and 1 m E₂/ml induced variability in GC response, and a maximal depression of 70%. The inhibition by E₂ of hamster GC function in vitro parallels that shown in vivo for both hamsters and monkeys, but contrasts with that shown for rats. Thus, hamsters may represent an appropriate model in which to study the atretogenic effects of E₂ directly on antral follicle development.     

The effects of dehydroepiandrosterone (DHEA) and its metabolites on the polycystic ovarian condition (PCO): cystogenic changes of rat granulosa cells in vitro

During mammalian folliculogenesis, granulosa cells (GCs) are initially steroidogenically quiescent, later proliferate, and subsequently commence to hormonally differentiate, first producing estrogen and later, in the preovulatory stage, secreting both estrogen and progesterone. In this study and elsewhere, we have used follicle-stimulating hormone with a combination of growth factors in vitro to simulate the above in vivo conditions. In a previous study, we used dehydroepiandrosterone (DHEA) to accomplish the polycystic ovary condition (PCO) in rats. In the latter model, there were high circulating levels of DHEA and its metabolite, androstenedione. In the present study, we investigated the effects of high levels of DHEA (10⁻⁵M) and its metabolites, androstenedione, androstenediol and dehydroepiandrosterone sulfate on the quiescent, proliferative, and steroidogenically differentiating stages of GCs cultured in a serum-free medium for up to 10 days. In addition to possessing the regularly occurring organelles, when cultured with the aforementioned androgens, the GCs acquired endoplasmic reticulum of the smooth variety which is associated with steroidogenesis. The radioimmunoassay data showed that GCs cultured in the quiescent and proliferative stages in the presence of the androgens, no longer remain in these stages but proceed to differentiate in a preovulatory direction by producing both estrogen and progesterone. This study supports our hypothesis that high circulating levels of DHEA and/or its metabolites have most effect during the quiescent and proliferative stages of granulosa cells, with regard to their structure and their steroidogenic activities.     

A role for somatomedin C as a differentiating hormone and amplifier of hormone action on ovarian cells: studies with synthetically pure human somatomedin C and swine granulosa cells

Isolated swine granulosa cells incubated in chemically defined medium in vitro responded to synthetically pure human somatomedin C/IGF-I in a dose and time-dependent fashion with increased pregnenolone, progesterone and estradiol biosynthesis. These stimulatory actions were not mimicked by growth hormone, proinsulin, desoctapeptide insulin, epidermal growth factor, or fibroblast growth factor. Moreover, somatomedin C/IGF-I augmented the steroidogenic response of granulosa cells to exogenously supplied sterol substrate in the form of low-density lipoprotein, and amplified the stimulatory actions of the classical ovarian effector hormones, estradiol and follicle-stimulating hormone, in a synergistic fashion. The ability of somatomedin C/IGF-I to stimulate estradiol production on the one hand, and to act synergistically with estradiol to stimulate progesterone biosynthesis on the other hand, suggests a unique intrafollicular mechanism for amplifying progestin biosynthetic capacity in granulosa cells.     

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.     

Estrogen dependent ciliogenesis in the chick oviduct

Both the hormone dependency and the morphological details of estrogen-dependent ciliogenesis in the shell gland of the chick oviduct were investigated. Ciliogenesis was initiated on day 3 of estrogen treatment, and progressively more cells became differentiated until, on day 10, 55% ciliation occurred with 17-estradiol (1 mg/day) and 75% ciliation occurred with diethylstilbestrol (1 mg/day). Simultaneous administration of progesterone with diethylstilbestrol (1 mg each/day for 10 days) caused a 50% depression in the number of ciliated cells on day 10. The rate of ciliogenesis was found to be affected by progesterone and the type of estrogen administered. The minimum stimulatory dose of estradiol was found to be between 0.01 mg/day and 0.05 mg/day. Ciliogenic cells were first recognized by the appearance of pro-basal bodies in the apical portion of the cell. Pro-basal body maturation and cilium formation were the same as those described for the chick trachea. Ciliogenesis in the chick was found to be homologous to estrogen-dependent ciliogenesis in various mammalian oviducts.     

Physiologic characterization of transformed and cloned rat granulosa cells

Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions.     

Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells

The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Mechanism of action of 2-hydroxyestradiol on steroidogenesis in ovarian granulosa cells: interactions with catecholamines and gonadotropins involve cyclic adenosine monophosphate

Previously we have shown that 2-hydroxyestradiol (2-OH-E2) synergizes with catecholamines to enhance progesterone production by porcine granulosa cells in vitro. The present studies were undertaken to determine if the synergistic effects of 2-OH-E2 and catecholamines were 1) modulated by gonadotropins, 2) unique to catecholamines, and 3) mediated by cyclic adenosine monophosphate (cAMP). Undifferentiated granulosa cells from 1- to 3-mm porcine follicles were cultured in serum-free medium for periods of 6-9 days. A 3-day pretreatment plus a 4-day cotreatment versus a 4-day cotreatment of granulosa cell cultures with follicle-stimulating hormone (FSH) did not significantly alter progesterone production stimulated by a saturating concentration of epinephrine (EPI; 2 micrograms/ml) but significantly reduced the effect of 4 micrograms/ml 2-OH-E2 on Day 7 of culture. Four-day cotreatment of either FSH or luteinizing hormone (LH) from Day 3 to 7 of culture dramatically enhanced progesterone production stimulated by 2-OH-E2 and estradiol (E2) but not by EPI when measured on Day 7 of culture. Progesterone production (expressed as "-fold of controls") stimulated by 4-day treatment of EPI, 2-OH-E2, or EPI-plus-2-OH-E2 was 1.4 +/- 0.2, 8.2 +/- 2.2, and 10.7 +/- 1.0, respectively, in the presence of LH (n = 5 experiments), and 1.9 +/- 0.1, 7.8 +/- 1.4, and 10.6 +/- 1.8, respectively, in the presence of FSH (n = 3 experiments). Similar to E2, 2-OH-E2 significantly enhanced the stimulating effect of the cAMP analog 8-bromo-cAMP (0.5 mM) on progesterone production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

Steroid production,cell proliferation,and apoptosis in cultured bovine antral and mural granulosa cells: Development of an in vitro model to study estradiol production

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [¹⁴C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P< 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells. Mol. Reprod. Dev. 50:170–177, 1998. © 1998 Wiley-Liss, Inc.     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Estradiol-17 beta and androgen secretion by isolated porcine ovarian follicular cells in vitro

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4-6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17 beta (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH. 0.25 microgram/mL) or luteinizing hormone (LH. 1 microgram/mL). Addition of testosterone or androstenedione (0.5 microM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3'.5'-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells

Vasoactive intestinal peptide (VIP) and VIPergic nerve fibers are present in the ovaries of several mammalian species, suggesting a possible ovarian action of VIP. We have investigated the direct effects of synthetic porcine VIP on rat granulosa cell steroidogenesis in vitro. The cells were obtained from immature, hypophysectomized, estrogen-primed rats, and cultured in a serum-free medium for 24 h in the absence or presence of varying amounts of VIP. Medium steroids were then determined by specific radioimmunoassay. Vasoactive intestinal peptide dose-dependently stimulated progesterone, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OH-progesterone), and estrogen production with an approximate ED50 value of 3 X 10(-8) M. Maximum steroid production induced by VIP ranged from 15% to 28% of that seen with maximal follicle-stimulating hormone (FSH) stimulation. In contrast to the ability of FSH to induce luteinizing hormone (LH) receptor formation, treatment with VIP did not increase [125I]iodo-human chorionic gonadotropin (hCG) binding to granulosa cells. The ability of several gastrointestinal peptides, having 17-44% sequence identity to VIP, to stimulate granulosa cell steroidogenesis was also tested. The most closely related peptide, PHM-27 was less effective than VIP, and the least closely related, secretin and glucagon, were ineffective at 10(-6) M. Vasoactive intestinal peptide seems to act at least partly through cyclic 3',5'-adenosine monophosphate (cAMP)-dependent processes: addition of a phosphodiesterase inhibitor significantly potentiated the VIP stimulation of granulosa cell steroidogenesis, and VIP was capable of producing a dose- and time-dependent increase in both intracellular and medium cAMP levels. Vasoactive intestinal peptide stimulation of estrogen production seemed to be a result of increased aromatase activity. The increased progesterone production was associated with increased pregnenolone production, increased rate of conversion of pregnenolone to progesterone via 3 beta-hydroxysteroid dehydrogenase, and decreased metabolism of progesterone via 20 alpha-hydroxysteroid dehydrogenase. These results indicate that VIP exerts a specific action on granulosa cells to increase estrogen and progestin production. The observed direct effects of VIP, coupled with its identification in the ovary, suggest that VIP may be a physiologically important regulator of ovarian activity.     

Correlation of plasma estradiol-17β and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char,Salvelinus leucomaenis

Summary Plasma estradiol-17 and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis. Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation.In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed ⁵, 3-hydroxysteroid dehydrogenase (3-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.Nanae Fish Culture Experimental Station Contribution No 14