Oxytocin and progesterone secretion by bovine granulosa cells of individual preovulatory follicles cultured in serum-free medium

To examine the secretion of oxytocin (OT) and progesterone (P) from a homogeneous population of cells during luteinization, we developed a serum-free culture technique for granulosa cells, obtained from individual preovulatory bovine follicles. Granulosa cells from earlier stages of the follicle development did not have the capability to secrete OT under the in vitro conditions used. For optimal stimulation of the cells the medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12) was supplemented with bovine serum albumin (BSA) and insulin. OT was detectable from day 1 of culture reaching a maximum level between days 2 and 4 and then declined towards day 5. In the absence of insulin OT declined from day 1 onwards and was undetectable from day 4. When cells were cocultured with theca tissue or theca-conditioned medium (TCM), there was an enhancement in OT secretion, but not in P secretion. Other tissues including liver, kidney, aorta, muscle and adrenal incubated with the cells induced a similar increase in OT production. In the presence of insulin progesterone secretion was increased and was correlated with OT production, but did not show a consistent pattern among follicles. We conclude that (a) culture of granulosa cells from an individual follicle in a serum-free medium can be used to study the secretion of OT and P from bovine granulosa cells, (b) insulin is essential for the optimal production of OT and P by these cells, and (c) the addition of theca or other tissues enhanced OT secretion by a mechanism not understood.     

AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles

AMP-activated protein kinase (AMPK) is a fuel sensor in glucose, lipid, and cholesterol metabolism. Using RT-PCR and Western blot, AMPK subunits mRNAs (alpha1/2, beta1/2, and gamma1/2) and proteins (alpha1/2 and beta1/2) can be found in the hen preovulatory follicles and precisely in both granulosa and theca cells. These preovulatory follicles are organized in a hierarchy according to their size (F5/6 to F1). The smallest number (F1) corresponds to the largest size and the latest mature stage. Phosphorylation of AMPKalpha on Thr172 and of acetyl-CoA carboxylase on Ser79 are higher in F4 and F3 than in F1 granulosa cells. However, they are not affected in F4-F1 theca cells. Treatment with 1 mM 5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, dose dependently increased phosphorylation of AMPKalpha on Thr172 in primary F3/4 and F1 granulosa cells. In the absence of FSH, AICAR treatment increased progesterone, P450 side chain cleavage and steroidogenic acute regulatory (StAR) production in both F3/4 and F1 granulosa cells. However, in the presence of FSH, AICAR treatment for 36 h increased progesterone secretion, StAR protein levels and reduced extracellular signal-regulated kinase (ERK)1/2 phosphorylation in F3/4 granulosa cells. Opposite data were observed in F1 granulosa cells. Adenovirus-mediated expression of dominant-negative AMPK totally restored the effects of AICAR on FSH-induced progesterone secretion, StAR protein production, and ERK1/2 phosphorylation in F3/4 and F1 granulosa cells. Using a specific inhibitor of ERK1/2 (U0126), we also showed that this kinase is a negative regulator of the FSH-induced progesterone secretion in F3/4 and F1 granulosa cells, suggesting that AICAR-mediated AMPK activation modifies FSH-induced progesterone secretion differently through the ERK1/2 signaling pathway in hen F3/4 and F1 granulosa cells.     

Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture

The effects of RU486 on progesterone synthesis were studied in human preovulatory granulosa cells in culture. No effect was observed at 1 and 10 micrograms/mL, but at 100 micrograms/mL, RU486 inhibited the simulation of progesterone secretion induced by LH and cAMP. It is suggested that the main target of RU486 is the cytochrome P450scc function [catalyzing the formation of pregnenolone (D5P) from cholesterol], since no accumulation of D5P or hydroxy derivatives of progesterone was observed. As RU486 is an antiglucocorticosteroid and antiprogesterone agent, the effects of dexamethasone and progesterone were also investigated. Dexamethasone did not modify progesterone secretion, but progesterone inhibited its own synthesis in both the presence and absence of LH. Thus, under these experimental conditions RU486 displayed a progesterone-like effect. However, since the effect of RU486 was observed only at a concentration around 10(-4) M, the mechanism of action may not involve a receptor pathway and may not apply to most clinical circumstances.     

Steroid secretion by cumulus cells isolated from human preovulatory follicles

The secretion of progesterone, testosterone, and oestradiol by intact human oocyte-cumulus complexes in vitro was examined in incubations lasting 6-24 h. The complexes were aspirated from preovulatory follicles in 32 women who, due to tubal disease, were participating in an in vitro fertilization program. In 12 of the women follicular maturation was induced with clomiphene and human chorionic gonadotrophin (hCG), in 13 women with human menopausal gonadotrophin (hMG) and hCG and in 7 women with a combination of clomiphene-hMG plus hCG. The net secretion of steroids into the fertilization medium was studied before (0-6 h) and after (6-24 h) the addition of sperm, by RIA of aliquots removed at specific times. A high and sustained secretion of progesterone was found both before and after insemination. Testosterone secretion remained at a low and constant level while a net release of oestradiol was found mainly during the first hours of incubation. The release of steroids, particularly progesterone, varied according to the mode of hormonal stimulation in vivo and was highest in complexes from clomiphene-hMG-treated women, probably reflecting different maturity of the aspirated follicles. In a second series of experiments the dispersed cumulus cells were recovered after fertilization and cultured as monolayers for 2-4 days. The cells underwent spontaneous luteinization and secreted high amounts of progesterone. These results extend previous work in animals showing that also in the human the periovulatory cumulus cells are steroidogenically active. The results also suggest a functional difference in the cumulus cells related to the mode of ovulation induction.     

The source of cholesterol for progesterone synthesis in cultured preovulatory human granulosa cells

There are three possible sources of cholesterol for immediate use in progesterone production by preovulatory human granulosa cells: follicular fluid high-density lipoprotein, de novo synthesis of cholesterol, and performed intracellular cholesteryl ester stores. In the present study these three alternatives were investigated. First, an in vitro model was established that mimics the preovulatory environment, including short-term cultures and use of autologous follicular fluid in the culture medium, instead of serum. Using this model it was found that the presence of high-density lipoprotein from follicular fluid in the culture medium did not affect the synthesis of progesterone by the granulosa cells. Next, addition of inhibitors of de novo sterol synthesis, like low-density lipoprotein, 25-OH cholesterol and compactin to the culture medium, did not reduce [14C]acetate incorporation into sterols and steroids by the cells. The sterol synthesis was accordingly interpreted to be at a low and therefore uninhibitable level. Finally, the content of free and esterified cholesterol in freshly isolated granulosa cells was found to be 50 +/- 7 and 52 +/- 13 pmol/mg cell protein, respectively. We suggest that neither follicular high-density lipoprotein nor endogenous synthesis is the immediate cholesterol source for the progesterone production in preovulatory human granulosa cells. However, granulosa cells have a large store of cholesteryl esters that may provide free cholesterol for the preovulatory progesterone production.     

Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin

Human granulosa-luteal cells were harvested from preovulatory Graafian follicles at the time of oocyte retrieval for in vitro fertilization after induction of follicle maturation by sequential injections of menopausal gonadotropins and hCG. Such highly differentiated granulosa cells produced large quantities of progesterone basally (6.8 pg/cell X 2 days) in monolayer culture. Human LH significantly increased progesterone biosynthesis after 6, 12, 48, 96, or 144 h in culture, with a maximal increase of 8- to 20-fold occurring at 96 h. The stimulatory effect of LH could be observed under serum-free conditions and was maximal in the presence of 4% serum. Human granulosa-luteal cells also exhibited significant stimulatory responses to hCG, prostaglandin E2, or the cAMP effectors 8-bromo cAMP, choleratoxin, or forskolin in serum-free incubations. Concentrations of 17 beta-estradiol that are attained physiologically in ovarian follicles in vivo markedly suppressed basal and LH (or cAMP)-stimulated progesterone production in vitro (maximal suppression, greater than 90%). The nonaromatizable androgen 5 alpha-dihydrotestosterone also inhibited progesterone production, but by no more than 45-50% even at supraphysiological concentrations. Estradiol's blockade of progesterone synthesis was associated with a corresponding increase in pregnenolone accumulation. The present studies indicate that human granulosa-luteal cells isolated from preovulatory follicles induced with exogenous gonadotropins and hCG secrete large quantities of progesterone in vitro. Such cells retain stimulatory responses to human LH, hCG, prostaglandin E2, and classical cAMP effectors in serum-free incubations. Moreover, physiological concentrations of 17 beta-estradiol suppress progesterone production, probably by inhibiting cellular conversion of pregnenolone to progesterone. Thus, the present in vitro system permits an investigation of hormone action in well differentiated, human granulosa-luteal cells isolated from preovulatory Graafian follicles that have a defined endocrine history of prior gonadotropin exposure in vivo.     

Androgens augment FSH-induced progesterone secretion by cultured rat granulosa cells.

Granulosa cells have been isolated from ovaries of estrogen-treated immature intact and hypophysectomized rats, and have been maintained in culture in a chemically-defined medium. Progesterone secretion by these cells was testosterone or 17beta-OH-5alpha-androstan-3-one (DHT), progesterone secretion was low or undetectable. However, the addition of testosterone or DHT together with FSH caused a dramatic 8- to 19-fold increase over that caused by FSH alone. On the other hand, luteinizing hormone (LH) alone had no effect on progesterone secretion, but produced a small stimulation when added together with testosterone. These results demonstrate synergism between androgens and FSH in the control of progesterone secretion by granulosa cells in culture.     

Stimulatory effect of luteinizing hormone upon relaxin secretion by cultured porcine preovulatory granulosa cells

Relaxin, a peptide hormone capable of causing connective tissue alterations, is produced by the corpus luteum and traditionally considered a hormone of pregnancy. We have cultured granulosa cells from preovulatory porcine follicles and have found that these non-pregnancy associated cells secrete relaxin, and that luteinizing hormone, which stimulates ovulation, enhances relaxin secretion by cells from large preovulatory follicles. These results suggest that relaxin secreted prior to ovulation may have a local ovarian effect, perhaps facilitating ovulation.     

Prolactin-mediated progesterone secretion by cultured rat granulosa cells: regulation by purified human glycoprotein hormones and their subunits

The effects of purified alpha- and beta-subunits of human glycoprotein hormones on initial luteinization and subsequent prolactin-mediated progesterone responses of cultured rat granulosa cells were studied. Granulosa cells, obtained from immature female rats 50 h after PMSG treatment, were incubated for 24 h in control medium lacking added hormones or in medium containing hCG or the alpha- or beta-subunit of human (h) FSH, LH, CG, or TSH at 0.1, 0.5, and 1.0 microgram/ml. Cultures were maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine PRL (bPRL), with medium changes every 48 h. Indices of luteotropic stimulation in response to bPRL were provided by 1) elevated progesterone concentrations determined by RIA of spent media samples, and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation of monolayers after 7 days of culture. Progesterone concentrations in media from cultures incubated in 0.5 or 1.0 microgram/ml hCG were 6-fold higher than in cultures incubated in control medium, while those in media from cultures incubated in 0.5 or 1.0 microgram/ml hFSH alpha, hLH alpha, hCG alpha, hTSH alpha, hLH beta, or hCG beta (but not in hFSH beta or hTSH beta) were from 2- to 4-fold higher than those in control cultures. This enhancement was not evident when subunits were added to the incubation media at the lowest concentration. Progesterone secretion corresponded directly with the degree of cytoplasmic osmiophilia. These results suggest that the alpha-subunit of each of the glycoprotein hormones as well as the beta-subunit of hLH and hCG have the ability to promote progesterone secretion during initial luteinization and to regulate subsequent PRL-mediated steroidogenesis by rat granulosa cells in vitro. Furthermore, these effects are greater than can be accounted for by potential contamination of subunit preparations with undissociated hormones, as demonstrated by dose-response curves.     

Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors

Human granulosa cells from Graafian follicles aspirated 3-4 h before the expected time of ovulation were incubated with various steroid substrates, including pregnenolone, androstenedione, testosterone and dehydroepiandrosterone (DHA). Steroid production after 3 and 10 h of incubation was determined by radioimmunoassay. Progesterone and 17alpha-hydroxyprogesterone were the major products of granulosa cells in control short-term cultures with endogenous substrates. The addition of pregnenolone increased the synthesis of progesterone and 17alpha-hydroxyprogesterone compared with the controls, although the response varied considerably between paired short-term cultures. Little or no oestradiol-17beta was produced from endogenous precursors or short-term cultures to which pregnenolone had been added; one follicle, however, produced similar amounts of oestradiol-17beta in the control cultures and after incubation with pregnenolone. When granulosa cells were cultured with various amounts of androstenedione, DHA or testosterone, large amounts of oestradiol-17beta were produced, especially in short-term cultures in which larger amounts of substrate were added. Progesterone production continued and progesterone was synthesized more rapidly or in greater amounts in some short-term test cultures than in the controls. The results indicate that human granulosa cells are one source of oestradiol-17beta during the preovulatory phase. The data support the two-cell theory for oestradiol synthesis, for granulosa cells do not appear to undertake steroid conversion via the 5-unsaturated pathway, but aromatize androgens known to be produced by thecal cells. It is also suggested that either androgens or oestradiol-17beta stimulate progesterone production by granulosa cells, at least in vitro.     

Effect of danazol on gonadotropin and cAMP stimulated progesterone synthesis in cultured human granulosa cells

The effect of danazol on progesterone formation was studied in cultured human granulosa cells obtained from 20 follicles in the mid- to late follicular phase of normally menstruating women. The cells were cultured for 2 to 6 days in the presence of danazol (0.01 to 5 mg/l) alone and in combination with one of several progesterone stimulatory agents. The medium was changed every other day and analysed for progesterone with radioimmunoassay. In all cases progesterone synthesis was markedly stimulated by human FSH (10 micrograms/l), human LH (10-100 micrograms/l), hCG (1000 IU/l), forskolin (1 mumol/l) or 8-Bromo-cAMP (1 mmol/l). In the presence of danazol (1 mg/l) the FSH-, LH- and hCG-stimulated progesterone synthesis was partially inhibited by 55 to 60%. The response to forskolin or 8-Bromo-cAMP was also inhibited by danazol although to a somewhat lower extent (35 to 50%). Basal progesterone synthesis was inconsistently inhibited in some cases. It was concluded that not only gonadotropin-stimulated steroid synthesis, as previously demonstrated for hCG, but also forskolin and 8-Bromo-cAMP-stimulated steroidogenesis is sensitive to danazol inhibition. This suggests that the effect of danazol in human granulosa cells is due, at least partly, to interference with steps distal to cAMP generation.     

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.     

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Met-enkephalin enhances follicle-stimulating hormone-dependent progesterone production from cultured granulosa cells

beta-Endorphin (beta-EP) and methionine-enkephalin (Met-Enk) have been detected in human follicular fluid in concentrations several times higher than those in plasma. These data stimulated us to study the possible physiological role of ovarian opioids. We, therefore, determined the effects of both beta-EP and Met-Enk, alone or in combination with naloxone, on FSH-induced progesterone (P) secretion by cultured granulosa cells. Granulosa cells were collected from follicular fluid recovered at laparoscopy in seven superovulated women. The cells were preincubated with RPMI-1640 medium containing 20% fetal calf serum in 5% CO2 for 48 h, followed by the addition of 100 mU purified FSH and the various test substances for 48 more h. beta-EP (10 nM to 1 pM) had no effect on P secretion either alone or in combination with FSH and/or naloxone. Micro- to picomolar amounts of Met-Enk increased FSH-induced P secretion up to 186.9 +/- 35.1% (+/- SEM). Met-Enk had no affect in the absence of FSH, and its action was significantly blunted by the concomitant addition of 10(-5) M naloxone. These data provide evidence for a dose-dependent naloxone-reversible synergistic action of Met-Enk and FSH on P secretion by cultured granulosa cells. This finding supports the hypothesis of the existence of an ovarian opioid system.     

Effect of sodium on progesterone production in granulosa cells of rapidly growing chicken ovarian follicles

The importance of extracellular sodium (Nao+) in the progesterone production in hen granulosa cells from the first (F1), second (F2), and third (F3) largest preovulatory follicles was investigated in short term incubations. Progesterone synthesis in the absence or presence of the gonadotropin increased with increasing Nao+ concentration. Luteinizing hormone (LH) caused an additional and significant increase in steroidogenesis at every Nao+ concentration tested. However, no significant difference in the ED50 of Nao+ among F1, F2, and F3 cells was observed whether in the absence or the presence of the gonadotropin. Furthermore, although LH provoked steroidogenesis dose dependently in F1, F2, and F3 granulosa cells, no significant change in the ED50 of LH was observed among F1, F2, and F3 cells. The Na+/H+ exchange inhibitor, amiloride, attenuated both basal and LH-stimulated steroidogenesis in a concentration-dependent manner in the presence of Nao+. Amiloride was ineffective in the absence of Nao+. The present studies indicate the importance of Nao+ in the modulation of steroidogenesis in hen granulosa cells. Since steroidogenesis was suppressed by amiloride only in the presence of sodium, it is suggested that Nao+ is important for the maintenance/regulation of intracellular pH by an exchange with intracellular H+. Our studies also suggest that the sodium modulatory mechanism may not be a determinant in the differential progesterone production observed among the three largest preovulatory follicles.     

Catecholestrogens stimulate progestin secretion by cultured porcine granulosa cells

The enzymatic metabolism of estradiol (E2) to the catecholestrogens, 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) in granulosa cells has been reported. Therefore, we evaluated the effects of these compounds and compared them to those of E2 on porcine granulosa cells cultured in serum-free medium. Cultures of granulosa cells were exposed to various treatments of E2, 2-OH-E2, 4-OH-E2 and(or) follicle-stimulating hormone (FSH) for 4 days and concentrations of progesterone in medium and cell numbers were determined. After 4 days of treatment, 2-OH-E2 and 4-OH-E2 stimulated basal progesterone production by granulosa cells, but 4-OH-E2 was less effective than 2-OH-E2. 2-OH-E2 (1 microgram/ml) stimulated progesterone production by 3.3 +/- 0.6-fold (n = 6 experiments), whereas E2 (1 microgram/ml) stimulated progesterone production 9.9 +/- 1.7-fold (n = 6 experiments). 2-OH-E2 at 4 micrograms/ml further stimulated progesterone production to 10.7 +/- 2.2-fold above controls (n = 9 experiments), whereas 4 micrograms/ml of E2 did not cause further stimulation of progesterone production. Thus, the average potency of 2-OH-E2 was less than E2. Concurrent treatment with 2-OH-E2 (4 micrograms/ml) and saturating concentrations of E2 resulted in further significant increases in progesterone production above the effects of either single treatment both in the absence and presence of FSH (200 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a gonadotropin-releasing hormone agonist on progesterone formation in cultured human granulosa cells

Human granulosa cells from follicles of natural cycles (13 women) in mid- or late follicular phase were cultured in modified Medium-199. Human luteinizing hormone (100 micrograms/l), follicle-stimulating hormone (100 micrograms/l) and gonadotropin-releasing hormone agonist (GnRHa) (10(-12) to 10(-6) mol/l) alone or in combination were added to the culture medium. Medium content of progesterone was analysed. In granulosa cells obtained in the mid-follicular phase, the basal progesterone formation averaged 0.5 pg.cell-1.(48 h)-1. FSH caused a 3-fold stimulation. GnRHa (10(-6) mol/l) had a variable influence on the basal progesterone formation, whereas it consistently inhibited (40-50%) the FSH response. In granulosa cells from late follicular phase basal progesterone formation averaged 5 pg.cell-1.(48 h)-1 and was stimulated 3- to 6-fold by LH. GnRHa (10(-6) mol/l) stimulated the basal progesterone formation and caused a tended to potentiate the LH response on progesterone formation. It is concluded that GnRHa at relatively high concentrations exerts direct effects on gonadotropin-stimulated progesterone formation of human granulosa cells and that these effects are different (inhibitory or stimulatory) dependent on the degree of follicular maturation, and/or type of gonadotropin used.