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.     

Insulin-like growth factor-I stimulates oxytocin and progesterone production by bovine granulosa cells in culture

The effect of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF) and nerve growth factor (NGF) on production of oxytocin and progesterone by cultured bovine granulosa and luteal cells was studied. Secretion of oxytocin was stimulated, in a dose-dependent manner, by IGF-I at 48 and 120 h of culture to levels much higher than those after stimulation with LH, FSH, EGF, FGF or NGF. A similar effect of IGF-I was observed for progesterone but, in contrast to oxytocin, secretion of progesterone was not increased by EGF, NGF or FGF. During primary culture, for 4 h, of dispersed bovine luteal cells obtained from corpora lutea between days 4 and 10 of the oestrous cycle, all the growth factors tested failed to stimulate secretion of oxytocin or progesterone. The data suggest the relevance of growth factors (especially IGF-I) for ovarian physiology and their possible importance for differentiation of follicles and luteinization.     

Role of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A reductase in progesterone production by cultured bovine granulosa cells

The relative contributions of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to progesterone production by bovine granulosa cells exposed to plasma or liquor folliculi (LF) were studied. LF did not contain and very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), or low density lipoprotein (LDL). These lipoproteins were present in the plasma at concentrations of 92 micrograms protein/ml for VLDL and IDL together and 139 micrograms protein/ml for LDL. In contrast, high density lipoprotein (HDL) was present in LF at a concentration (763 micrograms protein/ml) that was 59% of that in plasma (1293 micrograms protein/ml). Bovine granulosa cells exposed to human plasma produce progesterone in response to dibutyryl cAmP. Sixty-three percent of the progesterone released by the cells was dependent on LDL but not HDL derived from human plasma. When cells were exposed to bovine plasma, 75% of the progesterone release was dependent on the presence of lipoproteins in the medium. Both LDL and HDL of bovine origin were able to support progesterone production, although LDL was effective at concentrations (on a molar basis) 20-fold lower than HDL. The LF was able to support progesterone production 45% as well as bovine plasma. The differences between the greater ability of the whole fractions and the lesser ability of their respective lipoprotein-deficient derivatives to support progesterone synthesis were 4-fold for bovine plasma, 2.7-fold for human plasma, and 1.7-fold for LF. The relative abilities of equivalent concentrations of LDL to restore the rate of progesterone synthesis seen in the lipoprotein-deficient fraction toward that seen in the whole fraction were greatest in the LF, intermediate in human plasma, and least in bovine plasma. These observations taken together suggest that the low level of support of progesterone synthesis that is offered by LF is due to its deficiency in LDL. HMG CoA reductase, the regulated and rate-limiting enzyme of cholesterol synthesis, was induced (2- to 3-fold) by dibutyryl cAMP and was suppressed by both human and bovine LDL and to a lesser extent by bovine HDL. Compactin, a competitive inhibitor of HMG CoA reductase, inhibited progesterone production relatively little when cells were exposed to complete plasma or LF. However, when cells were exposed to a lipoprotein-deficient bovine plasma or LF, compactin was very efficient in reducing (by 76%) progesterone release. Bovine granulosa cells exposed to plasma primarily use cholesterol derived from LDL in order to produce progesterone. Their ability to produce progesterone when exposed to LF was limited, and the cells were probably more dependent on de novo cholesterol synthesis than cells exposed to plasma.     

Role of chloride ions in progesterone production by chicken granulosa cells.

The importance of chloride ions in luteinizing hormone (LH)-stimulated progesterone production by chicken granulosa cells from the two largest preovulatory follicles was investigated in vitro. Reduction of the extracellular chloride concentration from 147.8 mM to 2.8 mM, by substitution with equimolar concentrations of non-permeant glutamate and aspartate, inhibited the ability of LH to stimulate progesterone production and cAMP accumulation during a 4 h incubation. LH-stimulated granulosa cell progesterone production was also suppressed in a concentration-dependent manner by the chloride channel blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS; 10(-8)-5 x 10(-5) M) or 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS; 10(-8)-5 x 10(-5) M). The inhibitory effect was observed within 30 min of the addition of the blockers and was irreversible. DIDS appeared to act at a site(s) proximal to the generation of cAMP, since concentrations of DIDS (10(-8)-10(-6) M) which inhibited LH- and human chorionic gonadotropin-stimulated progesterone production, did not affect progesterone production stimulated by dibutyryl cAMP, 8-bromo cAMP or forskolin. In addition, concentrations of DIDS (10(-8)-10(-6) M) which attenuated LH-stimulated progesterone production also reduced the accumulation of extracellular cAMP. These studies suggest that chloride ions may play an important role in the stimulatory action of LH on chicken granulosa cell progesterone production.     

Cholinergic stimulation, through muscarinic receptors, of oxytocin and progesterone secretion from bovine granulosa cells undergoing spontaneous luteinization in serum-free culture

Bovine granulosa cells were cultured in a defined serum-free system to examine their responsiveness to acetylcholine (ACh). Continuous exposure to concentrations of ACh between 10(-8)-10(-4) M resulted in dose-dependent increases (up to 6.7-fold) in the secretion of oxytocin and progesterone, with an ED50 of 6.6 microM. Ascorbic acid (0.5 mM), a known stimulator of granulosa secretion, synergized with ACh, resulting in an increase in the amounts of hormone secreted and a 7-fold increase in cellular sensitivity to ACh (ED50 = approximately 0.9 microM). Treatment of cells with ACh for 24 h at various times during a typical 5-day culture resulted in a stimulation that persisted for up to 4 days after removal of ACh. Carbachol (10(-8)-10(-4) M), a receptor antagonist with both antimuscarinic and antinicotinic actions, had no distinct effect on hormone secretion by the cells, but the effects of 10(-5) M ACh could be completely abolished by equimolar or hypomolar concentrations of the specific muscarinic receptor antagonists atropine and scopolamine. Nicotine bitratrate (10(-8)-10(4) M), a dose-dependent nicotinic receptor agonist/antagonist, had no effect on the cells. It is concluded that bovine granulosa cells, exhibiting a luteinized phenotype in culture, are responsive to cholinergic agonists in a specific and saturable manner. The response of the cells is probably mediated through muscarinic receptors and has both medium and long term (persistent) components. These results indicate that cholinergic neurotransmitters may play a direct role in the regulation of ovarian function in the ruminant.     

Comparative effects of androgens and catecholestrogens on progesterone production by porcine granulosa cells

The objective of the present studies was to evaluate and compare the effects of 5 alpha-dihydrotestosterone (DHT) to those of 2-hydroxyestradiol (2-OH-E2) and 2-methoxyestradiol (2-MeO-E2) on progesterone production in cultured porcine granulosa cells. Granulosa cells were exposed to various treatments of DHT, 2-OH-E2 and 2-MeO-E2 in the absence or presence of follicle stimulating hormone (FSH) for 4 days and concentrations of progesterone in medium and cell numbers were determined. In the absence of FSH, maximally effective concentrations of DHT (1 micrograms/ml) and 2-OH-E2 (4 micrograms/ml) stimulated progesterone production (ng/10(5) cells/48 h) to 2.2 +/- 0.2- and 10.8 +/- 2.2-fold of controls (n = 4 experiments), respectively. In the presence of 200 ng/ml FSH, progesterone production stimulated by 1 micrograms/ml DHT and 4 micrograms/ml 2-OH-E2 was 5.4 +/- 1.1- and 15.5 +/- 6.0-fold of controls (n = 4 experiments), respectively. Thus, FSH appeared to enhance the response of both DHT and 2-OH-E2. The dose-response of DHT was biphasic in the presence and absence of FSH, such that progesterone production in the presence of 8 micrograms/ml DHT was similar to basal progesterone production. Concurrent treatment with saturating concentrations of 2-OH-E2 and DHT resulted in fully additive increases in progesterone production. Testosterone mimicked the effect of DHT. In comparison, concurrent treatment of saturating concentrations of 2-MeO-E2 and DHT or 2-MeO-E2 and 2-OH-E2 resulted in progesterone production that was only partially additive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms underlying endothelin's inhibition of FSH-stimulated progesterone production by ovarian granulosa cells

Protein kinase C (PKC) is involved in the ₁-adrenergic-potentiation of β-adrenergic stimulated cyclic nucleotide responses in rat pinealocytes. In the present study, the PKC isozymes expressed in rat pinealocytes and their regulation by norepinephrine (NE) were investigated. Western blot analysis identified PKC (a classical PKC isozyme), PKCδ and (novel PKC isozymes), and PKCζ: (atypical PKC isozymes). NE caused an increase in PKC, δ, and , but not PKCζ, in the particulate fraction. BAPTA-AM, which clamps intracellular Ca²⁺, reduced NE mediated translocation of PKC, δ, and . Subjecting the animals to stimulus deprivation, which altered adrenergic-stimulated cyclic nucleotide responses, had no effect on the expression of PKC, δ, , and ζ. Overnight treatment with 4β-phorbol 12-myristate 13-acetate, an activator of PKC, down-regulated PKC, δ, and , but not PKCζ. Our results indicate that all three classes of PKC isozymes (PKC, δ, , and ζ are expressed in the rat pineal gland. However, selective activation of these PKC isozymes does not appear to account for the differences in the pineal cAMP and cGMP responses to stimulation.     

Multihormone regulation of steroidogenesis in cultured porcine granulosa cells: studies in serum-free medium

FSH, LH, and estradiol are known to modulate ovarian follicular differentiation. However, the cellular site of action and relative importance of the three hormones have remained uncertain. The recent development of a serum-free system for the culture of immature porcine granulosa cells has enabled us to reinvestigate these issues with better control of pituitary peptides and gonadal steroids. Progesterone production in response to FSH was higher in cells cultured in serum-free complete medium than in those grown in the presence of 10% fetal calf serum [10-fold vs. 1.5-2 fold (control)]. Ovine LH alone was also able to stimulate progesterone production in serum-free free complete medium (6-fold); this effect could not be accounted for by FSH contamination. The LH stimulation, however, was enhanced by FSH. Insulin was required for both FSH and LH stimulation of progesterone production. Estradiol stimulated progesterone production per se (2- to 3-fold) and also enhanced FSH and LH actions. The estimated ED50 for estradiol in FSH-treated cells was 20 ng/ml. Maximal levels of progesterone after 6 days were observed when the combination of FSH, LH, and estradiol was present from the onset of the culture. Incubations carried out in the presence of 5-cholesten-3 beta-25-diol indicated that the hormonal interactions take place, at least in part, at the level of the side-chain cleavage enzyme. These results indicate that FSH is the most important hormonal stimulus for progesterone synthesis in immature granulosa cells. However, LH, estradiol, and insulin (or insulin-like growth factors) exert direct actions on the granulosa cell that may be required for the development of optimal steroidogenic potential.     

The role of gonadotropins and testosterone in progesterone production by human ovarian granulosa cells

Granulosa and theca cells obtained from patients were isolated and cultivated in a chemically defined medium containing gonadotropins and/or testosterone. Progesterone secretion by granulosa cells was consistently stimulated (2-40-fold) in all 5 patients by the addition of follicle-stimulating hormone (FSH, 0.25 micrograms/ml). In the presence of testosterone (0.5 micro M) alone, progesterone production was stimulated (2-8-fold) in 4 out of the 5 patients and cells of one patient showed a greater response to testosterone than to FSH alone. In 2 of the 5 patients, it was also noted that FSH and testosterone acted in a synergistic manner to stimulate the production of progesterone by granulosa cells. On the other hand, human chorionic gonadotropin (hCG, 1.0 IU/ml) alone failed to exert any significant effect. None of the treatments examined altered the production of progesterone by theca cells. These results suggest a role for FSH and testosterone in regulating progesterone biosynthesis by granulosa cells of the human ovary during follicular development.     

Proteoglycan production by bovine granulosa cells in vitro is regulated by calmodulin and calcium

Proteoglycan production by granulosa cells in vitro is regulated by gonadotropins. The objective of this study was to determine if FSH stimulation of proteoglycan synthesis was modulated by calmodulin or calcium. Assay for calmodulin using an ATPase assay dependent on calmodulin yielded concentrations of 7.7 microM. Bovine granulosa cells from follicles 1-9 mm diameter were incubated for 45 minutes in a chemically defined medium containing 5 microCi/ml 3H-glucosamine and various phenothiazine drugs which are inhibitors of calmodulin. In response to oFSH or rFSH at equivalent biological potencies, proteoglycan production decreased with increasing concentrations of phenothiazines from 1 to 50 microM. Addition of EGTA at 0.0, 0.5, 1.0 or 2.0 mM showed decreased proteoglycan production with increased amounts of the chelator. These data suggest that calmodulin and calcium are necessary for proteoglycan production by granulosa cells in response to FSH in vitro.     

Oxytocin stimulates progesterone production by bovine granulosa cells isolated before, but not after, the luteinizing hormone surge

Oxytocin and its mRNA have been detected in bovine granulosa cells, but the function of follicular oxytocin is not well understood. We have shown previously that oxytocin exerts a specific, dose-dependent, stimulatory effect on progesterone secretion by granulosa, but not theca cells isolated from bovine preovulatory follicles obtained 48 h after the initiation of luteolysis. The objective of the present study was to characterize the development of granulosa cell responsiveness to oxytocin during the follicular phase. Granulosa cells and theca interna were isolated form preovulatory follicles early in the follicular phase (24 h after the initiation of luteolysis) or after the luteinizing hormone (LH) surge and cultured in defined medium for 5 days with or without oxytocin and in the presence or absence of gonadotropins. Granulosa, but not theca cells obtained before the LH surge increased progesterone production 3.3-fold in response to oxytocin. However, late in the follicular phase, after the LH surge, granulosa cells did not respond to oxytocin (or to follicle-stimulating hormone (FSH) or LH). These findings suggest that the LH surge (1) stimulates granulosa cells to maximal progesterone secretion, so that they cannot be further stimulated, (2) abolishes the responsiveness of granulosa cells to oxytocin, or (3) stimulates granulosa cells to increase oxytocin production, so that exogenous oxytocin has no additional effect.     

Inhibition of hormone-induced steroidogenesis during cell proliferation in serum-free cultures of rat granulosa cells

Long term cultures of rat granulosa cells were grown in serum-free medium, consisting of Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient F-12 medium and supplemented with insulin, transferrin, hydrocortisone, and fibronectin (4F medium). In sparse cultures (10(4) cells/cm2), the granulosa cells were steroidogenically responsive to ovine FSH (NIADDK-oFSH-15) during days 1-2 and 10-14 (responsive periods). The major steroids produced were 20 alpha-hydroxyprogesterone (20 alpha-OH-P) and 5 alpha-pregnane, 3 alpha,20 alpha-diol (pregnanediol). However, as of day 3, the cells gradually lost their steroidogenic responsiveness which was inhibited by 88% at day 7 (refractory period). Nevertheless, from day 8 onward, the cells regained their responsiveness which was fully restored at day 12. The transient loss of responsiveness was uniquely associated with progestin biosynthesis, since FSH-induced aromatase activity declined to background levels within 12 days and was never restored again. The loss of progestin responsiveness was not due to lack of cAMP because FSH induced increasing levels of cAMP accumulation, reaching maximal values on day 7 in culture. On the other hand, the onset of the refractory period occurred concomitantly with the entry of the cultured cells into a synchronous proliferation phase, during which the cell population doubled. Thereafter, as DNA synthesis ceased, the cells regained their steroidogenic responsiveness. A deliberate arrest of cell replication, in the presence of excess thymidine or in high density cultures, prevented the temporal loss of activity. The data presented favor the notion that cell proliferation and expression of differentiated functions are inversely related. It is suggested that growth-related processes suppress steroidogenesis by an as yet unknown mechanism.     

Effect of mouse epidermal growth factor on DNA and protein synthesis, progesterone and inhibin production by bovine granulosa cells in culture

The effect of mouse epidermal growth factor (EGF) was investigated on DNA and protein synthesis, progesterone and inhibin production by bovine antral granulosa cells. When incubated for the whole period of culture, EGF inhibited inhibin production the second day of culture, progesterone the third and the fourth days whereas it stimulated DNA and protein synthesis only the fourth day of culture. Inhibition of progesterone and stimulation of DNA and protein were dose-dependent when treatment with EGF (pre-incubation) is followed by 24 h without EGF, a stimulatory effect on DNA and protein synthesis was observed after 48 and 72-h pre-incubation. Progesterone was reduced after 3 day pre-incubation and inhibin only after 2-day pre-incubation. Effects observed after 3-day pre-incubation were dose-dependent. These experiments demonstrated the stimulatory effect of EGF on growth of granulosa cells and its inhibitory action on hormonal production by these cells in vitro. The inhibitory effect on progesterone and inhibin production is more precocious than stimulatory effect on DNA and protein synthesis. The inhibitory action of EGF on granulosa cell production of progesterone and inhibin could thus be not directly dependent on its stimulatory action on DNA synthesis.     

Phorbol ester stimulates progesterone production by isolated bovine luteal cells

The tumor promoter, phorbol 12-myristate-13-acetate (PMA), is known to modulate the response of several steroidogenic tissues presumably by activating a Ca++- and phospholipid-dependent protein kinase (protein kinase C). The presence of this kinase has been demonstrated in bovine corpus luteum, although its role in steroidogenesis by these cells is unknown. We report here the effects of PMA on progesterone production by the enzymically dispersed bovine luteal cells in vitro. PMA (1-50 nM) produced a dose- and time-related increase in progesterone production by the luteal cells. The maximum stimulation was achieved with 10 nM PMA. Higher concentrations of PMA led to a decline of steroidogenesis close to the basal level. A nonpromoting derivative, 4 alpha-phorbol 12,13-didecanoate had no effect. The PMA-induced stimulation of progesterone production was not associated with a change in the cAMP level. PMA added together with suboptimal doses of human CG, 8Br-cAMP, cholera toxin, or forskolin significantly increased the amount of progesterone produced. PMA as well as human CG-induced steroidogenesis was sensitive to cycloheximide inhibition. The conversion of exogenous pregnenolone or 25-hydroxycholesterol to progesterone was not altered by PMA. We conclude that PMA at nanomolar concentrations is able to stimulate progesterone production by bovine luteal cells and that the site of action of PMA is distal to the formation of cAMP but before the formation of pregnenolone. The observed effects of PMA in luteal cells are probably linked to its ability to activate protein kinase C, since a diacylglycerol could mimic the steroidogenic action of PMA.     

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.     

Proteoglycan production by bovine granulosa cells in vitro occurs in response to fsh

Bovine granulosa cells from small (1-9 mm) or large (10-20 mm) follicles were incubated in a chemically defined medium containing 5 muCi/ml [3H]glucosamine, gonadotropins or polypeptide hormones, 8-Br-cAMP, theophylline or trifluoperazine (TFP). Radiolabeled proteoglycans were precipitated with 10% phosphotungstic acid. Maximum incorporation of isotope occurred in 45-60 min. Radiolabeled products were completely hydrolyzed with chondroitinase ABC. FSH, but not LH or hCG, yielded a significant log-dose response. High doses of hCG inhibited the ability of granulosa to respond to FSH. No other hormone altered the FSH dose-response curve. Addition of 8-Br-cAMP or theophylline mimicked the FSH response. The FSH effect was blocked by TFP, an inhibitor of calmodulin, but cAMP or theophylline overcame the effect of TEP. No distinct difference in response to these various compounds was noted between granulosa from small or large follicles. This system provides a biochemical marker for evaluating a mechanism of action for FSH.     

Expression of adrenomedullin by human granulosa lutein cells and its effect on progesterone production

OBJECTIVE: Adrenomedullin (AM) has diverse functions and is expressed in a variety of tissues. This study was conducted to investigate the expression of AM in the human ovary and its effect on progesterone production by human granulosa lutein cells. DESIGN AND METHODS: Follicular fluid and blood samples were obtained at the time of oocyte retrieval from patients undergoing in vitro-fertilization cycles. Concentrations of AM in follicular fluid and plasma were measured by RIA. Granulosa cells were isolated from follicular fluid and expression of AM mRNA was examined by RT-PCR. Granulosa lutein cells were cultured in vitro and secretion of AM by those cells was determined by immunoprecipitation followed by PAGE. Immunohistochemical staining with human ovaries was carried out, using a specific antibody to AM. Furthermore, the effect of AM on progesterone production by cultured granulosa lutein cells was studied. RESULTS: Concentrations of AM in follicular fluid collected just before ovulation were significantly higher than those in the plasma (P<0.01). AM mRNA was expressed in granulosa cells at the preovulatory stage. Cultured granulosa lutein cells secreted immunoreactive AM. With immunohistochemical staining, it was revealed that AM was most abundantly expressed in granulosa lutein cells at the midluteal phase. No appreciable staining for AM was observed in granulosa cells in primordial and preantral follicles, whereas immunolocalization of AM was noted in granulosa cells of dominant follicles although it was not as prominent as in granulosa lutein cells at the midluteal phase. Furthermore, addition of AM to cultured granulosa lutein cells augmented progesterone secretion in a dose-dependent manner. CONCLUSIONS: These results suggest that AM is transcribed and secreted in human granulosa lutein cells as a local factor to enhance progesterone production by those cells.     

Relationship between the production of prostaglandins and progesterone by luteinizing human granulosa cells.

Luteinizing granulosa cells synthesize high concentrations of progesterone, prostaglandin (PG) E(2) and PGF(2 alpha). The objective of this study was to explore the relationship between prostaglandin and progesterone output from human granulosa cells as they undergo functional luteinization in culture. Granulosa cells were partially purified from ovarian follicular aspirates and cultured at a density of 10(5) cells/ml in serum-supplemented DMEM:Ham's F(12) medium for 0, 1 or 2 days. Cells were then switched to serum-free medium for 24 h before measuring hormone concentrations in this spent medium by specific radioimmunoassays. Over the first 3 days in culture, PGF(2 alpha) and PGE(2) production declined progressively by up to 82+/-3% coincident with a 55+/-11% increase in progesterone output. In subsequent experiments, cells were treated for 24 h on the second day of culture with either 0.01 to 10 microM meclofenamic acid or with 10 microM and 100 microM aminoglutethimide. Meclofenamic acid inhibited synthesis of PGF(2 alpha) and PGE(2) by up to 70+/-9% and 64+/-7% respectively without affecting progesterone output. Likewise, 100 microM aminoglutethimide inhibited progesterone production by 62+/-6% without affecting concentrations of either PGF(2 alpha) or PGE(2). We have concluded that the progressive decline in prostaglandin production and the rise in progesterone output from luteinizing human granulosa cells occur independently of each other.     

Hyperglycosylated human chorionic gonadotropin does not increase progesterone production by luteinized granulosa cells

Context: Trophoblast-derived human chorionic gonadotropin (hCG) promotes corpus luteum progesterone (P4) production, and wide ranges of serum P4 levels are noted in various pregnancy outcomes, despite similar hCG concentrations. There are five unique biologically active hCG variants in human pregnancy urine, and previous studies of P4 production in response to hCG have used only preparations containing all isoforms. Understanding exactly which hCG variant is primarily responsible for stimulating corpus luteum steroidogenesis may have great clinical and diagnostic implications, including in the setting of ectopic pregnancy. Objective: Our objective was to delineate the role of the standard and hyperglycosylated (H)-hCG isoforms in stimulating P4 production by luteinized granulosa cells. Design and Setting: Cell culture, ELISA, and fluorometric-based protein assays were done at Duke University Medical Center. Patients: Patients were anonymous oocyte donors. Intervention: Cultured luteinized granulosa cells were treated with 0.25, 0.5, and 1.0 ng/ml total hCG, which contains all isoforms, purified standard hCG (37.1 kDa), and purified H-hCG (42.8 kDa). Main Outcome Measure: P4 produced per total cellular protein (nanograms per microgram) was measured via ELISA and fluorometric protein determination kits. Results: Both total hCG (P = 0.0003) and purified standard hCG (P < 0.0001) stimulated a dose-dependent increase in P4 production. Purified H-hCG did not change the P4 produced per total cellular protein response (P value not significant). Conclusions: Standard hCG stimulated P4 production by cultured granulosa cells and likely supports corpus luteum function via interactions with the LH/hCG receptor. In contrast, H-hCG did not increase P4 production, which indicates a nonsteroidogenic role for this protein during early gestation.