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

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

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

Luteal macrophage conditioned medium affects steroidogenesis in porcine granulosa cells

The purpose of the study was to examine the effect of luteal macrophage conditioned medium (LMCM) on progesterone and estradiol production by cultured granulosa cells. Porcine granulosa cells were cultured for 48 h with or without LMCM in the absence or presence of 100 ng/ml LH, FSH or prolactin. Progesterone and estradiol concentrations were measured by radioimmunoassay. Granulosa cells were analyzed histochemically and immunocytochemically for the activity and presence of Δ5, 3β-hydroxysteroid dehydrogenase (3β-HSD), respectively. LMCM stimulated basal and LH-, FSH- or prolactin-induced progesterone secretion. Similarly, LMCM augmented basal and stimulated activity of 3β-HSD in the examined cells. In contrast, LMCM decreased LH- and prolactin-induced estradiol secretion but increased FSH-induced estradiol secretion. These data demonstrate the clear stimulatory effect of LMCM on granulosal progesterone production. It is concluded that substances secreted by macrophages modulate gonadotropin effect on follicular progesterone secretion in a paracrine manner via 3β-HSD activity.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Effect of histone H2A on progesterone production by bovine luteal cells

Histone H2A competitively inhibits binding of GnRH to high affinity rat ovarian receptor sites and blocks gonadotropin-stimulated steroid and cAMP accumulation during culture of rat granulosal or luteal cells. The objective of our study was to examine the progesterone suppressive effects of histone H2A on bovine luteal cells. In the first study, luteal cells were treated at Time = 0 h with a partially purified preparation of bovine ovarian histone H2A (3 ng GnRH equivalents, 800 micrograms protein), equivalent amounts of GnRH (3 ng), or BSA (800 micrograms) and incubated for a total of 4 h. At Time = 2 h, cells were treated with 5 ng bovine LH (bLH) or with medium. Histone H2A completely blocked both basal and LH-induced accumulation of progesterone compared with untreated cultures or cultures treated with bLH. Neither BSA nor GnRH suppressed LH-induced progesterone accumulation. In the second study, histone H2A was added to cultures at Time = 0 h and bovine luteal cells were cultured for 8 h. After 2 h of treatment, histone H2A (3 ng GnRH equivalents) was removed from selected cultures and replaced with fresh medium. Four hours later cultures were treated with 5 ng bLH or medium. LH treatment of cultures from which histone H2A had been removed resulted in an increase in accumulation of progesterone compared with control cultures treated throughout the treatment period with histone H2A. The third study examined the effect of 9-181 pg GnRH equivalents (1.7-34 micrograms protein) of a highly purified preparation of bovine ovarian histone H2A on basal and LH-induced progesterone production during 2 or 3 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of cytokines on porcine granulosa cell steroidogenesis in vitro

Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.     

心房钠尿肽（ANP）对大鼠卵巢细胞生成雌激...

Isolated ovarian corpus luteal cells and granulosa cells of rat were employed to investigate the effect of alpha-ANP on the secretion of progesterone and estradiol. The contents of the steroid hormones are determined by RIA. The results showed that 0.1-10 ng/ml ANP promoted progesterone production in a dose dependent manner. alpha-ANP also enhanced progesterone production by granulosa cells, but not estradiol. It seems that the effect of alpha-ANP on ovarian steroidogenesis is a direct one.     

Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. Effects of these hormones on progesterone secretion in vitro

The relative levels of mRNAs for relaxin, prolactin, inhibin and oxytocin have been measured in porcine granulosa as well as luteal cells by hybridisation to single-stranded synthetic DNA. The likelihood of a paracrine function of oxytocin and prolactin in the porcine ovary was inferred from the in vitro effects of both hormones on progesterone secretion of ovarian cells. Both hormones were found to inhibit progesterone secretion of luteal cells. In contrast, only prolactin but not oxytocin stimulated progesterone secretion in granulosa cells.     

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.     

Prolactin, LH, and estradiol-17 beta in utilization of lipoprotein substrate by porcine granulosa cells in vitro

Porcine granulosa cells cultured under serum free conditions responded by increased progesterone secretion to the addition of the leuteotropic hormones, LH, prolactin, and estradiol. Provision of extracellular substrate for steroidogenesis in the form of porcine high density lipoprotein or low density lipoprotein enhanced progesterone accumulation by granulosa cell cultures. Estradiol, LH, and prolactin all greatly increased progesterone accumulation in the presence of either high or low density lipoproteins. Increases in progesterone accumulation following addition of prolactin or LH in combination with estradiol suggested the presence of a synergistic interaction among leuteotropins. Pre-exposure of granulosa cell cultures to estradiol increased the subsequent stimulatory effect of prolactin on lipoprotein utilization. It is concluded that all three leuteotropins function to enhance and may interact in the utilization of extracellular lipoprotein substrate for progesterone synthesis.     

Biochemical and hormonal characterization of follicles from follicular and luteal phase ovaries of goat and sheep.

Follicles from goat and sheep ovaries were characterized for their biochemical and hormonal parameters to investigate the effect of developmental stage of follicles on ovarian steroidogenesis. The follicles were isolated mechanically from follicular and luteal phase ovaries and divided in 6 morphologically different groups (small, medium and large follicular and small, medium and large luteal). Follicles were characterized for their contents of protein, DNA, estradiol-17 beta and progesterone and the activity of 3 beta-hydroxysteroid dehydrogenase. There was a progressive increase in the contents of all these biomolecules and activity of the enzyme as size of follicles increased in both the follicular and luteal phase ovaries. Follicles from follicular phase ovaries exhibited higher estradiol-17 beta content than those shown by luteal phase follicles. The reverse pattern was obtained for progesterone content. The results provide the basic data on biochemical and hormonal entities at different stages of follicular development in small ruminants which may be useful for in vitro studies on regulation of follicular development and steroidogenesis.     

Use of low-density and high-density lipoproteins in undifferentiated porcine granulosa cells

Granulosa cells aspirated from medium-sized porcine ovarian follicles (3-5 mm) in short-term incubation responded to the addition of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) with increased accumulation of progesterone. HDL was more potent than LDL in enhancing progesterone secretion. When granulosa cells were cultured under serum-free conditions for 72 h, HDL but not LDL exhibited a dose-dependent enhancement of progesterone secretion. Addition of insulin to the cells greatly potentiated the stimulatory effect of LDL on progesterone accumulation, while the response to HDL was only slightly increased. Granulosa cells in culture degraded LDL but not HDL. Addition of insulin enhanced LDL degradation. Exposure of cells in culture to chloroquine, an inhibitor of lysosomal function, completely blocked LDL degradation and LDL-induced stimulation of steroidogenesis. The stimulatory effect of HDL was not affected by chloroquine. We interpret these findings to indicate that granulosa cells derive cholesterol from LDL by means of lysosomal degradation, which is not required for use of cholesterol from HDL. Monensin, a carboxylic ionophore that interrupts recycling of LDL receptors, prevented LDL-enhanced progesterone accumulation but not HDL-induced stimulation. This provides evidence that HDL-induced stimulation of steroidogenesis does not involve LDL receptors. We conclude that HDL present in follicular fluid is capable of providing cholesterol to granulosa cells for steroidogenesis. The stimulatory effect of HDL is not due to the presence of apoprotein E, an HDL component that binds to the LDL receptor. A unique HDL pathway that does not involve LDL receptors and lysosomal degradation may operate in porcine granulosa cells.     

Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells.

Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.     

Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes

Vasoactive Intestinal Peptide (VIP), originally considered to be a gut hormone, has recently been found to increase estrogen and progesterone production by ovarian granulosa and luteal cells. Because several studies indicate that granulosa cells and oocytes are capable of producing plasminogen activators, we have studied the effects of VIP on plasminogen activator activity in cultured granulosa cells and cumulus-oocyte complexes collected from the ovaries of hypophysectomized, estrogen-treated immature rats. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a fibrin overlay technique to assess plasminogen activator activity, we observed that treatment with VIP stimulated the secretion of tissue-type plasminogen activator (tPA), but not urinary-type plasminogen activator (uPA), in a dose-dependent manner by cultured granulosa cells as well as by cumulus-oocyte complexes, but not by denuded oocytes. However, preparation of cumulus-free oocytes from cumulus-oocyte complexes which had previously been treated with VIP indicated substantial increases in tPA activity within the oocyte. The actions of VIP on tPA activity in granulosa cells were specific, because other closely related peptides (PHM-27 and glucagon) were ineffective. These effects of VIP, in addition to the previously observed effects on steroidogenesis, suggest that VIP may be an important regulator of ovarian function.     

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: Effects of prostaglandin F2α, protein kinase C and calcium

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progesterone concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P<0.05) for luteal cells from the late luteal phase. LH increased (P<0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P<0.05) GjIC between small luteal cells from the mid luteal phase and diminished (P<0.05) LH-stimulatory effects on GjIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P<0.05) the rate of GjIC between large and small, and between small luteal cells, and A23187 decreased (P<0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P<0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GjIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GjIC between small and between large and small luteal cells, whereas calcium ionophore decreases GjIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.     

Effects of androgen on progesterone secretion from granulosa cells obtained from antral follicles of rats

The effects of testosterone (T) on the secretion of progesterone (P) by ovarian granulosa cells obtained from immature rats pre-treated with pregnant mare's serum gonadotropin were examined in vitro. T (10 nM-10 microM) enhanced both basal and FSH- or cAMP-stimulated secretion of P in a dose-dependent manner. Furthermore, T augmented FSH-stimulated cAMP production. The biphasic secretory pattern of P induced by continuous superfusion of granulosa cells with FSH was much exaggerated in the cultures supplemented with T. A stimulatory effect of T on secretion of P was observed only in the medium that contained serum. T affected neither the basal nor the FSH-stimulated secretion on 20 alpha-dihydroprogesterone. Androsterone, a non-aromatizable and low-potency androgen, at a similar concentration as T mimicked the effects of T on the secretion of progesterone. These results indicate that androgen stimulates mature granulosa cells to enhance the secretion of P. This androgen action extends either up- or down-stream of cAMP in the process of steroidogenesis.     

Inhibin production by bovine ovarian tissues in vitro and its regulation by androgens

No detectable amounts of inhibin were produced by cultured ovarian stroma or luteal tissue. Follicular tissue produced inhibin in vitro and removal of the granulosa cells from the follicle wall caused inhibin production to fall by 80%. Granulosa cells alone had the greatest ability of any ovarian cell type to produce inhibin in vitro, and are probably the major site of follicular inhibin production. Cyproterone acetate at concentrations of 35 and 350 microM inhibited basal and testosterone (3.5 microM)-stimulated inhibin production by cultured intact follicle wall and granulosa cells. In addition, each concentration of cyproterone acetate inhibited progesterone but not oestradiol-17 beta production by the follicle wall and granulosa cell cultures. The synthetic, non-aromatizable androgens, methylestrenolone and mesterolone, at concentrations of 5 and 25 microM, mimicked the effect of testosterone and stimulated granulosa cell inhibin production, methylestrenolone being the more potent. These findings provide further evidence that androgens regulate follicular inhibin and progesterone production and that these may be receptor-mediated processes, and suggest that inhibin production may be a general property of androgenic compounds. Preliminary examination of the physicochemical characteristics of inhibin indicated that the inhibin activity of bovine granulosa cell culture medium was (a) retained by an Amicon XM100A filter with a nominal molecular weight cut-off point of 100 000; and (b) destroyed by heating to 80 degrees C for 30 min.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Morphological correlates of follicular fluid stimulation of steroidogenesis in immature porcine granulosa cells

Factors in porcine ovarian follicular fluid are known to influence steroidogenesis in cultured ovarian granulosa cells. This study examined whether ultrastructural changes characteristic of normal maturation and/or atresia accompany the steroidogenic alterations. Two and 5 day incubations of immature porcine granulosa cells were performed in media supplemented with either serum or follicular fluids (FFL) from mature follicles. Under these conditions both oestrogen and progesterone secretion were stimulated in FFL supplemented cultures as compared to serum supplemented cultures. Cells in serum exhibited increased size, number and volume of lysosomes and resembled in vivo atretic cells. In comparison, the FFL treated cells had greatly increased steroid output, numerous microvilli and increased size, number and volume of electron dense lipid droplets after 2 days of culture although the differences declined by day 5 of culture. This suggests that mature FFL contains a factor(s) stimulating granulosa maturation while inhibiting ultrastructural correlates of follicular atresia.     

In vitro responses of luteinizing rat granulosa cells to human thyroid-stimulating hormone

The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.