Basal Progesterone Research Articles

EFFECT OF EXOGENOUS OVINE PLACENTAL LACTOGEN ON BASAL AND PROSTAGLANDIN-STIMULATED PROGESTERONE PRODUCTION BY PORCINE LUTEAL CELLS

The ability of ovine placental lactogen (oPL) to stimulate progesterone secretion of porcine luteal cells isolated from ovaries in different stages of the oestrous cycle and to support the luteotropic action of PGE2 or to protect the corpus luteum (CL) against the luteolytic action of PGF2 alpha was investigated. oPL in all doses used had no effect on progesterone production of cells isolated from early developing corpora lutea while in doses of 1 and 10 ng/ml it increased oestradiol secretion by this type of cells. In doses of 1, 10 and 100 ng/ml it also increased progesterone secretion of cells isolated from mature corpora lutea in a dose-dependent manner. No influence on progesterone production of cells isolated from regressing corpora lutea was observed. oPL added to the culture media had no effect on PGE2-stimulated progesterone production by cells isolated from mature corpora lutea. However, it exerted a protective effect against the luteolytic action of PGF2 alpha observed in cultures treated with PGF2 alpha alone or in combination with PGE2 in a ratio of 4:1. These studies provide evidence that oPL is luteotropic and supports progesterone production in swine. The fact that oPL acted directly on ovarian steroidogenesis suggests that it may also play some role under non-pregnant physiological conditions. Future studies of structural and functional proteins secreted by the porcine conceptus will help resolve this uncertainty.

Activin A in JEG-3 cells: potential role as an autocrine regulator of steroidogenesis in humans.

Activin A has been shown to exert several regulatory functions on human placenta. In the present study, we tested the hypothesis that activin A is an autocrine regulator of trophoblast using a choriocarcinoma cell line, JEG-3, as a model. Messenger RNAs for activin beta(A) subunit, activin binding protein (follistatin), and various activin receptors, including ActR-IA, ActR-IB, ActR-IIA, and ActR-IIB, were detected in JEG-3 cells by reverse transcription-polymerase chain reaction. The expression of activin A in JEG-3 cells was further confirmed by Western blot analysis using an antibody against activin beta(A) subunit. Using Northern blot analysis, Smad-2 and Smad-4 mRNAs were also observed in JEG-3 cells. These data suggest that JEG-3 cells produce activin A and express activin binding proteins and receptors, as well as potential downstream signals. In cultured JEG-3 cells, basal progesterone production was stimulated by activin A but inhibited by follistatin-288. Similarly, in the presence of androstenedione, estradiol production was enhanced by activin A but decreased by follistatin-288. On the other hand, neither activin A nor follistatin affected JEG-3 cell growth. Taken together, these findings strongly suggest that activin A is an autocrine factor that is involved in the regulation of progesterone and estradiol production in JEG-3 cells.

High Ovulatory Rates With Use of Troglitazone in Clomiphene-Resistant Women With Polycystic Ovary Syndrome

This preliminary report reviews our experience with 18 infertile patients with clomiphene-resistant polycystic ovary syndrome (PCOS). In the first treatment cycle, troglitazone was administered alone. During cycles 2-5, clomiphene was added with increments of 50 mg (up to 200 mg/day) if the previous cycle was anovulatory. Basal body temperature charts and serum progesterone were obtained to confirm ovulation. In a total of 66 treatment cycles, ovulation occurred in 44 (67%) and pregnancy in seven (11%). There were no significant changes in body weight, waist:hip ratio or liver enzymes during treatment. Troglitazone, alone or with clomiphene, induced ovulation in 15 of 18 patients (83%) and seven (39%) of them achieved pregnancy. This is the first report on ovulatory rates in clomiphene-resistant women with PCOS when troglitazone was used alone or with clomiphene. Recently, metformin and clomiphene were successfully used in women with PCOS. However, our patients represent a more resistant population of women with PCOS, with each patient serving as her own historical control by previous resistance to clomiphene. Although the pregnancy rate (39%) was promising for clomiphene-resistant women with polycystic ovary syndrome, it does not seem to have a definite advantage over gonadotrophins.

Placental lactogen as a regulator of luteal cells function during pregnancy in sheep

The luteotropic activity of ovine placental lactogen (oPL) on different days of gestation in ewes was assessed using in vitro methods. Corpora lutea (CL) harvested on Days 45, 70, 95, 120 and 135 of gestation and during parturition were enzymatically dispersed and plated on multiwell plates. After 48 h of incubation, all cultures were terminated and media were frozen for further steroid analysis. Cells were cultured in control medium, with addition of oPL alone, or in combination with PGE 2 or PGF 2α. Supplementation of culture media with oPL increased basal progesterone secretion by cells isolated on Days 45 and 70 of gestation. There was no effect on progesterone secretion by cells isolated on other days of gestation; PGE 2 added to the culture media increased progesterone production only by cells isolated on Day 70 of pregnancy. Simultaneous oPL treatment with PGE 2 had a statistically significant and stimulatory effect on progesterone production by luteal cells collected on Days 70 and 95 of pregnancy. In contrast, PGF 2α alone in culture media decreased progesterone secretion by cells isolated on Days 45, 70 and 95 of gestation, while oPL plus PGF 2α on Days 70 and 95 of gestation protected against luteolytic action of PGF 2α. The results showed 1) a direct effect of the oPL on luteal cells isolated on Days 45 and 70 of gestation; 2) synergism between PL and PGE 2 in progesterone production; by cells isolated on Day 70; 3) and a luteoprotective effect of oPL against the luteolytic action of prostaglandin F (PGF 2α) observed on Days 70 and 95 of gestation.

Nitric oxide synthase activity and progesterone release by isolated corpora lutea of rabbits in the early and mid-luteal phases of pseudopregnancy are modulated differently by prostaglandin E-2 and prostaglandin F-2alpha via adenylate cyclase and phospholipase C.

By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2alpha (PGF-2alpha) induced in the corpora lutea (CL) of pseudopregnant rabbits, we have demonstrated that these prostaglandins modulate luteal nitric oxide synthase (NOS) activity and progesterone production differently, depending on the age of the CL. On CL obtained on day 4 of pseudopregnancy (day-4), PGE-2 was found to depress NOS total activity to 13% of control and to significantly increase basal progesterone secretion by 61%, while PGF-2alpha had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2alpha caused a 2.5-fold increase of NOS activity and a marked decrease in progesterone production. Using specific inhibitors, we found that the regulatory actions of PGE-2 in vitro are mediated via the adenyl cyclase/protein kinase A (PKA) second messenger system, while the PGF-2alpha-induced luteolytic effects on day-9 CL depend upon activation of the phospholipase C/protein kinase C (PKC) system. The different responsiveness of day-4 and day-9 CL to PGE-2 and PGF-2alpha could depend on receptor availability for these two prostaglandins, even if other cellular mechanisms cannot be excluded. The present study supports a functional role for NOS in regulating the steroidogenic capacity of rabbit CL, and reveals a novel interaction between a stimulatory G-protein-coupled receptor and PKC/PKA-mediated signal transduction modulating NOS activity.

Variant estrogen receptor-alpha messenger RNA expression in hormone-independent human breast cancer cells.

T5-PRF cells are insensitive to the growth-stimulatory effects of estrogen while still retaining expression of estrogen receptor-alpha (ER-alpha). In the apparent absence of ligand, T5-PRF cells have a 3. 6+/-0.5 (s.e.m.)-fold increased basal ER-alpha activity and elevated basal progesterone receptor levels compared with the parent, estrogen-sensitive, T5 cells. Long-range ER-alpha reverse transcription-PCR was performed to characterize variant ER-alpha mRNA expression in the two cell lines. An increased relative expression of an exon 3/4-deleted ER-alpha mRNA variant was found in T5-PRF. Recombinant expression of this ER-alpha variant resulted in significantly increased estrogen responsiveness, as well as a trend to increased basal ligand-independent activity when expressed with wild-type ER-alpha in ER-negative cell lines, as well as significantly increasing both ligand-independent and estrogen-induced ER-alpha transcriptional activity when expressed in parental T5 cells. These results suggest a role for altered variant ER-alpha in ligand-independent activation of ER-alpha which may contribute to hormone independence in breast tumors.

Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.

Pulsatile output of prostaglandin F(2alpha) does not increase around the time of luteolysis in the pregnant goat.

Prostaglandin (PG) F(2alpha) secreted from the uterus is the luteolysin of the estrous cycle and is also believed to be responsible for luteolysis in the pregnant doe at term. We have reported that basal progesterone concentrations decrease before basal PGF(2alpha) concentrations increase, which is inconsistent with this view. In this study we investigated whether luteolysis is associated with increased frequency or amplitude of pulsatile PGF(2alpha) secretion in does over the last 2 wk of gestation. Progesterone concentrations decreased approximately 1 wk before parturition. There was no accompanying increase in PGF(2alpha) concentrations or pulse frequency, and those pulses that were observed were of lesser amplitude and duration than those that have been associated with luteolysis in cycling ewes. A small increase in PGF(2alpha) pulse frequency was identified during the 3 days before parturition, but this was not associated with any change in progesterone concentrations. The biological significance of these small changes in PGF(2alpha) pulse frequency is obscure, although the high concentration of this eicosanoid at labor may have been related to the final, precipitous decline in plasma progesterone concentrations. These findings do not support the notion that PGF(2alpha) is the principal luteolysin in the pregnant doe at term.

Cell characteristics and function of two enriched fraction of human luteal cells prolonged culture.

Two subpopulations of steroidogenic cells exist in the corpus luteum of most species. The aims of the present study were to characterize these cells and to study their function during long-term culture. Human corpora lutea from early and late luteal phases were treated by mechanical and enzymatic digestion, followed by density sedimentation. Five distinct cell bands were obtained, two of which produced large amounts of progesterone. These were characterized according to density, size, steroidogenic enzymes, and numbers. More than 75% of cells expressed immunoreactive 3beta-hydroxydehydrogenase (3beta-HSD). Cells of higher density/smaller size were obtained in increasing numbers during the luteal phase and were more numerous compared with large cells. Under basal, human chorionic gonadotrophin (HCG)-, and prostaglandin E(2)-stimulated culture conditions, progesterone synthesis was greater in large cells of the early, but not late, luteal phase. Both cell fractions obtained from late, in contrast to early, luteal phase increased their basal progesterone production during the culture period of 9 days. We conclude that this technique for luteal cell isolation in the human yields two distinct subpopulations of steroidogenic cells, which respond differently to luteotrophic stimuli. We also conclude that cells of late luteal phase readily increase their progesterone synthesis over a period of 9 days, indicating a transition to longevity.

Inhibitory effects of TNFα on mouse tumor Leydig cells: possible role of ceramide in the mechanism of action

TNFα is reported to inhibit steroidogenesis in mouse Leydig cells. In primary cells this inhibition resulted mainly from a reduced expression of Cyp-17 gene. Mouse tumor Leydig cells, MA-10, being free of macrophages and lacking Cyp-17, appear to be an excellent model to investigate those effects of TNFα which are independent of either macrophages or Cyp-17. We report here that TNFα receptors are expressed in this cell line. Treatment of the cells with TNFα had no effect on basal progesterone production. In contrast, LH-, 8Br-cAMP and forskolin-stimulated progesterone production was inhibited by TNFα. Neither enzymes involved in the conversion of cholesterol to pregnenolone nor hormone-induced hydrolysis of [ 14C] cholesterol-ester were affected by TNFα. The hormone- induced expression of StAR protein was diminished in mitochondrial fractions from TNFα-treated cells. Also cell permeable ceramides markedly inhibited StAR protein levels. We show further that TNFα was able to induce [ 14C]-ceramide accumulation in MA-10 cells and suggest that this sphingolipid may be considered as a transmitter of TNFα signals to the StAR protein.

Functional dopamine-1 receptors and DARPP-32 are expressed in human ovary and granulosa luteal cells in vitro.

The catecholamines norepinephrine and dopamine (DA) are present in the human ovary; in particular, in follicular fluid. Norepinephrine activates ovarian alpha- and beta-adrenergic receptors and modulates ovarian steroidogenesis, but the significance of ovarian DA is unclear. We examined whether a DA receptor of the D1-subtype (D1-R) is present in human ovary and in cultured human granulosa luteal cells (GC). Using RT-PCR, we cloned complementary DNAs from adult human ovarian and GC messenger RNAs, which are identical to the human D1-R sequence. In ovarian sections, D1-R protein was identified (by immunohistochemistry) in granulosa cells of large antral follicles, cells of the corpus luteum, as well as in cultured GC. An immunoreactive band of approximately Mr 50,000 was found in cultured luteinized GC using the same antiserum in Western blots. The D1-R in these cells was functional, because DA, alone or in the presence of the beta-receptor antagonist propranolol, caused cellular contraction. The selective D1-R agonist SKF-38393 induced a similar change in cytomorphology and increased the levels of media cAMP. SKF-38393 failed, however, to significantly affect basal and hCG-stimulated progesterone release in vitro, indicating that the activation of the D1-R was not directly linked to synthesis of progesterone, the major steroid of human GC. Estradiol synthesis likewise was not affected. Using RT-PCR and immunohistochemistry, we found that GC express DA- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), a protein typically associated with neurons bearing the D1-R. In cultured GC, DA and SKF-38393 induced increased threonine-phosphorylation of DARPP-32, even in the presence of propranolol but not in the presence of D1-R antagonist SCH-23390. Taken together, the presence of DA and a functional DA receptor and DARPP-32 indicate that a novel, physiological regulatory pathway involving DA exists in the human ovary.

In vitro culture of hamster ovarian primary interstitial cells: effect of serum.

The function of ovarian interstitial cells has been largely addressed using rat theca-interstitial cell culture. However, this preparation is primarily enriched with theca and secondary interstitial cells, which make it difficult to address selectively the function of the primary interstitial cells. We have developed an in vitro culture of hamster ovarian primary interstitial cells. Cells were isolated from postnatal hamster ovaries by collagenase digestion and purified over a Percoll gradient. The preparation contained 90% viable, pure interstitial cells, which anchored to the plastic and glass culture surface in the presence of fetal bovine serum. Cell proliferation was noted in the presence of serum dosages higher than 0.2%; however, reduction of serum concentration to 0.1% or complete serum starvation did not affect cell viability but almost completely abolished cell proliferation as determined by [3H]thymidine incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage immunoreactivity, which corresponded to basal progesterone and androstenedione accumulation. Replacement of serum to starving cells resulted in the induction of the "S" phase and "M" phase specific cyclins, and resumption of cell proliferation. Our results indicate that hamster primary interstitial cells can be cultured in vitro as a monolayer, and the anchorage and proliferation of these cells depend on serum supplement; however, a viable monolayer can be maintained for several days without serum. This model will be useful for addressing the mechanisms of differentiation of ovarian interstitial cells.

Endothelin-1: expression and role in human corpus luteum.

Several recent data suggest an involvement of endothelin (ET)-1, a powerful vasoconstrictor peptide, in reproductive function. This study was designed to investigate the presence and role of ET-1 in human corpus luteum. Purified luteal cells were incubated for different times with ET-1 or ET-3 alone or associated with human chorionic gonadotropin. In another set of experiments cells were treated with ET-1 and BQ485, an ET-A receptor antagonist, or with phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C. ET-1 reduced both basal and human chorionic gonadotropin-induced progesterone production at all examined times, similarly PMA inhibited basal progesterone synthesis. BQ485 prevented the inhibitory effect of ET-1, while no effect was observed with ET-3. Finally, ET-1 mRNA was detected in the luteal cells. ET-1 is expressed by human luteal cells and reduces basal and human chorionic gonadotropin-induced progesterone synthesis through the ET-A receptors and the protein kinase C pathway. Conversely, ET-3 does not affect luteal steroidogenesis.

Luteotropic hormone receptors in the ovary of the mink (Mustela vison) during delayed implantation and early-postimplantation gestation.

The reproductive cycle of the mink displays rigid seasonality and obligate embryonic diapause. After ovulation, the corpus luteum (CL) involutes, and it secretes basal progesterone until activated prior to implantation. To study changes in the relative abundance of luteal prolactin and LH receptor mRNA through gestation, ovaries and serum were collected from pregnant female mink at 2-day intervals (n = 3 per date) through embryonic diapause and CL activation (March 19-31) and at 5-day intervals through implantation and early-postimplantation gestation (March 31-April 15). To determine the effect of endogenous prolactin, mink received Alzet osmotic minipumps releasing 2 mg/day 2-bromo-alpha-ergocryptine (bromocriptine) or saline on March 19. Ovaries and serum were taken from 3 animals every 2 days until March 31. Prolactin receptor mRNA in ovaries was low during CL activation but increased 3-fold through embryo implantation. Its abundance correlated with prolactin binding to ovarian membranes and with circulating prolactin. Bromocriptine suppressed endogenous prolactin levels and prevented the increase in prolactin receptor mRNA. There was a transient peak in LH receptor mRNA in the ovaries at March 19-23, which declined to basal levels by March 25 and remained constant through midgestation. Bromocriptine prevented the preimplantation peak in LH receptor mRNA and reduced its abundance below pretreatment levels. The results suggest that prolactin up-regulates its receptor and maintains the LH receptor in the mink CL. The pattern of LH receptor mRNA argues for a role for LH in CL reactivation and termination of embryonic diapause in mink.

Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen

During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum. Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied. We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH). Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001). In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001). In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed. These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.

Effects of LH, prostaglandin E2, 8-bromo-cyclic AMP and forskolin on progesterone secretion by pig luteal cells.

The present study examined the effects of LH, prostaglandin E2 (PGE2), 8-bromo-cyclic AMP (cAMP) and forskolin on progesterone secretion by small and large pig luteal cells. Corpora lutea were isolated from gilts (n > or = 3 per day) on days 9, 12 and 14 of the oestrous cycle and days 9, 12, 14 and 30 of pregnancy. After enzymatic dissociation of the corpora lutea, small and large luteal cells were obtained by elutriation. Culture plates (24-well) were then seeded with 150,000 small luteal cells or 30,000 large luteal cells per well in 1 ml M199 medium in the absence or presence of LH, PGE2, LH plus PGE2, 8-bromo-cAMP or forskolin. After 12 h of incubation, culture plates were centrifuged, and the supernatant collected and frozen for subsequent assay of progesterone. Differences within day were not detected between cyclic and pregnant gilts, and thus, results were combined for days 9, 12 and 14. Basal progesterone secretion by small luteal cells was less (P < 0.05) on days 14 and 30 than days 9 and 12. Treatment with LH, PGE2, 8-bromo-cAMP or forskolin increased (P < 0.05) progesterone secretion by small luteal cells on days 9 and 12; however, treatments had no effect on days 14 and 30. Basal progesterone production by large luteal cells was less (P < 0.05) on day 30 compared with other days. PGE2 stimulated (P < 0.001) progesterone production by large luteal cells at all days. In contrast, 8-bromo-cAMP and forskolin inhibited progesterone production by large luteal cells on day 12 (P < 0.05), and day 14 (P < 0.001). These data show that pregnancy status does not alter luteal cell response to the aforementioned secretagogues. However, regulation of progesterone secretion differs between small and large luteal cells, and the age of the corpora lutea. Also, it is unlikely that the stimulatory actions of PGE2 involve increased cAMP production in pig large luteal cells.

Partial Purification and Characterization of Two Non-FSH Steroid-Modulating Factors in Rat Thymic Epithelial Cell-Conditioned Medium (TCM)

Previously, we have reported that unknown factor(s) in rat thymic epithelial cell-conditioned medium (TCM) stimulates basal and follicle-stimulating hormone (FSH)-induced steroid hormone production and aromatase enzyme activity in cultured rat granulosa cells. Here we report the partial purification and characterization of two of these activities. Thymic epithelial cells were prepared from immature female rats and used for TCM production. Lyophilized aliquots of TCM were reconstituted with distilled water at 25% of the original volume, applied to a gel filtration column, and column fractions were tested for their stimulation of steroidogenesis in granulosa cells prepared from immature diethylstilbestrol-treated rats. Two distinct biologically active regions were identified that corresponded to apparent molecular weights of approximately 22,000 and less than 1,000. The <1 kDa activity (“TCM-1”) stimulated (P < 0.01) basal production of progestins [progesterone and 20α-hydroxypregn-4-en-4-one (20α-OH-progesterone)] and estrogen, and also induced dramatic morphological changes on the rat granulosa cells. In contrast, the ∼22 kDa activity (“TCM-22”) stimulated (P < 0.01) only basal progestins, and had no effect (P < 0.05) on basal estrogen production or morphology of the cultured rat granulosa cells. In the presence of 100 ng/ml FSH, TCM-1 stimulated (P < 0.01) estradiol and progesterone production, whereas TCM-22 stimulated (P < 0.01) progesterone, but inhibited (P < 0.01) estradiol production. When both activities were assayed together, they were synergistic in stimulating (P < 0.01) basal progesterone production, but TCM-22 antagonized (P < 0.01) TCM-1-induced estradiol production. The biologic and physico-chemical characteristics of TCM-1 and TCM-22 were distinct from one another, as well as from FSH. When subjected to C 8 reverse-phase HPLC, TCM-1 retained its characteristic biologic properties and was eluted (54% acetonitrile) as A 214-absorbing moiety with a peak retention time of 92–93 minutes. The elution of TCM-22 was not correlated with an identifiable protein peak. These results suggest that ovarian steroid production may be modified by non-FSH factors produced by thymic epithelial cells although amino acid sequencing of TCM-1 was unsuccessful. This highlights a potential role of the thymus gland in regulating ovarian function.

Recombinant luteinizing hormone as a survival and differentiation factor increases oocyte maturation in recombinant follicle stimulating hormone-supplemented mouse preantral follicle culture.

The effects of recombinant luteinizing hormone (rLH) and recombinant follicle stimulating hormone (rFSH) both separately and together were analysed on the in-vitro maturation of a well-defined class of mouse early preantral follicles. Metaphase-II (MII) oocytes were only obtained when rLH or rFSH or a combination of both hormones was added to a standard rich culture medium at constant doses throughout the culture period. Without gonadotrophins only 8% of the follicles reached day 12 of culture. Follicle survival was optimal (between 90 and 100%) when rFSH was used; significantly fewer follicles were kept intact when rLH was added as the only supplement (54%; P < 0.001). Theca cells played a fundamental role in follicle survival when rLH was the only supplement. Presence of rLH significantly influenced the formation of antral-like cavities: on day 12, 59% of follicles cultured with rLH + rFSH had a large cavity, as compared with only 21% in cultures with rFSH only (P < 0.0001). The rHCG stimulated germinal vesicle breakdown rate from the different gonadotrophin-supplemented cultures on day 12 was not significantly different (FSH: 89%; LH: 70%; FSH/LH: 83%). However, addition of rLH to rFSH did significantly improve (P < 0.05) the completion of the first meiotic division up to the MII stage (FSH: 46%; LH: 39%; FSH/LH: 76%). Interfollicle differences in steroid production were very large and depended on the presence of theca cells and on the kind of gonadotrophin supplemented. Without theca cells, oestrogen concentrations in rFSH-supplemented cultures were 100 times lower than their theca-containing counterparts during the entire culture period. From culture day 9 onwards most conditioned media had increased basal progesterone concentrations. This study points up the in-vitro role of LH on the differentiation of follicle-like structures and the oocyte's meiotic maturation and suggests an important role for theca cells in providing modulators of in-vitro gonadotrophin action.

Beta-endorphin inhibition of progesterone secretion by porcine granulosa cells during follicle development.

Exogenous beta-endorphin or the opioid antagonist, naloxone, was added to the culture medium of control or LH-treated granulosa cells to elucidate the role of beta-endorphin in follicular steroidogenesis. beta-endorphin decreased basal progesterone (P4) production by Gc isolated from small and medium follicles and had no effect on P4 production by cells from large follicles. beta-endorphin decreased P4 production by LH-treated Gc isolated from small, medium and large follicles. Naloxone increased basal P4 production by cells from small and medium follicles and had no effect on P4 production by Gc from large follicles. Neither agent had any effect on basal oestradiol (E2) secretion by Gc from any of the follicles. However, E2 secretion by LH-treated Gc from all sizes of follicles was inhibited by beta-endorphin, with the greatest effect on cells from small follicles. We conclude that opioid peptides act in the follicle in a paracrine manner to prevent excessive basal progesterone secretion by Gc and may also be involved in the regulation of LH action on granulosa cells steroidogenesis during follicle development.

Effects of estradiol and an aromatase inhibitor on progesterone production in human cultured luteal cells

The human corpus luteum produces both estradiol and progesterone. It is well known that there are both autocrine and paracrine systems for the regulation of the corpus luteum and that estradiol regulates the progesterone production of the corpora lutea of some other species. To assess the direct effects of estrogen on human luteal function, we performed cell culture experiments.A low concentration of estradiol, almost equal to the amount of estradiol produced by human cultured luteal cells, directly stimulated progesterone production. 4-Cyclohexylaniline, an aromatase inhibitor, significantly reduced both progesterone production and estradiol production.Levels of estradiol higher than the levels that cultured human luteal cells themselves produced significantly reduced basal progesterone production and also significantly reduced human chorionic gonadotropin (hCG), forskolin and dibutyryl-cyclic AMP-stimulated progesterone production. According to these data, high doses of estradiol produced a luteolytic action which widely inhibited the steroidogenesis process.In conclusion, our results indicated that estradiol in part regulates progesterone production physiologically and blocks progesterone production in a pharmacological or pathological state in the human corpus luteum.