Luteal Steroidogenesis Research Articles

Are human luteinizing granulosa cells a site of action for progesterone and relaxin?

Specific nuclear staining for progesterone receptor (PR) was detected by immunocyto-chemistry in human granulosa cells (GCs) obtained from in vitro fertilization protocols. The percent of PR-positive cells (60% to 80%) remained unchanged during 7 days of culture in media containing fetal calf serum, in the absence or presence of human chorionic gonadotropin (hCG) or the progesterone antagonist RU486. Progesterone (P) production by GCs cultured on extracellular matrix from bovine corneal endothelial cells was stimulated by hCG and prostaglandin E 2 (PGE 2 ). However, addition of RU486 or human relaxin had no effect on control, hCG-, or PGE 2 -stimulated P production. Thus, the receptor data are consistent with an autocrine action of P in luteinizing GCs, but initial experiments in cell culture did not define a role for P or relaxin in modulating luteal steroidogenesis.

Localization of superoxide dismutase in the ovary and uterus of mice during pregnancy with special reference to steroidogenesis and implantation.

The distribution of the enzyme Cu-Zn superoxide dismutase in the ovary and uterus during early pregnancy has been dealt with in this histochemical study. Cu-Zn superoxide dismutase is found to be present in membrana granulosa of Graafian follicles and ovulated follicles, corpora lutea and blood vessels of the ovary. This presence has been attributed a functional significance in the process of luteal steroidogenesis. The marked low levels of uterine Cu-Zn superoxide dismutase and its noticeable absence in the endometrium, the site of blastocyst attachment, at 5.00 A.M. on Day 5 of pregnancy is evident. The possible involvement of these lowered levels in implantation is discussed.

Effect of gonadotrophin-releasing hormone and related analogue on human luteal cell function in vitro

The possible direct effect of gonadotrophin-releasing hormone (GnRH) and GnRH agonist (GnRH-A; buserelin) on basal and human chorionic gonadotrophin (HCG)-stimulated progesterone (P) and cyclic AMP (cAMP) production by cultured human luteal cells was examined. Luteal cells from the early or mid-luteal phase were incubated in long-term cultures. They responded to HCG stimulation with a 2- to 3-fold increase in P production and a 2-fold increase in cAMP production. The addition of GnRH (10(-7) and 10(-5) M) or GnRH-A (10(-7) and 10(-5) M) to the medium had no effect on either basal or HCG-stimulated secretion. These results indicate that both GnRH and GnRH-A have no direct effect on human luteal steroidogenesis in vitro.

Correlation of plasma progesterone concentrations to ovarian H-type lipase activity during pseudopregnancy in the rat.

Conditions for extraction and assay of hepatic type (H-type) lipase from rat ovaries were studied. An alkaline buffer with protease inhibitors and detergents gave the most efficient extraction. The specificity of the assays was ascertained using antiserum to H-type lipase from heparin perfusates of rat livers. H-type lipase activity was determined in ovarian compartments during pseudopregnancy (1-13 days) as well as during the ensuing period of luteal regression (Day 17). The activity was low in the luteal compartment immediately after ovulation, increased 6-fold to a maximum between Day 5 and Day 8 and then decreased again. This is similar to previously known changes in blood flow. There was a significant correlation between luteal H-type lipase activity and plasma progesterone regardless of luteal age. In contrast, neither the activity in the remainder of the ovary nor the activity in plasma changed during the luteal phase or correlated to plasma progesterone. Injection of heparin at the height of the luteal cycle (Day 8) caused a pronounced decrease in luteal lipase and in plasma progesterone. These studies suggest that the H-type lipase activity has an important role in luteal steroidogenesis, probably to facilitate uptake of cholesterol from lipoproteins.

Cellular basis of luteal steroidogenesis in the human ovary.

A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20 alpha-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.

A possible role of superoxide anion radical in the process of blastocyst implantation in [formula omitted

Superoxide anion radical and superoxide dismutase, the enzyme responsible for dismutating it, are both present in the overy and uterus of Mus musculus during early pregnancy. The detectable, stable levels of superoxide radical and the constant high levers of superoxide dismutase in the ovary during early pregnancy suggests that these may be involved in the regulation of extended luteal steroidogenesis for the maintenance of pregnancy. An inverse correlation between the levels of superoxide anion radical and superoxide dismutase in the uterus is shown. The high levels of superoxide anion radical in the uterus on the early morning of Day 5 of pregnancy point towards a probable role for this radical in the act of implantation and in mediating the increased vascular permeability at the initiation of implantation.

Regulation of steroid hormone synthesis by human corpora lutea: failure of follicle-stimulating hormone to support steroidogenesis in vivo and in vitro

AbstractThe authors studied the role of follicle-stimulating hormone (FSH) in luteal steroido-genesis by replacing gonadotropin-releasing hormone (GnRH) infusion with pure FSH 48 hours after ovulation in two hypogonadotropic patients. Plasma progesterone (P) and estradiol (E2) decreased after FSH administration. Human luteal cells were cultured for 48 hours in the presence and absence of FSH, human chorionic gonadotropin (hCG), testosterone (T), or dibutyryl cyclic adenosine monophosphate (Bu2cAMP). In the presence of T, E2 synthesis increased significantly, indicating an active aromatase system in these cells. Human chorionic gonadotropin as well as Bu2cAMP significantly increased E2, T, and P synthesis. Follicle-stimulating hormone did not stimulate luteal E2, T, or P synthesis. The authors conclude that FSH does not sustain luteal steroidogenesis. Moreover, the in vitro findings reveal that hCG modulation of luteal E2 synthesis is mediated principally by an increase in androgen precursors. These in vivo and in vitro results confirm a crucial role for luteinizing hormone (LH) in the maintenance ofluteal steroidogen-esis.

Bovine luteal cell activity in culture. Maintenance of steroidogenesis by high density lipoprotein containing high or low beta-carotene concentrations

Beta-carotene is transported in bovine blood in association with high density lipoprotein (HDL) and is also found in high concentrations within the bovine corpus luteum. We have examined the effect of HDL, containing high or low β-carotene concentrations, upon cyclic AMP-stimulated luteal cell steroidogenesis in culture. Cells were cultured in defined medium containing a low (1%) supplement of lipoprotein-deficient serum. In all experiments, addition of HDL (200 μg cholesterol/ml), irrespective of β-carotene concentration, markedly enhanced luteal progesterone synthesis and in most experiments maintained luteal cell steroidogenesis for at least 2 weeks in culture. In approximately 50% of experiments, β-carotene significantly enhanced progesterone synthesis, although no effect was seen in the other experiments. This inconsistency may be due to variations between cells from different corpora lutea since the same HDL samples had different effects on four cultures prepared from individual corpora lutea. These data show (1) that trophic-stimulated luteal cell steroidogenesis can be maintained at a high level in culture if sufficient substrate is provided and (2) that β-carotene can enhance luteal steroidogenesis although the effect is variable in vitro.

EFFECTS OF GnRH ON LUTEINIZING HORMONE RELEASE AND ONSET OF CYCLIC OVARIAN ACTIVITY POSTPARTUM IN PELIBUEY EWES

Synthetic gonadotropin releasing hormone (GnRH) was administered to Pelibuey ewes to determine its effect on luteinizing hormone (LH) release and on cyclic ovarian activity during the first 3 – 4 wk postpartum. Two GnRH doses were given (10 and 100 μg) on days 4 and 5, 7 and 8, 14 and 15, and 21 and 22, after parturition, respectively. In three trials, 11, 8 and 7 ewes were used. For each trial 5, 3 and 2 ewes were used as controls, respectively; in trial 3, only the 100-μg dose was given once. Results from the three trials indicated that LH release always occurred after GnRH treatment, relative to the saline-treated controls. In trials 1 and 2, the 10-μg dose induced a higher (P < 0.001) LH release than the 100-μg dose; the LH release in response to the 100-μg dose in the three trials was of similar magnitude. Release of LH in response to both doses was lower (P < 0.05) during days 4 – 5 postpartum, than during the following 3 wk. Two types of luteal activity or steroidogenesis were observed in the GnRH-treated ewes, relative to the saline-treated ewes; (1) within 2 – 4 d after each GnRH injection small peaks of progesterone appeared, and (2) within 5 – 15 d after the last GnRH injection there were elevations of progesterone in peripheral plasma to concentrations and of durations characteristic of the luteal phase of the ovine estrous cycle. These results indicate that the pituitary of postpartum Pelibuey ewes regains the ability for maximal release of LH in response to GnRH challenge within 7 d postpartum. Further, treatment with GnRH induces earlier return to cyclic ovarian activity. Key words: Hair sheep, postpartum, GnRH, luteal activity

Effect of a GnRH-analogue and of the phorbol ester PMA on rat luteal cell steroidogenesis

In the rat, the hypothalamic peptide GnRH acts not only at the pituitary but can also modulate ovarian function directly [1, Review]. Recently, a role for a phospholipid- and Ca2+-dependent protein kinase (PK-C) has been suggested in the action of GnRH on the pituitary [2]. GnRH is known to reduce gonadotrophin-stimulated progesterone (P) production by rat luteal cells through the inhibition of hormone-responsive adenylate cyclase [3]. The tumor-promoting phorbol-ester, phorbol 12-myristate 13-acetate (PMA), a well-known activator of PK-C, has been shown to reduce steroidogenesis by mouse Leydig cells through inhibition of hormone-stimulated cAMP formation [4]. In order to determine whether the inhibitory action of GnRH on luteal steroidogenesis is effected through activation of PK-C, we compared the effects of a GnRH agonist, Des-10-Gly-(6Trp)-GnRH-ethylamide (GnRH) on steroidogenesis and cAMP formation by rat luteal cells with those of PMA.

Nonreceptor Binding of Human Chorionic Gonadotropin (hCG): Detection of hCG or a Related Molecule Bound to Endo me trial Tissue during Pregnancy Using Labeled Monoclonal Antibodies that Bind to Exposed Epitopes on the Hormone*

When hCG adsorbs to surfaces, including membranes from tissues that lack specific hCG receptors, it adsorbs with a particular orientation. Some sites on the alpha- and beta-subunits project away from the surface and can be detected with radiolabeled monoclonal antibodies. Other epitopes, which are located on a region on the hormone that presumably contacts the surface, lose their ability to bind antibody. Using antibodies specific for epitopes on hCG which remain exposed and can be detected when the hormone is adsorbed to rat brain homogenates, we found hCG or closely related substances bound to progestational decidual tissues. Immunologically reactive material adsorbed to the decidual tissue increased and decreased in parallel with the serum levels of hCG throughout pregnancy. Binding of labeled monoclonal antibody to substances similar or identical to hCG in other tissues, including placenta and fetal lung, but not red cells, also was identified. Unlike material adsorbed to decidual tissues, receptor-bound hCG was not recognized by any of our alpha-subunit-specific antibodies. This finding suggests either that the adsorbed hormone has a different orientation than receptor-bound hormone or that the adsorbed hormone has dissociated into subunits. These studies represent the first detection of nonreceptor binding of hCG or related molecules to tissues lacking receptors or presumed not to synthesize the hormone. The role of nonreceptor-bound hCG, if any, is unknown. Other than its effects on stimulation of luteal steroidogenesis during early pregnancy, the role of hCG during most of pregnancy has not been determined. Conceivably, the nonreceptor binding we identified is related to a role for hCG in pregnancy that is not associated with an action on the ovarian LH receptor.

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models.

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.

Stimulation of rat ovarian cell steroidogenesis by high density lipoproteins modified with tetranitromethane.

Human high density lipoprotein (devoid of apo-E) was modified by nitration of tyrosine residues with tetranitromethane. As a result of extensive cross-linking, monomeric apo-A-I was markedly depleted, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the modified HDL did not effectively bind to high-affinity sites present on dispersed rat ovarian cells and isolated rat ovarian membranes. Nonetheless, the modified HDL retained the ability to stimulate steroidogenesis by both dispersed rat ovarian cells and cultured rat granulosa cells to a degree at least equal to that of native HDL. Modified HDL stimulated luteal steroidogenesis under basal conditions and when cells were stimulated with luteinizing hormone or 8-bromo-cAMP. Although modified HDL did not effectively bind to high-affinity sites, it exhibited substantial "nonspecific" or "low-affinity" binding which was not displaceable by native HDL. These data suggest that high-affinity binding is not an essential event in the "HDL pathway" and that HDL can deliver its sterols through low-affinity cellular associations.

Do catecholamines play a physiologic role in regulating corpus luteum function in the pseudopregnant rabbit?

In these studies the beta-adrenergic receptor antagonist propranolol was administered to estrogen-treated hypophysectomized pseudopregnant rabbits in vivo, and serum progesterone concentrations were measured to monitor luteal function. In Experiment 1, which was designed to determine an effective dose of propranolol, 1 mg/(kg X h) s.c. propranolol for 3 h (integral of 80 ng/ml in serum) gave an adequate level of beta-adrenergic receptor blockade, i.e., a 1000-fold inhibition of the blood pressure/isoproterenol dose-response relationship. In Experiment 2, "acute" administration of propranolol (P; 1 mg/(kg X h) s.c.) or saline (control, C) for 24 h on Days 7-8, 10-11, and 13-14 of pseudopregnancy did not produce any marked differences in serum progesterone concentrations in P or C animals on any of the days tested, although hourly fluctuations were observed. In Experiment 3, "chronic" (4-day) treatment with propranolol was achieved by the use of propranolol-containing pellets placed s.c. (integral of 200-600 ng/ml in serum), on Days 13-17. Control animals received pellets of vehicle only. Serum progesterone concentrations were very similar in P and C animals throughout the period of treatment (Days 13-17) and on Days 18 and 20. We conclude that endogenous catecholamines play no major role in regulating luteal steroidogenesis or corpus luteum regression in the pseudopregnant rabbit.

Contrasting effects of prolactin on luteal and follicular steroidogenesis.

To determine whether prolactin affects both luteal and follicular production of testosterone and oestradiol, pseudopregnant rats, either intact or hypophysectomized on day 8, were injected daily between days 8 and 9 with 1.5 i.u. human chorionic gonadotrophin (hCG), 250 micrograms prolactin or a combination of both. Control rats were given vehicle. On day 9, blood was obtained from the ovarian vein and corpora lutea and follicles were isolated and incubated in vitro for 2 h. Administration of hCG to intact rats increased ovarian secretion of testosterone and oestradiol dramatically, but did not affect progesterone secretion. Hypophysectomy on day 8 of pseudopregnancy was followed by a drop in ovarian steroid secretion. Prolactin treatment of hypophysectomized rats markedly enhanced progesterone production but had no stimulatory effect on either testosterone or oestradiol. In contrast, hCG dramatically enhanced ovarian secretion of both testosterone and oestradiol without affecting progesterone secretion. Prolactin administered together with hCG antagonized the stimulation of both testosterone and oestradiol secretion by hCG, yet increased progesterone production. When the specific effects of hCG and prolactin administration on follicles and corpora lutea were studied separately, it was found that hCG treatment in vivo greatly stimulated testosterone and oestradiol production by both tissues in vitro. Since hCG only marginally affected aromatase activity in the follicle, had no effect on aromatase activity in luteal cells and did not increase progesterone synthesis, it appears that hCG acts to increase the formation of androgen substrate for oestradiol biosynthesis. Prolactin, administered with or without hCG, inhibited both basal and hCG-stimulated testosterone and oestradiol synthesis by the follicle. In sharp contrast to its inhibitory effect on follicular production of steroids, prolactin appears to be essential for LH stimulation of testosterone and oestradiol by the corpus luteum. In the absence of prolactin, luteal cells gradually ceased to respond to LH and decreased their output of testosterone and oestradiol. Prolactin administration to hypophysectomized rats did not affect luteal cell production of either steroid. However, corpora lutea of rats treated with prolactin responded to the hCG challenge with an increase in testosterone and oestradiol synthesis. In summary, results of this investigation demonstrate that prolactin affects follicular and luteal production of testosterone and oestradiol in opposite ways.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of high-density lipoprotein on estradiol stimulation of luteal steroidogenesis.

The aim of this investigation was to determine whether luteal cells utilize cholesterol derived from high-density lipoprotein (HDL) for steroidogenesis and whether estrogen enhances luteal utilization of exogenous sterol. Incubation of Day 15 corpora lutea (CL) with different doses of human HDL resulted in a dose-dependent increase in progesterone production. HDL in vitro enhanced the overall steroidogenic capacity. However, the percentage of increases in 17 alpha-hydroxyprogesterone, testosterone and estradiol were significantly less than that of progesterone. Day 12 hypophysectomized and hysterectomized pregnant rats were treated with either estradiol, testosterone or vehicle for 72 h. Serum pregnenolone and progesterone were markedly increased by the steroid treatment, yet in vitro production of progesterone by CL in all the groups was similar. However, in the presence of HDL in the media, only luteal tissues from steroid-treated rats increased their progesterone output. The reduced production of progesterone by luteal cells of vehicle-treated rats was not due to an accumulation of pregnenolone but to an overall reduction in exogenous sterol utilization. In summary, results of this investigation suggest 1) luteal cells of pregnant rats effectively utilize cholesterol from HDL for maximal steroidogenesis, and 2) estradiol may stimulate luteal steroidogenesis, at least in part, by affecting the incorporation or utilization of cholesterol from HDL into the cell.

Endocrine role of the decidual tissue: decidual luteotropin regulation of luteal adenylyl cyclase activity, luteinizing hormone receptors, and steroidogenesis.

We have recently reported that the decidual tissue of pseudopregnant and pregnant rats produces a luteotropic substance, decidual luteotropin, which can sustain luteal function in the absence of PRL. However, the luteotropic activity of the decidua depends on the presence of LH in the circulation. The objective of the present investigation was to determine whether decidual luteotropin sustains luteal function by maintaining the luteal cell content of LH receptors and LH-responsive adenylyl cyclase. Pseudopregnant adult rats had their uteri scratched on day 5 to induce decidual tissue formation. Both day 5 hysterectomized and intact pseudopregnant animals were used as controls. On day 7, rats were injected with 0.4 mg 2-bromo-ergocryptine (CB-154) to suppress PRL secretion. Within 48 h, CB-154 administration to either control group not possessing decidual tissue induced a dramatic decrease in progesterone production, LH receptor content, and LH-stimulated adenylyl cyclase in luteal cells. In contrast, suppression of PRL secretion in rats with decidualized uteri had no effect on progesterone secretion, luteal cell LH receptor content, or LH-stimulated adenylyl cyclase. Removal of the decidual tissue immediately after CB-154 treatment caused a significant decline in LH receptors, whereas administration of PRL to control rats without decidual tissue reversed the detrimental effect of CB-154 on both receptor content and LH-stimulated adenylyl cyclase. Examination of the time course (0, 24, and 48 h) of CB-154 action in rats lacking decidual tissue indicated that the decrease in serum progesterone preceded any detectable loss in luteal LH receptor content and LH-stimulated adenylyl cyclase activity. The results also suggest that the decline in LH-stimulated cyclase activity is largely due to the decrease in LH-binding sites and not to an alteration in the cyclase system. PRL withdrawal does not decrease but, rather, enhances epinephrine-stimulated cyclase activity. In summary, results of these investigations have revealed that decidual luteotropin can maintain the luteal content of LH receptors and LH-responsive cyclase and can sustain luteal cell production of progesterone in the absence of PRL; the synergism between decidual luteotropin (or PRL) and LH on luteal steroidogenesis involves more than the maintenance of LH receptors; and PRL withdrawal has opposite effects on LH- and catecholamine-responsive adenylyl cyclases, decreasing the former and stimulating the latter.

Differential action and secretion of rat placental lactogens.

The abilities of the two different mol wt forms of rat placental lactogen (rPL) to maintain luteal steroidogenesis in pregnant rats in the absence of PRL were determined. The two forms of rPL present in day 12 pregnant rat serum were separated by gel chromatography, lyophilized, and reconstituted in a volume of saline equal to the original volume of serum. Rats were injected with 0.4 mg ergocryptine (ECO) on day 6 of pregnancy to suppress PRL and were then treated with a preparation of the large mol wt (LMW) hormone, the small mol wt (SMW) hormone, or both molecules (2 ml/day). Control animals received saline. Twice daily injections of the amount of LMW hormone contained in 1 ml day 12 pregnant rat serum reversed the abortifacient effect of ECO. In contrast, administration of the SMW hormone did not maintain either pregnancy or progesterone levels. Administration of both mol wt forms of rPL was also capable of maintaining luteal function. Sera obtained from day 18 pregnant rats containing only the SMW hormone had no luteotropic activity when administered, yet treatment with sera of day 12 pregnant rats sustained progesterone synthesis and fetal survival after ECO treatment. rPL were measured in the peripheral circulation and in the uterine vein throughout pregnancy by radioreceptor assay (RRA) using particulate membranes from either rabbit mammary gland or rat ovaries. Two peaks of activity were observed in the peripheral circulation by the two RRAs: one between days 11-14 and another between days 17-21, with a decline in activity between days 14-16. In contrast, levels of rPL in the uterine vein remained elevated throughout pregnancy. Concentrations of the LMW placental luteotropin were 5-10 times higher by ovarian RRA (O-RRA) than by mammary gland RRA (MG-RRA) between days 11-13, but concentrations of the SMW placental lactogen were found by the two RRAs to be similar in the later stages of pregnancy. Gel filtration of day 12 pregnant rat serum revealed two peaks of PRL-like activity. The O-RRA detected 19 times more LMW placental luteotropin than did the MG-RRA in the first peak of activity, yet measured equivalent amounts of the SMW placental lactogen in the second peak. Similar to results found in the peripheral circulation, levels of LMW placental luetotropin in the uterine vein measured by MG-RRA were significantly lower than those determined by O-RRA until day 14 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the effect of gonadotropin-releasing hormone and its agonist on human luteal steroidogenesis in vitro

The possible direct effect of gonadotropin-releasing hormone (GnRH) and a potent GnRH agonist [( imBzl )-D- His6 -Pro9-NEt]-GnRH on basal and human chorionic gonadotropin (hCG)-stimulated progesterone, androstenedione, and estradiol production by cultured human luteal cells was examined. Luteal cells from the early or midluteal phase of the menstrual cycle responded to hCG stimulation with two to fivefold increases in steroid production in both short-term (4 hours) and long-term (up to 144 hours) culture in chemically defined medium without serum. After 48 hours in this system, levels of androstenedione and estradiol were very low, and progesterone was the predominant steroid produced. The addition of GnRH or a potent GnRH agonist to the medium had no effect on either basal or hCG-stimulated steroid secretion. When luteal cells were cultured longer (for up to 10 days) in the presence of serum, GnRH agonist caused no significant alteration of either basal or hCG-stimulated progesterone production. Collectively, these results support the conclusion that GnRH and its potent agonist do not act directly on human corpora luteal cells to modulate steroidogenesis.

Further studies on in vitro steroidogenesis by luteal cells from long-term hypophysectomized rats.

Immature pregnant mare's serum gonadotropin-treated rats were hypophysectomized on the day of ovulation (Day 1 of luteal function), and luteal steroidogenesis and human chorionic gonadotropin (hCG) and prolactin (Prl) binding sites were determined on Days 5, 10, 20 and 30 (H5- H30 ) compared with intact rats on Days 5 or 10 (C5 or C10). On H5, dispersed luteal cells secreted large amounts of progesterone (P), 20 alpha-dihydroprogesterone (20 alpha-DHP), 17 alpha-hydroxyprogesterone (17 alpha-OHP), and small amounts of testosterone (T) and estradiol-17 beta (E2), but between H10 and H30 , reduced levels of all steroids were produced except for 20 alpha-DHP. Addition of large amount of pregnenolone (P5) or P (100-1000 ng) to dispersed luteal cells increased production of P and 20 alpha-DHP in C5 and H5 rats. The higher serum levels and basal in vitro production of 20 alpha-DHP from H5 to H30 indicates that 20 alpha-oxidoreductase persists in the corpora lutea (CL) at high levels and that 3 beta-ol-dehydrogenase is also present but with P rapidly shunted into its principal metabolite. From H5 to H30 , addition of 10 ng P to luteal cells increased the production of 17 alpha-OHP and addition of 10 ng androstenedione (A) or T enhanced production of T and E2, indicating that 17 alpha-oxidoreductase, 17 beta-hydroxysteroid dehydrogenase and aromatase also persist in the CL. In vitro addition of 10 ng LH significantly stimulated production of P from luteal cells on C5 and H5, whereas on C10 and H10, 100 ng LH was required and on H20 and H30 , 1 microgram LH produced a minimal increase in P.(ABSTRACT TRUNCATED AT 250 WORDS)