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The present study was undertaken to assess the effects of 5-hydroxyeicosatetraenoic acid (5-HETE) on steroidogenesis and prostaglandin (PG) production in cultured luteal cells derived from human corpora lutea in the mid-luteal phase. The luteal cells were cultured with 5-HETE at 10, 100, 500, or 1,000 ng/ml in the absence or presence of hCG at 100 ng/ml for 10 days. The addition of 5-HETE to the culture media did not affect growth curves of cultured luteal cells. 5-HETE significantly inhibited progesterone (P) production by cultured luteal cells in a dose-related fashion on day 2. P production stimulated by exposure to hCG was also reduced significantly in response to 5-HETE. The addition of 5-HETE did not affect estradiol production by cultured luteal cells. The production of 6-keto-PGF1 alpha by cultured luteal cells in the presence of 5-HETE was slightly but not significantly less than that observed in the absence of 5-HETE. However, 5-HETE affects neither PGF2 alpha nor PGE2 production by cultured luteal cells throughout the culture period. The present study demonstrates that 5-HETE inhibits P production in cultured luteal cells by a mechanism(s) other than through PG production. These data suggest the involvement of a lipoxygenase pathway in the synthesis of P and 6-keto-PGF1 alpha of luteal cells.

Kinetics of protein synthesis in the human granulosa lutein cell during pregnancy were investigated using the electron microscopic autoradiography of 3H-leucine. The corpus luteum of early pregnancy was chased at 10 and 30 min, 1, 3, 6, 12, 24 and 48 h after pulse labelling (10 min) with 100 microCi of 3H-leucine. Although the whole silver grains were very few at 10 min postpulse, silver grains were localized mainly over the rough endoplasmic reticulum (r-ER, 34.3%) and there were no silver grains over the smooth endoplasmic reticulum (s-ER). At 30 min postpulse, the ratio of the grain number to r-ER is decreased (18.7%) and silver grains begin to appear over the s-ER (5.4%). The total number of silver grains over the cell increases with the lapse of time, the ratio of the grain number over the r-ER slightly increases and then decreases afterwards, while that over the s-ER continues to increase gradually. From these facts, it is felt that some protein synthesized on the r-ER might be transported to the s-ER in the human granulosa lutein cell during pregnancy showing steroidogenic activity.

Luteal cells from the corpora lutea of rats on day 15 of pregnancy were suspended in DMEM-F12 and progesterone accumulation was measured by radioimmunoassay. Progesterone accumulation increased steadily over eight hours and then gradually decreased. Progesterone accumulation was seen to increase with the addition of BSA and 25-hydroxycholesterol. At doses of 12 ng/ml and 600 ng/ml hCG, progesterone accumulation increased, but was seen to decrease at a dose of 30 micrograms/ml hCG. However, the addition of cyclohexamide alone had no effect on progesterone accumulation, but the simultaneous addition of cyclohexamide and hCG blocked the effects of hCG in a dose related manner. These data indicate that progesterone production by rat luteal cells depends on cholesterol availability. The results reveal that hCG stimulates progesterone production by a process requiring new protein synthesis.

The effects of human chorionic gonadotropin (HCG), dibutyryl cyclic adenosin-3',5'-monophosphate (d-cAMP) on cyclic AMP (cAMP) accumulation and estradiol (E2), progesterone (P) productions in human corpora lutea were investigated by the method of short term incubation of human corpora lutea of menstrual cycle and pregnancy (6th-12th week). Thus the luteotropic effects of administered HCG on human corpora lutea were studied. (1) Elevated E2, P and cAMP concentrations in corpora lutea tissues of the menstrual cycle and comparable their levels of pregnancy were observed in the incubation with HCG. (2) In the incubation with d-cAMP, the concentrations of E2, P in luteal tissue were significantly elevated in menstrual cycle and pregnancy. (3) High doses of HCG were administered to patients during luteal phase. In the incubation of their luteal tissue, E2, P and cAMP concentrations were elevated by exogenous d-cAMP, but not HCG. These results indicate that cAMP accumulation and steroidogenesis in the corpora lutea were not activated by exogenous HCG. Furthermore, the behaviour of the intra-cellular cAMP in the corpora lutea was not different between menstrual cycle and pregnancy.

The present study was undertaken to assess the ability of cultured luteal cells from human corpora lutea (CL) in the mid luteal phase and the early pregnancy to secrete steroids and prostaglandins (PGs). Luteal cells responded to hCG with a significant increase (2- to 4-fold) in progesterone (P) production. The addition of hCG to the culture media did not stimulate estradiol (E2) production. In contrast, both P and E2 secretion by luteal cells in early pregnancy were significantly lower than those found in the mid luteal phase. Exposure to hCG did not affect P production by luteal cells in early pregnancy. Arachidonic acid (AA) significantly stimulated PGE2 synthesis by luteal cells in the mid luteal phase in a dose-dependent manner. Both basal PGE2 production and the responsiveness to AA were maintained for the duration of the culture. However, hCG did not affect AA-stimulated PGE2 production. Both PGF2 alpha and 6-keto-PGF1 alpha production abruptly declined as the culture proceeded. PG synthesis by cultured luteal cells in early pregnancy was significantly lower than in the mid luteal phase. The ultrastructural characteristics of luteal cells in early pregnancy, which contained lipid droplets, granular and agranular endoplasmic reticulum and large spherical mitochondria, were maintained after 10 days in culture. The present results demonstrate that P and PGE2 production by cultured luteal cells predominate during the mid luteal phase. These data suggest that PGE2, but not PGF2 alpha, may be involved in the regulation of CL function in the menstrual cycle.

To study the luteotropic effect of human chorionic gonadotropin (hCG) on human corpus luteum in early gestation, serum pregnenolone, 17 alpha OH-pregnenolone, dehydroepiandrosterone, progesterone, 17 alpha OH-progesterone, androstenedione, 20 alpha-dihydroprogesterone and estradiol levels in both ovarian and peripheral venous blood measured by RIA before and after administration of hCG. Seven patients selected for this experiment were 6 to 16 weeks of gestation complicated with fibromyoma. On hysterectomy, 1,000 or 2,000 I.U. of hCG was directly injected into the ovary with corpus luteum to the patients of 6, 12 and 16 weeks of gestation, or 100,000 I.U. of hCG was infused intravenously to the patients of 9 and 14 weeks of gestation. Before 12 weeks of gestation, all free steroid levels except for conjugated steroids in ovarian vein increased promptly 1.8 to 8.9 fold as compared with control levels and stayed high levels for 25 to 60 minutes after administration of hCG. However no marked alteration of these steroids was observed in peripheral vein. In cases studied after 14 weeks of gestation, no marked change of steroid levels in ovarian vein was noticed, while a moderate increase of hCG was observed. These results demonstrated that hCG stimulates the steroidogenesis of corpus luteum in early pregnancy. It was demonstrated that extremely high doses of hCG was required to stimulate steroidogenesis corpus in luteum gravidarum of early gestation.

The present study was designed to investigate whether or not prostaglandins(PGs) were produced by human luteal cells(HLC) and their effects on the luteal cells by monolayer culture. The following results were obtained. Cultured HLC secreted progesterone(P), prostaglandin F(PGF) and prostaglandin E(PGE) into a medium at concentrations of 276.6 +/- 38.6, 1.95 +/- 0.36, 2.44 +/- 0.45 ng/ml/1 X 10(5) cells/day (mean +/- SE), respectively. Cultured HLC was able to convert 14C-arachidonic acid to 14C-PGF2 alpha, 14C-PGE2. These two results indicated that HLC had the ability to produce PGF and PGE. Cultures were carried out in the presence of indomethacin (Ind), PGF2 alpha and PGE2 alone as well as in a combination. P production by HLC was reduced in the presence of Ind. P production in the presence of Ind+PGE2 was more than that in the presence of Ind alone. There was no significant difference in P production between the presence of Ind and Ind+PGF2 alpha. It was concluded that HLC had the ability to produce PGs and that PGE2 significantly stimulated P production in as low concentrations as HLC could produce physiologically while PGF2 alpha did not.

In order to elucidate the mechanism of anovulation and luteal insufficiency that occur in hyperprolactinemia, the direct effect of prolactin (PRL) on luteal cells in vitro was investigated using human luteal cells in a monolayer cell culture. 1) The direct effect of PRL (that was kindly provided by the National Institute of Arthritis, Metabolism and Digestive Diseases) as seen in the production of P was such that when PRL concentrations were 1 through 100ng/ml the luteal stimulatory action was demonstrated, but in concentrations above 100ng/ml the stimulatory action was attenuated, and at 1 microgram/ml inhibitory effects were observed. 2) There was no significant difference between the PRL-added group and non-added group on the production of E2 and 17OHP. 3) Production of P in the HCG 100ng group increased to about 4 times that of the non-added group. Production of P in the group with simultaneous addition of PRL 1 microgram-HCG 100ng also showed a similar degree of increase. From the above, it is concluded that PRL alone exerts a direct effect on luteal cells that is luteotropic and luteolytic, depending on the different concentrations of PRL. These facts suggested that the direct inhibitory effect of PRL on the ovary was one of the causes of anovulation and luteal insufficiency during hyperprolactinemia in vivo.

On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CLs) were dissected and dissociated into single cell suspensions by enzyme treatments. To assess 3 beta-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. The number of cells positive for 3 beta-HSD in CL were 148.0 +/- 13.7, 130.1 +/- 25.4, 134.0 +/- 23.5 and 116.8 +/- 13.5 X 10(3) cells on days 5, 10, 15 and 20 of pregnancy, respectively. The 3 beta-HSD positive cells increased in size from 18.5 +/- 0.28 microns on day 5 to 35.7 +/- 0.50 microns on day 20 of pregnancy. The suspended luteal cells were incubated in serum-free DME-F12 for 20 hours with or without human chorionic gonadotropin (hCG, 100 ng/ml) or dibutyryl cyclic AMP (dbcAMP, 10mM) to test their functionality, and progesterone accumulation was determined by radioimmunoassay. Progesterone secretion from the 3 beta-HSD positive cells was maintained at the same levels until day 15 and decreased significantly on day 20 of pregnancy. However the response to hCG stimulation of the 3 beta-HSD positive cells decreased significantly on day 20, the 3 beta-HSD positive cells maintained the same responsiveness to dbcAMP stimulation on progesterone secretion throughout pregnancy. These data suggest that the steroidogenic rat luteal cells may be regulated by morphologically and functionally different mechanisms, and that progesterone may be secreted by at least two different pathways.

The purpose of the experiments covered by this report was to see whether or not cell cultures reflect conditions in vivo. We have used mainly the primary cell culture technique of human corpora lutea in TC199 containing 20% fetal calf serum. Steroid concentrations were determined by RIA. As compared with relative low production of estradiol and 17 alpha-hydroxyprogesterone, progesterone (P) production showed a peak of 27.5 +/- 8.7 ng/10(5) cells/ml/2 days on about the sixth day. When the cells were cultured in the presence of hCG, the P concentration (83.9 +/- 13.4) significantly increased and an inhibition in the growth rate of luteal cells was observed. When a drop of suspension of isolated cells was examined under a phase contrast microscope, the cells appeared spherical. In Sudan Black fat staining preparations, numerous lipid granules could be seen in the cytoplasm. Also electromicroscopic studies induced that the cells contained numerous mitochondria and well developed, smooth endoplasmic reticula. The cells exhibited strongly positive 3 beta-hydroxysteroid dehydrogenase activity and the cells on the tenth day culture showed the 46, XX karyotype. As mentioned above, we could show that this in vitro system, namely; monolayer cell culture reflected the conditions in vivo, mainly through P production by luteal cells and the effect of hCG.

It is known that Prostaglandins (PGs) have a specific effect on the corpus luteum function. In this paper we have studied the physical and chemical characteristics of PGE2 receptor in human corpora lutea to explain the mechanism on corpus luteum function. The presence of PGs binding sites in corpora lutea has been reported, but hardly any papers are available on the presence of PGE2 binding sites in human corpora lutea. The present study was undertaken to determine, using the proportion graph method, whether PGE2 receptors are present in human corpora lutea or not. The number of binding sites and association constant of PGE2 receptor to human corpora lutea of the luteal phase of the cycle and gestation were examined. There is evidence (1) that PGE2 receptors were present in human corpora lutea, and the binding sites have one specific and one non-specific binding system. (2) That the number of binding sites in early, mid and late luteal phase corpora lutea were respectively 105-157, 190-196, 162-190 f moles/mg protein. Association constants were 2.0-12.0 x 10(11) M-1. (3) That the number of binding sites of 6th week gestation corpora lutea were 98-100 f moles/mg protein, and those of 12th week gestation were 40-85 f moles/mg protein. Association constants of the gestation corpora lutea were 17.0-20.0 x 10(11) M-1.

The purpose of the present study was to define the effect of low density lipoprotein (LDL) on progesterone (P) synthesis and on intracellular cholesterol content using monolayer cultured human luteal cells. When cultured in TC199 containing 10% lipoprotein poor serum (LPPS), P production decreased as culture time passed. In the presence of hCG, it also decreased, but slowly. In the presence of LDL, P production was kept at a higher level. Intracellular cholesterol content in luteal cells was measured by gas chromatography. The cholesterol content of luteal cells in a 4 day LPPS culture was higher than that in a 10 day LPPS culture. The cholesterol content in a 10 day LPPS culture with LDL was higher than that in a 10 day LPPS culture. The cholesterol content in a 10 day LPPS culture with hCG had a tendency to be lower than that in a 10 day LPPS culture, but there was no statistical difference (p less than 0.1). It was concluded that LDL was utilized in supplying intracellular cholesterol to luteal cells as a substrate for P synthesis. And in a state of LDL depletion in the medium, the luteal cells produced a small amount of P utilizing intracellular cholesterol. It was estimated from the results of the present study that hCG enhanced utilization of intracellular cholesterol.

The present investigation was designed to study the effect of synthetic ACTH on the synthesis of progesterone, 20 alpha-OH-progesterone and adenosine-3', 5'-monophosphate (cAMP) in isolated luteal cells. Experiments were conducted on rats from three groups. Group I: PMS primed immature rats. Group II: PMS plus hCG primed immature rats. Group III: pregnant rats. Luteal cells were isolated by the Kumai Method (7) using a sucrose density gradient after digestion with trypsin and collagenase. Luteal cells were divided into three fractions; S-1, S-2 and S-3. Following an 18 hr-incubation of S-1 cells of day 7 after PMS injection, a significant increase in progesterone release by ACTH lasted from 6 to 18 hr, whereas 20 alpha-OH-progesterone decreased significantly by ACTH from 8 to 18 hr. There were no differences in progesterone and 20 alpha-OH-progesterone releases from S-2 cells of day 7 between media with and without ACTH. Progesterone release without ACTH reached the peak on day 7 in Group I and on day 6 in Group II. ACTH stimulated progesterone and inhibited 20 alpha-OH-progesterone releases in Groups I and II. The maximum effect of ACTH in both Groups was noted on days 6 and 7. There were no differences in the Effective Dose (ED50) and the Inhibitory Dose (ID50) of ACTH between Group I and Group II. In Group III, releases of progesterone and 20 alpha-OH-progesterone from S-1 cells to media with or without ACTH decreased remarkably after 11 days of gestation. On days, 3, 5 and 8 of gestation, a significant increase in progesterone release from S-1 cells was noted by the ACTH addition. 20 alpha-OH-progesterone release from S-1 cells to media with or without ACTH decreased as the pregnancy advanced. Total cAMP of intra- and extra-cellular S-1 cells (Group II) on day 5 after PMS injection was assayed. ACTH induced an increase in intracellular cAMP. The present results indicate the following: (1) In Group I and Group II, progesterone release increased with the age of the luteal cells and ACTH dose. In Group III, ACTH stimulated progesterone release in a dose-dependent manner in early gestation. (2) 20 alpha-OH-progesterone release decreased by ACTH dose in Group I and Group II, and decreased in Group III as the gestation advanced. (3) Intracellular cAMP increased by ACTH in Group II.

Extensive studies have recently been conducted on the expression of hormone actions, especially on its mechanisms. Sutherland, et al. identified the function of C-AMP as an intracellular messenger of peptide hormones. Recent studies by several investigators including ours have concluded that the role of C-GMP and calcium are as important as that of C-AMP. In this report we present out findings on the role of Ca ions in the acceleration of steroid production in isolated porcine cells of the corpora lutea. (1) The incubation time needed to compare the production of progesterone and that of C-AMP is at least 120 mins. (2) Production rates of both C-AMP and progesterone in the presence of HCG were maximum at 10(-7) g/ml HCG. Inclusion of Ca ions in the medium increases this production rate. (3) When Ca ions are included in the medium, the accelerated rate of progesterone production takes on its highest value at 1.2mM Ca. (4) Verapamil and cycloheximide inhibit the production of progesterone. (5) The binding capacity of 125I-HCG combined with corpora lutea cells is independent of the Ca concentration in the medium. (6) Ca uptake into corpora lutea cells increases according to the concentration of HCG until it reaches a plateau at 10(-7) g/ml HCG. This acceleration by HCG is affected by Ca and is remarkably inhibited by verapamil. These results indicate the important role of Ca ions in the acceleration of progesterone production compared to that of C-AMP.

Although considerable information is available concerning steroidogenesis in the human fetus, the function of the different steroids formed during pregnancy and the factors regulating this delicate hormones balance are poorly understood. During human pregnancy, the placenta synthesizes large quantities of progesterone, estradiol, estrone and estriol and secretes these hormones into both the maternal and fetal circulations; progesterone from maternal lipoprotein-cholesterol, estradiol and estrone from maternal and fetal dehydroepiandrosterone sulfate (DHAS), and estriol largely from fetal 16 alpha-OH-DHAS. It has been demonstrated that preimplantation blastocysts of several animal species have the capacity to accumulate steroids to pregnenolone to progesterone, and to interconvert estrone and estradiol. Estetrol (E4), 15 alpha-hydroxy derivative of estriol is an interesting compound, since its formation is relatively unique to fetal liver function. Of special interest is that placental sulfatase deficiencies result in an extension of the gestation, and Cesarean section has to be done. This raises the question of the role of estrogens in determining the onset of labor, much as in the case of anencephaly. In general, progesterone may decline prior to an abortion, but there has not been a direct application to clinical practice. Estrogen levels during pregnancy are influenced by factors other than fetal well-being and include fetal weight, placental enzyme function, fetal adrenal function, maternal intestinal flora, maternal renal excretion and maternal liver function. Although not yet extensively utilized, such a dynamic test as the infusion of DHAS may yield useful information within a short period in otherwise complicated cases related to fetal and placental function.

To evaluate the direct inhibitory action of luteinizing hormone-releasing hormone (LH-RH) on the steroidogenesis of the human ovary, the primary cultured human corpus luteum cells were investigated. The following were the effects of the addition of LH-RH: Estradiol (E2) and progesterone were produced and secreted in the cultured corpus luteum cells. In the cytoplasm of the cultured corpus luteum cells, E2 and P-antibody complexes were observed as fine granules by the immunohistochemical staining method. The progesterone production of these cells was not inhibited in the cells cultured with LH-RH 10(-8) Mol alone. The progesterone production of the cells was stimulated in the cells, cultured with gonadotropins (LH, HCG and HMG). The gonadotropin stimulated progesterone production was inhibited by LH-RH administration in the cells. In the short term incubation of the human corpus luteum cell suspension, the cyclic adenosine monophosphate (c-AMP) accumulation of the cells incubated with LH-RH alone did not change, but the gonadotropin-stimulated c-AMP accumulation of the corpus luteum cells was significantly inhibited by LH-RH. Concerning these results, it is concluded that LH-RH inhibits the gonadotropin stimulated progesterone production directly in vitro. It is suggested that the mechanisms of these inhibitory actions of the LH-RH are related to the gonadotropin receptor-adenyl cyclase systems, c-AMP metabolizing enzyme and/or progesterone metabolizing enzyme.

To investigate the interrelation between steroidogenic function of granulosa and theca lutein cells of the human corpus luteum, twelve corpora lutea of menstruation and pregnancy were divided into inner and outer layers, each of which was histologically confirmed to be composed of pure granulosa lutein cells (GL) or GL plus theca lutein cells (TL), respectively. Slices of each layer were incubated with radioactive pregnenolone, 17-hydroxyprogesterone and androstenedione respectively, and the metabolites were analysed by reverse dilution technique with recrystallization to constant specific activity. Both slices converted pregnenolone-14C to progesterone, 17-hydroxyprogesterone, androstenedione, estrone and estradiol-17 beta, with progesterone being the major steroid formed. Formation of progesterone was significantly pronounced in the case of slices from the inner layer, while conversion into the other steroids was significantly higher in the case of slices from the outer layer. Formation of androstenedione from 17-hydroxyprogesterone-14C was also greater with the outer slices than the inner slices. However, conversion of androstenedione-14C to radioactive estrone and estradiol-17 beta was more efficient with the inner slices than the outer slices. These results suggest that the activity of aromatizing enzymes is higher in granulosa lutein cells, while that of 17 alpha-hydroxylase and C17-C20 lyase is higher in theca lutein cells. Therefore, cooperation of the two cell types would be necessary for the fulfillment of the function of estrogen biosynthesis by human corpus luteum.

Isolation of progesterone secreting cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotropin (PMS) was conducted by a simple procedure which combined the collagenase digestion with a density gradient method. After digestion of the ovarian tissue slices by the enzyme, the residue was gently pressed and placed on a sucrose density gradient. Three bands appearing in the tube after centrifugation were designated as S-1, S-2 and S-3 from the top to the bottom, respectively. The S-1 cells from the ovaries at 6 days after PMS secreted the greatest amount progesterone, i.e. approximately 430 ng per 10(5) cells during the 18th incubation. Progesterone secretion from the S-2 cells was less than 48% of that from the S-1 cells. A physiological interrelation between the S-1 and S-2 cells remains unexplained by the present experiment. The luteal cells were yellow, spheroidal and 15 to 40 mus in diameter. Many vesicle-like particles were found on the cell surfaces. Progesterone secretion from the prepared cells was stimulated significantly by hCG in vitro. This result indicates that progesterone secreting cells isolated by the collagenase-sucrose density gradient preserve their native function as luteal cells. The procedure for preparation of luteal cells in the present report may provide a suitable model for in vitro studies on the corpus luteum.

FSH secretion from the pituitary is now generally believed to be controlled only by LH-RH. However, since 1971 I have been publishing lines of evidence suggesting that FSH is controlled by both LH-RH and FSH-RH distinct from LH-RH. Recent accumulated reports suggest that lots of data cannot be explained by the one-RH theory. The one-RH theory means that both LH and FSH are regulated by a sole releasing factor, LH-RH. In 1985 we succeeded in purification of porcine and bovine follicular inhibin for the first time in the world. Its molecular weight was 32,000, which consisted of alpha-subunit (mw 20,000) and beta-subunit (mw 13,000). The site of production of inhibin was confirmed to be granulosa cell by immuno-histochemical technique and to be corpus luteum by RIA. Secretion of inhibin was stimulated by FSH, insulin, platelet extract, dibutyryl c-AMP, cholera toxin, forskolin, and inhibited by cortisol. Testosterone augmented FSH action. In the pituitary cells, inhibin inhibited synthesis of FSH, so cycloheximide could mimicked inhibin action in vitro. Inhibin did not inhibit LH release from the pituitary, but LH release stimulated by LH-RH was also inhibited by inhibin in vitro. Using anti-porcine inhibin antibody and anti bovine inhibin antibody prepared by us, we could measure blood inhibin levels during rat, pig, cow and goat sexual cycles, human menstrual cycle, human pregnancy, and clomiphene-HMG stimulated menstrual cycle. We found maximum blood inhibin levels in the luteal phase and during pregnancy. These high levels of inhibin were demonstrated to be derived from the corpus luteum, pregnant corpus luteum and placenta.(ABSTRACT TRUNCATED AT 250 WORDS)