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A novel method for purifying dispersed porcine theca cells, with less than 3% granulosa cell contamination, was developed by the repeated use of mechanical and enzymatic procedures. The steroidogenic criteria used for the identification and purity evaluation of both theca and granulosa cells were also improved. Purified theca and granulosa cells from medium-sized follicles displayed steroidogenic differences when they were cultured in the presence of 10% fetal bovine serum: (1) the theca cells synthesized oestradiol (239.1 +/- 35.1 pg ml-1 per 2.5 x 10(5) cells in 40 h), but the granulosa cells did not synthesize it unless aromatizable androgens were added; (2) theca cells synthesized androstenedione (73.2 +/- 14.4 ng ml-1 per 2.5 x 10(5) cells in 40 h), but granulosa cells did not; (3) FSH did not affect progesterone production in theca cells; (4) the theca cells secreted androstenedione for up to 48 h; and (5) FSH significantly stimulated progesterone production in granulosa cells during a culture for 40 h (P < 0.05), but not during culture for 12 h. The lack of response to FSH was used as a reliable, functional indicator of the purity of porcine theca cells. However, this criterion proved not to be useful for cells cultured for 12 h; porcine FSH had no effect on the progesterone production of theca cells co-cultured for this time with as many as 20% granulosa cells. However, after co-culturing for 40 h, this criterion resulted in the detection of only 3% granulosa cell contamination.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of GnSI/AF was measured in pig follicular fluid (pFF) from 58 individual follicles of various sizes, by bioassay using rat pituitary cells, to investigate the relationship between gonadotrophin surge inhibiting/attenuating factor (GnSI/AF) activity and follicular development. In addition, the correlation between GnSI/AF and inhibin activities and the content of sex steroids (oestradiol, progesterone and testosterone) of follicles was examined. The activity of GnSI/AF in pFF varied significantly (0.155-1.69 U microliter-1) with size of the follicle. The activities (mean +/- SEM) were intermediate and constant in follicles with diameters from 3 to 5 mm (0.583 +/- 0.080 U microliter-1, n = 24), were higher and reached the highest value in follicles with diameters between 6 and 8 mm (0.863 +/- 0.068 U microliter-1, n = 21), and were lower, reaching the lowest value in follicles with diameters of 9 and 10 mm (0.401 +/- 0.089 U microliter-1, n = 13). In contrast, inhibin activity was almost constant during the development of follicles, although individual values varied from 0.9 to 2.5 U microliters-1. For follicles with diameters of 4-8 mm, inhibin activity was 1.754 +/- 0.042 U microliters-1 (n = 39); activity was higher in the smallest follicles with diameters of 3 mm (2.063 +/- 0.015 U microliters-1, n = 6) and was lower in follicles with diameter of 9 mm, reaching the lowest value in follicles with diameter of 10 mm (1.176 +/- 0.068 U microliters-1, n = 7); inhibin activity was not significantly correlated with GnSI/AF activity.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate endocrine pathophysiology of luteal phase deficiency (LPD), GnRH/TRH stimulation tests were performed in the early follicular (EFP) and midluteal phases (MLP) of the menstrual cycle in 52 infertile women with a history of short luteal phase, in whom pituitary responsiveness to GnRH/TRH and steroidogenic competency of the corpus luteum were analyzed. Twelve women with either elevated basal-LH or exaggerated PRL response to GnRH/TRH in EFP were eliminated, and the remaining 40 women were studied. Basal-FSH in EFP inversely correlated with steroidogenic parameters in MLP, indicating that compromised folliculogenesis causes LPD. In a fraction of LPD women, decreased basal-LH in MLP was associated with decreased basal-progesterone (p), in spite of normal steroidogenic potential of the corpus luteum, suggesting that aberrant LH secretion is another progenitor of LPD. The other group of LPD women showed shortening of high phase period and/or extravagant discrepancy in endometrial dating without apparent abnormal endocrine parameters, suggesting that unknown factors are involved in establishment of LPD. From the diagnostic point of view, they were discriminated into three groups, normal, incomplete LPD and complete LPD groups, with a modified classification of LPD; 1) shortening of high phase period < 11 days, 2) delay in histological to chronological dating of the endometrium > 2 days, and 3) decreased max-P in MLP < 10 ng/ml. Normal (n = 14) and complete LPD (n = 7) groups consisted of women having all the criteria of classification within and out of the cut-off values, respectively. The remainders were enrolled into incomplete LPD group (n = 19). Complete LPD group mainly consisted of women having compromised folliculogenesis as a cause of LPD. In contrast, incomplete LPD group appeared a mixture of heterogeneous populations as to the genesis of LPD. GnRH/TSH stimulation test, especially when performed in MLP, would unveil endocrine pathophysiology of LPD and provide an accurate standard for diagnosis of LPD.

To examine whether or not dehydroepiandrosterone sulfate (DHAS) is a substrate for steroidogenesis in the corpus luteum, we studied 17 women in the luteal phase, the follicular phase, and after castration. Following suppression of adrenal function with dexamethasone, DHAS was administered intravenously and the serum levels of DHAS, dehydroepiandrosterone (DHA), androstenedione (ADS), testosterone (T), 17 beta-estradiol (E2) and progesterone (P) were measured serially for 24 h. An obvious increase in the serum levels of all steroids except for E2 and P was observed in each subject for at least 8 h after DHAS administration. To evaluate the effect of DHAS on the serum levels of the steroid hormones, the integrated response area (IRA) was calculated for each hormone in all the subjects. The IRA values for ADS, T and E2 (at 2 and 4 h) in the luteal phase group were significantly higher than in the other DHAS treated groups, and the IRA values for DHA and P tended to be higher than in the other groups. These results suggest that the corpus luteum utilizes serum DHAS as a substrate for steroidogenesis.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.

The expression of aminopeptidase-N and neutral endopeptidase in human ovarian tissue was examined using specific monoclonal antibodies for each of these peptidases and histochemical staining for enzyme activity. Aminopeptidase-N is a membrane-bound metalloprotease catalyzing the removal of N-terminal amino acids from peptides and was detected by immunofluorescence staining on theca interna cells in secondary follicles and on luteinized thecal cells in preovulatory follicles and corpora lutea. However, aminopeptidase-N was not detected on granulosa cells. Peptidase activity was also detected by histochemical staining on theca interna cells and luteinized thecal cells. Luteinized granulosa cells showed peptidase activity, despite the lack of aminopeptidase-N. Neutral endopeptidase was not detected in ovarian granulosa and thecal cells. These observations indicate that aminopeptidase-N can be a useful surface marker for thecal cells.

Objective: To re-evaluate the concept of polycystic ovarian syndrome (PCOS) in view of androgenic function., Design: Patients were studied endocrinologically and ultrasonographically., Setting: This study was performed as a clinical investigation., Patients, Participants: Sixty-nine euprolactinemic anovulatory patients with luteinizing hormone (LH) hypersecretion and 18 normal volunteers were selected., Interventions: One hundred micrograms of LH-releasing hormone were administered., Main Outcome Measure(s): It was possible to divide PCOS patients into three types., Results: Patients with neither hirsutism nor elevation of serum androstenedione (delta 4) and/or testosterone (T) were designated as type I PCOS (n = 20). Patients without hirsutism but with elevated delta 4 and/or T were referred to as type II PCOS (n = 33). Patients with both hirsutism and elevation of delta 4 and/or T were defined as type III PCOS (n = 16). Concentrations of delta 4 appeared gradedly increasing in types I, II, and III in that order, whereas T concentrations were significantly higher in types II and III than in control. Ultrasonographically, cystic ovaries were found in 88%, 84%, and 100% of types I, II, and III patients, respectively., Conclusions: It is postulated that each type may represent a subset of whole spectrum of PCOS from Stein-Leventhal syndrome to simple anovulation with LH hypersecretion.

Endothelin (ET), a novel vasoconstrictor peptide containing three isopeptides [ET-1, ET-2, and ET-3 (ETs)], has various biological effects including vasoconstriction, mitogenesis, and steroidogenesis. We examined the ET-1-like immunoreactivity level in porcine follicular fluid and culture medium of porcine granulosa cells by RIA. The ET level in the follicular fluid was 9.4-14.2 pg/ml. These levels were within 0.42 to 0.62-fold of the porcine plasma level (22.7 +/- 3.1 pg/ml) (mean +/- SE). ET was detected in the culture medium of granulosa cells with and without LH treatment at the concentration of 56 +/- 9.3 and 4.9 +/- 1.2 pg/10(6) cells.h, respectively. We also examined whether ETs affect the luteinization of granulosa cells. ETs inhibited the LH-stimulated progesterone and cAMP accumulation in cultured porcine granulosa cells in a dose-dependent manner with an EC50 of 5 x 10(-11) M. ET-1, ET-2, and ET-3 (5 x 10(-8)M inhibited progesterone accumulation by 62.3 +/- 1.8, 59.8 +/- 4.0, and 63.3 +/- 5.7% in 6-day cultures, respectively, and significant inhibition was observed within 24 h of culture. ET-1, ET-2, and ET-3 (5 x 10(-8) M) inhibited the LH-stimulated cAMP accumulation in granulosa cells by 54.8 +/- 2.3, 55.4 +/- 7.1, and 55.5 +/- 6.2%, respectively, whereas they did not affect basal cAMP levels. As well as progesterone accumulation, ETs partially inhibited LH-stimulated morphological transformation of granulosa cells. In this study, we demonstrated that ET exists in follicular fluid and in the culture medium of granulosa cells, and that ET inhibited LH-induced progesterone accumulation, morphological transformation, and cAMP accumulation in cultured porcine granulosa cells. These findings suggest that ET acts as a modulator of steroid metabolism in preovulatory follicles.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.

We have examined the effect of LH on the regulation of the progesterone receptor (PR) in cultured porcine granulosa cells. In this study we used the RNase protection assay to evaluate the PR mRNA levels with a porcine cDNA clone isolated by the polymerase chain reaction (PCR) method. This clone was regarded as part of the porcine PR cDNA because of its 98.3% and 95.7% homology to the hormone-binding domain of human PR cDNA in amino acid and nucleotide sequences, respectively. Treatment with LH (500 ng/ml) increased porcine PR mRNA to a maximum level of 8.6 +/- 1.1-fold (mean +/- SE) after 3-h exposure. This induction was mimicked by (Bu)2cAMP as well as by FSH and hCG, and the increased PR caused by LH and (Bu)2cAMP occurred in a dose-dependent manner. Basal and LH-induced PR mRNA levels were not affected by progesterone (100 ng/ml), estrogen (100 ng/ml), and RU 486 (10 ng/ml) at 3 h. The mechanism of the increased PR mRNA levels was studied in the presence of actinomycin-D and cycloheximide. While inhibition of RNA synthesis with actinomycin-D blocked LH-induced PR mRNA expression, inhibition of protein synthesis with cycloheximide increased basal and LH-induced PR mRNA levels. These results indicate that the expression of PR mRNA is positively regulated by LH, and this induction does not require ongoing protein synthesis. There may be a cycloheximide-sensitive mechanism that modulates PR mRNA stability. From our results we suspect that progesterone modulates ovarian function through LH-induced PR in granulosa cells.

We present a case of solitary secondary gynecologic lymphoma, in which MR imaging contributed to the diagnosis. The finding was a region of uterine enlargement consisting of ill-defined nodules of variable signal intensity diffusely mixed with the myometrium.

Effects of interleukin-1 (IL-1) on FSH-induced differentiation of immature porcine granulosa cells in vitro were examined in short-term (48-h) cultures. IL-1 inhibited FSH induction of aromatase activity and of LH-stimulated cAMP accumulation by granulosa cells. Both these inhibitory actions of IL-1 were concentration-dependent. Significant inhibitory effects were observed with as low as 0.05-0.25 ng/ml of IL-1, with maximal effects at 25 ng/ml. IL-1 also significantly inhibited increases in [125I]iodo-LH binding and progesterone secretion induced by FSH, as well as reducing basal levels of aromatase activity and LH-stimulated cAMP accumulation. Studies on the mechanisms of IL-1 actions on FSH-induced differentiation of immature porcine granulosa cells revealed that IL-1 reduced cAMP accumulation by the cells in response to FSH in a time- and concentration-dependent manner. IL-1 also inhibited induction of aromatase activity and LH-stimulated cAMP accumulation induced by dibutyryl cAMP, suggesting that IL-1 also affects the steps distal to cAMP generation. In contrast, IL-1 had no effect on progesterone secretion induced by dibutyryl cAMP, suggesting that post-cAMP steps of progesterone secretion were unaffected by IL-1.

The image diagnosis of a gynecologic tumor is progressing with the propagation of magnetic-resonance imaging (MRI), fast X-ray computed tomography (CT), and transvaginal probe. We introduce you the role which an image diagnosis should play in the decision of the therapeutic plan and the evaluation of the therapeutic effect of a uterine cervical cancer, a uterine endometrial cancer, and an ovarian cancer. In a uterine cervical cancer, MRI is useful to grasp the stage and the tumor size, and contributes toward determining a therapeutic plan. In addition, we may guess the response to the therapy, and judge the existence of a residual tumor during or immediately after an irradiation or an intraarterial injection. In a uterine endometrial cancer, MRI will contribute toward demonstrating a cervical invasion and toward assessing a myometrial invasion, therefore may help us to modify a therapeutic plan. To stage an ovarian cancer, it is necessary to visualize a small disseminated lesion. However, as all imaging modalities has a limitation to reveal the small disseminated nodules, they will not replace probe laparotomy. The probability of X-ray CT to take the place of second-look operation by an improvement has been suggested. MRI is poor at the visualization of a dissemination, however it is sometimes helpful for the evaluation of the therapeutic effect of a localizing tumor with demonstrating the changes of the signal intensity. We suppose that also an image diagnosis leaves room for expectation to contribute toward deciding the therapeutic plan of an ovarian cancer.

Immunohistochemistry was used to determine the distribution of oestrogen receptors (ER) and progesterone receptors (PR) in the human ovary during folliculogenesis. Primordial and preantral follicles did not contain ER or PR. The granulosa cells of antral follicles had ER, but negligible PR, before the LH surge. In contrast, at the time of LH surge, these cells of the dominant follicle contained PR, but not ER. On the other hand, granulosa cells of the non-dominant follicles had ER, but not PR. After ovulation, the PR persisted in the luteinized granulosa cells and in the corpus luteum during early pregnancy. The theca interna and surrounding stromal cells were ER-negative and PR-positive throughout the menstrual cycle. Thus, the results show that ER and PR are not expressed simultaneously in the granulosa cells, the thecal cells, or the stromal cells during folliculogenesis. Mechanisms controlling the expression of steroid receptors during the normal menstrual cycle and in early pregnancy are discussed.

In an attempt to understand the molecular mechanism underlying the depressive effect of glucocorticoids on corticotropin production, the level of corticotropin messenger RNA activity in rat pituitaries was measured with the use of the cell-free protein-synthesizing system derived from wheat germ. The large translation product of corticotropin messenger RNA was identified and quantitated by indirect immunoprecipitation with antibody against corticotropin. The level of corticotropin messenger RNA activity was increased 3- to 6-fold by adrenalectomy. Dexamethasone administration to adrenalectomized rats resulted in a marked suppression of corticotropin messenger RNA activity. Cortisol and corticosterone also exhibited a suppressive effect but were less effective than dexamethasone. In contrast, nonglucocorticoids such as progesterone and aldosterone had no suppressive effect. These results indicate that at least part of the glucocorticoid effect on corticotropin production in the pituitary is exerted at the pretranslational level.

It has been noticed that hyperprolactinemia can cause luteal insufficiency as well as anovulation in women. In order to explore the mechanism underlying this disorder, hyperprolactinemia was induced in baboons (Papio cynocephalus) by daily administration of sulpiride during follicular and early luteal phases. In hyperprolactinemic baboons, the plasma progesterone level was suppressed without notable changes in plasma estradiol, LH and FSH levels. When corpora lutea from these baboons were examined in vitro to investigate their ability to convert 14C-pregnenolone into various steroids, there was progressive inhibition of steroid metabolism related to the plasma levels of prolactin. These findings strongly suggest, although do not actually prove, that an elevated level of prolactin could directly impair luteal function by adversely affecting 3 beta-hydroxysteroid dehydrogenase activity.

To elucidate the mechanisms of the inhibitory effect of interleukin-1 (IL-1) on LH-stimulated progesterone secretion by cultured porcine granulosa cells, we examined which steps of the LH-stimulated, cAMP-mediated, progesterone biosynthetic pathway were affected by IL-1. Pretreatment of the cells for 48 h with IL-1 reduced intra- and extracellular cAMP accumulation in response to LH by 73% and 83%, respectively. The inhibitory effects of IL-1 were time and concentration dependent. Significant inhibition was observed at as low as 50-250 pg/ml, and the effect was maximal at 100 ng/ml (ID50; 2 ng/ml). The lowest concentration of IL-1 used (0.5 pg/ml), in contrast, showed a tendency to stimulate LH-induced cAMP accumulation. Effect of IL-1 on the specific binding of [125I]LH to granulosa cells was then examined, which showed that IL-1 (100 ng/ml) significantly reduced the specific binding by 36%. IL-1 also significantly reduced intra- and extracellular cAMP accumulation by the cells in response to forskolin (50, 150 microM) by 28-46%, indicating that IL-1 can directly inhibit adenylate cyclase activity. Contrary to these inhibitory actions of IL-1 on LH-stimulated cAMP generation, IL-1 did not significantly reduce progesterone secretion induced by (Bu)2cAMP. These results indicate that the inhibitory effect of IL-1 on LH-stimulated progesterone secretion is due to its actions at at least two different sites along the LH-stimulated, cAMP-mediated, progesterone biosynthetic pathway, the LH receptor level and adenylate cyclase systems. The post-cAMP steps of progesterone production, in contrast, did not seem to be affected by IL-1.

Increasing evidence suggests that functions of the immune system and gonads are closely related with each other. In cultures of granulosa and luteal cells, macrophages have been shown to modulate steroidogenic functions. In this paper we present the modulatory effects of interleukin-1, a cytokine produced predominantly by activated macrophages, on gonadotropin-induced differentiation, as well as growth of cultured porcine granulosa cells.

The influence of sulpiride-induced hyperprolactinemia on the hypothalamic-pituitary-ovarian function of the baboon (Papio cynocephalus) was investigated. Plasma levels of prolactin, LH, FSH, estrone, estradiol, 17-hydroxyprogesterone, progesterone and 20 alpha-dihydroprogesterone in control and consecutive treatment cycles (sulpiride i.m. injections 100 mg/day) were determined serially. The hormonal changes indicate that the ovary is the most sensitive site to the direct inhibitory action of sulpiride-induced hyperprolactinemia.

Total RNAs extracted from amnion, chorion and decidua of the human second trimester placentas were translated in the cell-free translation system, followed by immunoprecipitation with antiserum to human prolactin (Prl) and electrophoresis in sodium dodecyl sulfate slab gel. A single immunospecific protein with an apparent molecular weight of approximately 25,500, about 3000 daltons larger than authentic Prl, was formed only with RNA from decidua, and it competed with unlabeled Prl but not with unlabeled human placental lactogen (hPL) for binding to the antibody. The electrophoretic patterns of the fragments formed by partial enzymatic proteolysis of it and authentic Prl were similar.

Polyadenylate-containing RNA prepared from the membrane fraction of bovine anterior pituitary gland was shown to direct the synthesis of a large translation product related to corticotropin (adrenocorticotropic hormone) in a cell-free system derived from wheat germ. This product was identified by immunoprecipitation with specific antibody against corticotropin, followed by electrophoretic analysis of the dissociated immunoprecipitate on sodium dodecyl sulfate-polyacrylamide gel. Further evidence for the identity of the translation product was provided by the presence of a common peptide in the chymotryptic digest of [35S]methionine-labeled cell-free product and in that of authentic corticotropin. The molecular weight of the translation product was estimated to be approximately 35,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that corticotropin messenger RNA directs the cell-free synthesis of a product that contains the amino acid sequence of corticotropin but is much larger than this hormone.

Interleukin-1 (IL-1) significantly inhibited basal progesterone secretion and influenced the cell number in high density cultures of porcine granulosa cells harvested from small (1-2 mm) or medium (3-5 mm) follicles. These two effects of IL-1 showed similar but inverse dose-response relationships: Significant effects were observed at concentrations higher than 50 pg/ml, and the maximal effective concentration was 5 ng/ml, which reduced progesterone secretion by 53.5 +/- 2.1%. In contrast, IL-1 neither inhibited progesterone secretion nor did it influence cell number in cultures of fully mature granulosa cells harvested from the largest (8-11 mm) follicles. The growth-promoting effect of IL-1 on granulosa cells was further examined in the low density cultures, which revealed that IL-1 significantly stimulated proliferation of the less mature granulosa cells at concentrations higher than 50 pg/ml, and that at a maximal stimulatory concentration (25 ng/ml) it increased cell number to 206 +/- 11% of control. Although IL-1 increased cell number and reduced progesterone secretion of fully mature granulosa cells after 5 days in low density cultures, it seemed that the proliferating population were not the cells with luteinized morphology and positive 3 beta-hydroxysteroid dehydrogenase staining. The proliferating population of cells appeared to have less mature morphology and were negative for 3 beta-hydroxysteroid dehydrogenase staining.

Enriched small and large cell fractions were prepared from mature corpora lutea from 15 women in the midluteal phase by enzymatic dissociation, followed by Percoll gradient centrifugation. The steroidogenic function of each cell type was assessed by measuring the gonadal steroids released into the incubation medium. The large cell fraction was estimated to be 97% pure, with minimal contamination by small cells, whereas the small cell fraction was approximately 68% pure, being contaminated with 10% large cells and 22% nonsteroidogenic cells. In the unstimulated state, large cells were approximately 2-fold more potent in progesterone formation and aromatase activity, but only half as potent in androstenedione and testosterone formation as an equal number of small cells. When stimulated with hCG, the small cells responded with significant increases in progesterone, androstenedione, and testosterone release, but the large cells did not. Both cell types secreted estrone and 17 beta-estradiol in the presence of androgen substrate, but the addition of FSH significantly stimulated aromatization only in large cells. Thus, small and large human luteal cells have steroidogenic properties similar to those exhibited by follicular thecal and granulosa cells, respectively.

Interleukin-1(IL-1), a cytokine predominantly produced by activated macrophages, has been shown to possess a wide range of biological functions as well as to play a role as an immune mediator. As it has been reported that homogenates of peritoneal macrophages contained substances which stimulated progesterone production by luteinized granulosa cells of mice, we examined whether IL-1 could modulate the steroidogenic functions of granulosa cells. Porcine granulosa cells obtained from medium-sized follicles were cultured with IL-1 in the presence or absence of LH, and the effects of IL-1 on LH-stimulated as well as basal progesterone production by these cells were examined. Contrary to our expectation, IL-1 markedly inhibited the LH-stimulated progesterone production in a dose-dependent manner. The morphological luteinization of the granulosa cells induced by LH was also inhibited by IL-1. In addition to effects on LH-induced differentiation, IL-1 exhibited an inhibitory effect on basal (unstimulated) progesterone production as well. The ovarian granulosa cell was identified as one of the possible targets for IL-1, and it was shown for the first time that IL-1 can modulate steroidogenic functions of mammalian cells in culture.

The distribution of the mRNA coding for the common precursor of corticotropin and beta-lipotropin among different parts of the bovine pituitary has been investigated by quantifying the mRNA activity with the use of a cell-free protein-synthesizing system. The results obtained have demonstrated that this mRNA activity is located both in the anterior lobe and in the intermediate lobe, while it is essentially not detectable in the neural lobe nor in the stalk. The structural identity of the translation products of corticotropin/beta-lipotropin mRNA from the anterior and from the intermediate lobe has been indicated by their molecular weight as well as by the electrophoretic patterns of the peptide fragments formed from them upon partial enzymatic proteolysis or upon cyanogen bromide cleavage. The specific activity of corticotropin/beta-lipotropin mRNA in the intermediate lobe is about 20-fold higher than that in the anterior lobe, and the total activity of this mRNA in the former is about 2-fold higher than that in the latter. In the intermediate lobe, the translation product of corticotropin/beta-lipotropin mRNA amounts to almost one-third of the products encoded by total translatable mRNA. These results indicate that corticotropin/beta-lipotropin mRNA represents a major mRNA species in intermediate lobe of the pituitary, thus suggesting that this lobe may perform a highly specialized function in producing a large amount of the common precursor of corticotropin and beta-lipotropin.

Ten clomiphene-resistant, normoprolactinemic women with polycystic ovary syndrome (PCOS) were treated by continuous and increasing administration of bromocriptine (Brc), and changes in hormonal profiles as well as therapeutic efficacy were examined. Ovulation was restored in four patients (responders), and two of them became pregnant, whereas the other six patients did not ovulate with Brc alone (non-responders). Endocrine analyses revealed distinct differences between responders and non-responders: 1) pretreatment serum levels of dehydroepiandrosterone sulfate (DHAS) in non-responders were significantly higher than those in responders: 2) exaggerated LH secretion was definitely aggravated with Brc therapy in non-responders, but unchanged or slightly reduced in responders: 3) basal PRL secretions showed a marked reduction in both groups, whereas this response to TRH in responders decreased more markedly than in non-responders with the therapy. It is concluded that low DHAS group patients of PCOS are likely to respond to Brc, whereas high DHAS group patients appear contraindicated for this treatment due to its aggravating effect on LH secretion.

Very recently, it has been reported that interleukin-1 (IL-1) has an inhibitory effect on progesterone production by porcine granulosa cells in vitro. In the present study we investigated the presence of IL-1 or IL-1-like activity in porcine ovarian follicular fluids (FF) as the first step in elucidating the physiological role of IL-1 in follicular growth and maturation. Since IL-1 and IL-1-like substances have interleukin-2 receptor (IL-2R)/p55(Tac)-inducing activity (TIA), we determined the TIA in the FF by means of a highly sensitive TIA assay using flow cytometry. TIA was significantly higher (P less than 0.01) in the FF of small follicles than in those of medium-sized and large follicles. A significant negative correlation (P less than 0.05) was apparent between TIA and 17 beta-estradiol concentration in the FF. The conditioned media of porcine granulosa cells also showed TIA. Of these conditioned media, those from small follicles exhibited higher TIA than those from medium-sized and large follicles. TIA in the conditioned media decreased rapidly as the culture period was extended. Sex steroids such as 17 beta-estradiol, progesterone, testosterone, and androstenedione had no effect on IL-2R/p55(Tac) induction. These results indicate that porcine granulosa cells produce the IL-2R/p55(Tac)-inducing factor, the activity of which decreases in association with the maturation of the follicles. Because of the heterogeneity of IL-2R-inducing factors, the relationship between TIA in the FF and IL-1 should be elucidated. We discuss the possibility that this factor may play a role in follicular maturation and that enhancement of IL-2R/p55(Tac) expression by this factor may contribute to the local defense mechanism in ovarian follicles.

The expression of renin and angiotensinogen genes in the human placenta and related tissues has been examined by RNA blot hybridization analysis with specific human complementary DNA (cDNA) probes. Renin mRNA was detectable in the chorion throughout pregnancy and in the hydatidiform moles, but not in the decidua, amnion or myometrium. The relative concentration of renin mRNA in the chorion was at the highest level in early pregnancy and decreased thereafter, while the total amount contained in the whole placenta was at the lowest level in early pregnancy, and increased thereafter, reaching at term about one-sixth of the total renin mRNA in the kidney. Hydatidiform moles had an even higher concentration of renin mRNA than the early chorion. There was no significant difference in either the relative concentration or the total renin mRNA content in the placentae from 4 normal and 4 toxemic pregnancies. Angiotensinogen mRNA was undetectable in any of the placental tissues, hydatidiform moles or myometrium. These results show that renin is synthesized in the placenta, possibly to play some physiological role locally by utilizing angiotensinogen which is abundantly present in the maternal systemic circulation.

A bolus iv injection of 150 IU purified human urinary FSH (hFSH) or saline was given to 21 normal women during 3 different luteal phases (early luteal phase, n = 7; midluteal, n = 8; late luteal, n = 6), and the responses of ovarian steroids and gonadotropins were analyzed sequentially for 24 h after hFSH injection. A rapid and dramatic increase in serum FSH concentrations occurred after each hFSH injection, followed by a gradual decrease. Serum LH concentrations, including hourly fluctuations, were significantly higher after FSH administration than after NaCl injection at the same time during the preceding cycle. Neither serum progesterone nor testosterone levels were affected by the hFSH injection in any of the 3 luteal phases, but in the early and midluteal phases serum estradiol levels were higher than the preinjection values and those during the control cycles. These results indicate that FSH stimulates aromatization of luteal tissue in vivo as it does in vitro. Thus, FSH in addition to LH as the indispensable luteotropin may have a permissive role in the regulation of corpus luteum function.

In an assay for thyroid-stimulating antibodies, in which FRTL-5 thyroid cells were incubated with crude immunoglobulin (Ig) fractions precipitated from serum with 15% polyethylene glycol, significant increase in cAMP production was elicited by the samples from 25 (35.7%) out of 70 pregnant women. The highest value was 529.5%. There was a close correlation between thyroid stimulating activities and serum hCG concentrations (r = 0.708, P less than 0.001). When 125I-hCG was added to serum from pregnant women, about 20% of the radioactivity was incorporated into the Ig fractions. hCG preparations within a range of concentrations of 30-300 IU/m elicited 2.3-16.5 times increase in cAMP in a dose-dependent manner. Nine pregnant women with serum TSH concentrations less than the lower limit of the normal range (less than 0.25 mU/l) displayed significantly higher values for both thyroid stimulating activities and serum hCG concentrations (P less than 0.001, respectively) compared with those who had normal TSH levels in serum. These data suggest that hCG or its variant may stimulate the thyroid sufficiently to suppress secretion of TSH from the pituitary in some pregnant women.