Stimulatory Effect Of hCG Research Articles

Upregulation of adamts9 by gonadotropin in preovulatory follicles of zebrafish

Previously we had identified adamts9 as a downstream target of Pgr, which is essential for ovulation in zebrafish. The primary goal of this study is to determine whether human chorionic gonadotropin (hCG, LH analog) also regulate adamts9 expression prior to ovulation. The expression of adamts9 was induced by hCG in a dose and time dependent manner in zebrafish preovulatory follicles in vitro. Interestingly, the stimulatory effect of hCG on adamts9 expression was not blocked in pgr−/− follicles but blocked in lhcgr−/−. This effect of hCG was via Lhcgr and its associated cAMP and PKC signaling pathways. Reduced fecundity and reduced expression of adamts9 were also found in lhcgr−/− females in vivo. Therefore, we have provided the first evidence of gonadotropin (hCG) regulated adamts9 in zebrafish, which could be important for ovulation.

Thyroid hormone dysfunction during pregnancy: A review

Thyroid dysfunctions such as hypothyroidism, thyrotoxicosis and thyroid nodules may develop during pregnancy leading to abortion, placental abruptions, preeclampsia, preterm delivery and reduced intellectual function in the offspring. Epidemiological data have shown the significant role of maternal thyroid hormone in fetal neurologic development and maternal health. It has been suggested that the deleterious effects of thyroid dysfunction can also extend beyond pregnancy and delivery to affect neuro-intellectual development in the early life of the child. Pregnancy poses an important challenge to the maternal thyroid gland as hormone requirements are increased during gestation as a result of an increase in thyroid- binding globulin, the stimulatory effect of HCG on TSH receptors, and increased peripheral thyroid hormone requirements. Maternal thyroid dysfunction is associated with increased risk for early abortion, preterm delivery, neonatal morbidity and other obstetrical complications. Early diagnosis for thyroid dysfunction of pregnant women and treatment of thyroid dysfunction during pregnancy is important and cost effective to avoid both fetal and maternal complications secondary to thyroid dysfunction. Therefore the aim of this review was to assess the thyroid function changes occurring during pregnancy, the different disorders with their maternal and fetal implications, the laboratory diagnosis and the best ways of management of these conditions.

Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells: involvement of Ca2+ entry via the T-type Ca2+ channel

Intracellular cAMP and Ca(2+) are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone (LH) and human chorionic gonadotropin (hCG). However, the identification of Ca(2+) entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca(2+) channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil (a putative T-type Ca(2+) channel blocker) on steroidogenesis were assessed using reverse transcription (RT)-polymerase chain reaction analysis for the steroidogenic acute regulatory protein (StAR) mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L(-1) extracellular Ca(2+), hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion (P < 0.05), and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner (P < 0.05). Moreover, the hCG-induced increase in testosterone production was completely removed when external Ca(2+) was omitted, implying that Ca(2+) entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca(2+) currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca(2+) entry carried out by the T-type Ca(2+) channel in the Leydig cells of mice.

Periovulatory Expression of Hyaluronan and Proteoglycan Link Protein 1 (Hapln1) in the Rat Ovary: Hormonal Regulation and Potential Function

ABSTRACT Periovulatory follicular matrix plays an important role in cumulus-oocyte complex (COC) expansion, ovulation, and luteal formation. Hyaluronan and proteoglycan link protein 1 (HAPLN1), a component of follicular matrix, was shown to enhance COC expansion in vitro. However, the regulatory mechanisms of periovulatory expression of Hapln1 and its role in periovulatory granulosa cells have not been elucidated. We first determined the periovulatory expression pattern of Hapln1 using pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed immature rat ovaries. Hapln1 expression was transiently induced both in intact ovaries and granulosa cells at 8 h and 12 h after hCG injection. This in vivo expression of Hapln1 was recapitulated by culturing preovulatory granulosa cells with hCG. The stimulatory effect of hCG was blocked by inhibition of protein kinase A, phosphatidylinositol dependent kinase, p38 MAPK, epidermal growth factor signaling, and prostaglandin synthesis, revealing key mediators involved in LH-induced Hapln1 expression. In addition, knockdown of Runx1 and Runx2 expression by small interfering RNA or inhibition of RUNX activities by dominant-negative RUNX decreased hCG or agonist-induced Hapln1 expression. Chromatin immunoprecipitation assays verified the in vivo binding of RUNX1 and RUNX2 to the Hapln1 promoter in periovulatory granulosa cells. Luciferase reporter assays revealed that mutation of the RUNX binding sites completely obliterated the agonist-induced activity of the Hapln1 promoter. These data conclusively identified RUNX proteins as the crucial transcription regulators for LH-induced Hapln1 expression. Functionally, treatment with HAPLN1 increased the viability of cultured granulosa cells and decreased the number of the cells undergoing apoptosis, whereas knockdown of Hapln1 expression decreased granulosa cells viability. This novel finding indicates that HAPLN1 may promote periovulatory granulosa cell survival, which would facilitate their differentiation into luteal cells.

Periovulatory Expression of Hyaluronan and Proteoglycan Link Protein 1 (Hapln1) in the Rat Ovary: Hormonal Regulation and Potential Function

Periovulatory follicular matrix plays an important role in cumulus-oocyte complex (COC) expansion, ovulation, and luteal formation. Hyaluronan and proteoglycan link protein 1 (HAPLN1), a component of follicular matrix, was shown to enhance COC expansion in vitro. However, the regulatory mechanisms of periovulatory expression of Hapln1 and its role in periovulatory granulosa cells have not been elucidated. We first determined the periovulatory expression pattern of Hapln1 using pregnant mare serum gonadotropin/human chorionic gonadotropin (PMSG/hCG)-primed immature rat ovaries. Hapln1 expression was transiently induced both in intact ovaries and granulosa cells at 8 h and 12 h after hCG injection. This in vivo expression of Hapln1 was recapitulated by culturing preovulatory granulosa cells with hCG. The stimulatory effect of hCG was blocked by inhibition of protein kinase A, phosphatidylinositol-dependent kinase, p38 MAPK, epidermal growth factor signaling, and prostaglandin synthesis, revealing key mediators involved in LH-induced Hapln1 expression. In addition, knockdown of Runx1 and Runx2 expression by small interfering RNA or inhibition of RUNX activities by dominant-negative RUNX decreased hCG or agonist-induced Hapln1 expression. Chromatin immunoprecipitation assays verified the in vivo binding of RUNX1 and RUNX2 to the Hapln1 promoter in periovulatory granulosa cells. Luciferase reporter assays revealed that mutation of the RUNX binding sites completely obliterated the agonist-induced activity of the Hapln1 promoter. These data conclusively identified RUNX proteins as the crucial transcription regulators for LH-induced Hapln1 expression. Functionally, treatment with HAPLN1 increased the viability of cultured granulosa cells and decreased the number of the cells undergoing apoptosis, whereas knockdown of Hapln1 expression decreased granulosa cells viability. This novel finding indicates that HAPLN1 may promote periovulatory granulosa cell survival, which would facilitate their differentiation into luteal cells.

Paracrine regulation of the resumption of oocyte meiosis by endothelin-1

Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus–oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge.

Expression, Regulation, and Function of Paired-Box Gene 8 in the Human Placenta and Placental Cancer Cell Lines

Pax proteins are transcriptional regulators that control a variety of developmental decisions in vertebrates. During development, the paired-box gene 8 (PAX8) is expressed in the thyroid, kidney, and several areas of the central nervous system. It is also expressed in the adult thyroid gland, in which it mediates TSH-induced modulation of the expression of important genes, such as those encoding thyroglobulin, thyroperoxidase, and the sodium/iodide symporter (NIS). Thus far, placental expression of PAX8 has been described only in mice. In the present study, we show that PAX8 is also expressed in the human placenta at term. In an in vitro model of placental cancer, the JAR choriocarcinoma cell line, human chorionic gonadotropin (hCG) increased levels of PAX8 mRNA and protein, and gel retardation assays indicated that the up-regulation of PAX8 protein expression is associated with an increase in its DNA-binding activity. The effects of hCG were mimicked by forskolin, indicating that they are cAMP dependent. Levels of mRNA for the Wilms' tumor 1 (WT1) and NIS genes were increased in JAR cells by hCG treatment, whereas overexpression of PAX8 increased only levels of WT1 mRNA. In cells transfected with PAX8-specific small interfering RNA, the stimulatory effects of hCG on WT1 mRNA levels were abolished, but hormonal enhancement of NIS mRNA levels was unchanged. These findings indicate that, in JAR cells, hCG activates a cAMP-dependent pathway that can up-regulate WT1 expression through PAX8.

LH/hCG-Stimulated Androgen Production and Selective HDL-Cholesterol Transport Are Inhibited by a Dominant-Negative CREB Construct in Primary Cultures of Rat Theca-Interstitial Cells

Theca-interstitial (T-I) cells synthesize androgens that are converted to estrogen by the granulosa cells. In rat ovary, T-I cells primarily utilize HDL-derived cholesteryl esters (CE) as a precursor for androgen synthesis. The HDL-CE is delivered to steroidogenic cells by a process termed "selective" uptake in which CE is internalized without the simultaneous uptake of apolipoprotein(s). This process is mediated by an HDL receptor, scavenger receptor class B, type I (SR-BI) and is stimulated by trophic hormone (LH/hCG), which also activates the cAMP cascade. In this study, we tested whether the adenoviral (Ad)-mediated introduction of a dominant-negative analog of cyclic AMP response element binding protein (A-CREB) inhibits the stimulatory effect of LH/hCG on the selective uptake of high-density lipoprotein (HDL)-cholesterol and androgen production in primary cultures of rat T-I cells. Androstenedione production by cultured T-I cells was stimulated by hCG and by the adenoviral overexpression of wtCREB. Additionally, the stimulatory effect observed with hCG was amplified in the presence of HDL. Androgen synthesis was increased 17-fold in the presence of HDL and hCG but the stimulatory effect of hCG was inhibited by Ad A-CREB by approx 70%. In the selective up-take studies, cell-surface association of the labeled HDL was significantly enhanced by hCG treatment, and this effect was inhibited by Ad A-CREB. The selective uptake of HDL-cholesterol was also enhanced by hCG but exposure to Ad A-CREB also abrogated this effect. It is concluded that CREB plays an intermediary role in the stimulatory action of LH/hCG on androgen synthesis and the selective uptake of HDL-cholesterol in T-I cells.

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

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

Functional assessment of the calcium messenger system in cultured mouse Leydig tumor cells: regulation of human chorionic gonadotropin-induced expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein, a 30-kDa mitochondrial factor, is a key regulator of steroid hormone biosynthesis, facilitating the transfer of cholesterol from the outer to the inner mitochondrial membrane. StAR protein expression is restricted to steroidogenic tissues, and it responds to hormonal stimulation through different second messenger pathways. The present study was designed to explore the mechanisms of extracellular calcium (Ca2+) involved in the hCG-stimulated expression of StAR protein and steroidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracellular Ca2+ (1.5 mmol/liter) enhanced the hCG (50 microg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 +/- 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immunoblotting. The potentiating effect of Ca2+ on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In accordance, omission of Ca2+ from the extracellular medium by specific Ca2+ chelators, EDTA or EGTA (4 mmol/liter each), markedly diminished the hCG-stimulated P production. The Ca2+ effect on hCG-induced StAR mRNA expression was dramatically suppressed by 10 micromol/liter verapamil, a Ca2+ channel blocker. The Ca2+-mobilizing agonist, potassium (K+; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca2+ antagonists, further supporting the involvement of intracellular free Ca2+ ([Ca2+]i) in these responses. The interaction of Ca2+ or K+ with hCG accounted for a clear increase in the StAR protein level (1.4-1.8-fold; 4 h) compared with that after hCG stimulation. Inhibition of protein synthesis by cycloheximide (CHX) drastically diminished the hCG-induced StAR protein content, indicating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca2+ was increased by 26% with hCG and was strongly inhibited by verapamil. [Ca2+]i moderately augmented the response to hCG in fura-2/AM-loaded mLTC-1 cells within 30-40 sec, reaching a plateau within 1-3 min. Interestingly, the calcium ionophore (A23187) clearly increased (P < 0.01) StAR mRNA expression, in additive fashion with hCG. Northern hybridization analysis revealed four StAR transcripts at 3.4, 2.7, 1.6, and 1.4 kb, with the 1.6-kb band corresponding to the functional StAR protein; all of them were up-regulated 3- to 5-fold upon hCG stimulation, with a further increase in the presence of Ca2+. The mechanism of the Ca2+ effect on hCG-stimulated StAR expression and P production was evaluated by assessing the involvement of the nuclear orphan receptor, steroidogenic factor 1 (SF-1). Stimulation of hCG significantly elevated (2.1 +/- 0.3-fold) the SF-1 mRNA level, which was further augmented in the presence of Ca2+, whereas EGTA and verapamil completely abolished the increase caused by Ca2+. Cells expressing SF-1 marginally increased StAR expression, but coordinately elevated StAR mRNA levels in response to hCG and hCG plus Ca2+ compared with those in mock-transfected cells. On the other hand, overexpression of the nuclear receptor DAX-1 remarkably diminished (P < 0.0001) the endogenous SF-1 mRNA level as well as hCG-induced StAR mRNA expression. In summary, our results provide evidence that extracellular Ca2+ rapidly increases [Ca2+]i after hCG stimulation, presumably through opening of the transmembrane Ca2+ channel. Neither extracellular Ca2+ nor K+ alone has a noticeable effect on StAR expression and steroidogenesis, whereas they clearly potentiate hCG induction. The Ca2+-mediated increase in hCG involved in StAR expression and P production is well correlated to the levels of SF-1 expression. The stimulatory effect of hCG that rapidly increases [Ca2+]i is responsible at least in part for the regulation of SF-1-mediated StAR expression that consequently regulates steroidogenesis in mouse Leydig tumor cells.

Regulation of prostaglandin F2 alpha receptor expression in cultured human granulosa-luteal cells.

PGF2 alpha is a metabolite of arachidonic acid that triggers regression of the corpus luteum. Recent animal studies have indicated that PGF2 alpha (FP) receptor messenger ribonucleic acid (mRNA) is expressed in the corpus luteum. To understand the regulation of the FP receptor in the ovary we have cloned a partial complementary DNA (cDNA) sequence of the FP receptor from human granulosa cells obtained from women undergoing in vitro fertilization. The sequence of this cDNA is identical to the previously reported FP receptor sequences obtained from human uterine and placental cDNA libraries. Low levels of the FP receptor mRNA were observed in freshly isolated granulosa cells or in cultured granulosa-luteal (GL) cells, as detected by reverse transcriptase-PCR. hCG and 8-bromo-cAMP increased the steady state levels of the FP receptor mRNAs after incubation for 24-48 h, as detected by Northern blot hybridization. The stimulatory effect of hCG was concentration and culture stage dependent. Further, hCG and 8-bromo-cAMP increased binding of radiolabeled PGF2 alpha to intact GL cells. In contrast, phorbol 12-myristate 13-acetate inhibited basal as well as hCG- and 8-bromo-cAMP-induced FP receptor mRNA expression and binding of the radiolabeled ligand. In summary, hCG, 8-bromo-cAMP, and phorbol 12-myristate 13-acetate modulate the expression of the FP receptor in human GL cells, which may represent a mechanism to regulate the responsiveness of the ovary to PGF2 alpha.

Monoclonal antibodies specific for the binding site on the LH receptor alter progesterone production in cultured granulosa cells.

Monoclonal antibodies (mAbs) specific for the LH receptor (LHR) were generated through a modified auto-anti-idiotypic approach in which human chorionic gonadotropin (hCG) was used as the immunogen followed by cyclophosphamide to induce anti-idiotypic antibodies. The purpose of this study was to investigate the effectiveness of these antibodies to alter progesterone production in porcine granulosa cells in vitro. Anti-LHR mAbs were incubated with granulosa cells in the presence or absence of a stimulatory dose of hCG. Progesterone output by treated cells was measured using a RIA procedure. Most of the mAb could inhibit stimulated progesterone production by cultured granulosa cells. It was speculated that two possible mechanisms may cause the inhibition effect observed. Several of the antibodies appeared to block hCG binding thus removing the stimulatory effects of hCG. However, the most potent inhibiting mAbs for progesterone production had little or no effect on hCG binding, suggesting that some other mechanism was responsible for the observed inhibition. In addition, several of the antibodies were found to have a stimulatory effect on progesterone production by granulosa cells even in the absence of a stimulating dose of hCG. It is proposed that these antibodies were able to mimic hCG.

Regulation of corpus luteum function in early human pregnancy

To investigate the capacity of the human corpus luteum (CL) of pregnancy to form cyclic adenosine-3',5'-monophosphate (cAMP) and P in vitro in response to hCG and prostaglandin (PG) E2. Six women undergoing sterilization concomitant with legal abortion and eight women undergoing surgery for ectopic pregnancy were included. Human chorionic gonadotropin was analyzed preoperatively in two serum samples. The CL were excised and luteal specimens were incubated for 2 hours in the presence or absence of hCG or PGE2. The tissue concentrations of cAMP and P concentrations in the incubation media were measured. The in vitro results were correlated to the preoperative daily change in serum hCG. In CL from pregnancies with normally rising serum hCG levels, the addition of hCG in vitro did not affect luteal cAMP or P production. In pregnancies with plateauing and/or decreasing serum hCG levels, the addition of hCG in vitro significantly stimulated cAMP and P formation, and this stimulatory effect correlated significantly with the preoperative daily change in serum hCG. In contrast to hCG, PGE2 had the same stimulatory effect in vitro on CL specimens from both normal and pathological pregnancies. This effect did not correlate with the preoperative daily change in serum hCG. Prostaglandin E2 stimulates all CL, irrespective of any changes in preoperative serum hCG levels. In contrast, the stimulatory effect of hCG in vitro does not correlate with changes in serum hCG, and thus CL from pregnancies with normally rising serum hCG levels are refractory to hCG in vitro.

Regulation of the Activin-Binding Protein Follistatin in Cultured Human Luteinizing Granulosa Cells: Characterization of the Effects of Follicle Stimulating Hormone, Prostaglandin E2, and Different Growth Factors1

Regulation of the activin-binding protein follistatin (FS) by recombinant human (rh) FSH, prostaglandin E2 (PGE2), and several polypeptide growth factors was examined in cultures of human granulosa-luteal (GL) cells obtained from in-vitro fertilization patients at oocyte retrieval. Northern and dot blot hybridization analyses demonstrated that both rhFSH and PGE2 caused stimulatory effects on FS mRNA levels in a culture stage-, time-, and concentration-dependent manner. An 8-h stimulation with rhFSH (100 ng/ml) significantly increased FS mRNA levels on Days 5 and 7 of culture and PGE2 (10(-6)M) on Days 2, 4, and 7. The stimulatory effect of rhFSH and PGE2 on FS mRNA levels were rapid and transient. Maximal inductions occurred 8 h after stimulation, whereas weak or no stimulatory effects were seen at 24 or 48 h. PGF2 alpha did not affect FS mRNA levels at any time point studied. Treatment of the cells with the protein synthesis inhibitor cycloheximide prior to rhFSH stimulation did not inhibit the rapid induction of FS mRNAs, but it prevented the decline at 24 h. Both rhFSH and PGE2 clearly also increased the levels of secreted FS proteins are detected by immunoprecipitation studies with a specific antibody. The effects of the polypeptide growth factors epidermal growth factor (EGF); transforming growth factor beta 1 (TGF beta 1), and activin A on FS mRNA levels were also examined. TGF beta 1 and activin A had no effect on basal FS expression at any concentration or time point studied. An 8-h stimulation with EGF increased FS mRNA levels, but the effect was weaker than those caused by rhFSH and PGE2. We conclude that rhFSH and PGE2 induce FS mRNA and protein in human cultured GL cells. EGF is able to induce FS mRNA to a lesser extent than are rhFSH and PGE2, whereas PGF2 alpha, TGF beta 1, and activin A do not affect basal FS mRNA levels in human cultured GL cells. This study together with our previous report on the stimulatory effect of hCG on FS levels suggest that in the luteal phase of the human menstrual cycle, FS expression in granulosa cells is likely to be positively controlled by luteotropic factors such as gonadotropins and PGE2. Consequently, elevated FS levels may support the survival of the human CL since FS is known to prevent the antisteroidogenic effects of activin in human GL cells.

Regulation of inhibin alpha- and beta A-subunit messenger ribonucleic acid levels by chorionic gonadotropin and recombinant follicle-stimulating hormone in cultured human granulosa-luteal cells.

We studied the effects of recombinant human FSH (rhFSH) and purified hCG on the steady state messenger ribonucleic acid (mRNA) levels of inhibin alpha- and beta A-subunits in cultured granulosa-luteal cells of preovulatory ovarian follicles obtained from women undergoing in vitro fertilization. Specific mRNA transcripts for the alpha- and beta A-subunits were detected in Northern and dot blot filter hybridization analyses, and the levels of these mRNAs were induced by rhFSH and hCG in a distinct concentration- and time-dependent manner. The basal and hCG-stimulated alpha-subunit mRNA levels were first determined at 2- to 3-day intervals over a 3- to 10-day culture period after the initiation of the cultures. Both the basal and hCG-stimulated alpha-subunit mRNA levels declined steadily during culture, but the maximal relative stimulatory effect of hCG was observed on day 7 of culture. All subsequent experiments, therefore, were performed on days 6-8 of culture. Both gonadotropins induced alpha-subunit mRNA levels with slower kinetics than those of the beta A-subunit. Varying between experiments, rhFSH and hCG increased the expression of the alpha-subunit with a maximal effect of 2.5- to 5.7-fold and 1.7- to 7.2-fold, respectively, above basal levels 24-48 h after stimulation. rhFSH and hCG induced beta A-subunit mRNA levels with 3.0- to 5.8-fold and 2.3- to 8.6-fold increases above basal levels, respectively, at 2 h; thereafter, only moderate or no stimulation of the beta A-subunit mRNA levels could be detected at 7-48 h. Treatment of the cells with the RNA synthesis inhibitor actinomycin-D prevented the induction of alpha-subunit mRNA levels by hCG, and no significant differences were detected in the stability of alpha-subunit mRNA transcripts in hCG-treated cells vs. untreated cultures. This indicates that hCG induces transcription of the alpha-subunit gene rather than maintains the levels of preexisting transcripts. As the kinetics of induction of alpha- and beta A-subunit mRNAs by gonadotropins were different, we examined how the inhibition of protein synthesis affects the induction of alpha- and beta A-subunit mRNAs by hCG. Cycloheximide had no effect on basal alpha-subunit mRNA levels at 2 or 24 h. However, it inhibited at 24 h the induction of the alpha-subunit by hCG.(ABSTRACT TRUNCATED AT 400 WORDS)

Increasing 125I-human chorionic gonadotrophin specific binding in human granulosa cells by follicle-stimulating hormone and follicular fluid*

It has been reported that success at in-vitro fertilization/embryo transfer (IVF/ET) treatment is increased by follicular fluid (FF) re-injected into the abdomen. In the present study a possible direct effect of FF on human granulosa cell (GC) progesterone (P4) secretion and LH/human chorionic gonadotrophin (HCG) receptor content was studied in the presence and absence of FSH. Human GC cultured for 8 days in medium alone showed a 40-fold decrease in P4 secretion. Addition of human FSH increased P4 secretion and [125I]HCG specific binding by 12- and 8-fold, respectively, compared to human GC cultured in medium alone. The effect of FF was evaluated in a heterologous system by the addition of FF from large antral porcine follicles (LFF) to human GC in culture. The decline in human GC-P4 secretion after 8 days of culture was not altered by either porcine serum alone or porcine LFF alone. However, the concomitant addition of FSH and LFF significantly increased [125I]HCG specific binding, but did not alter the FSH-induced P4 secretion when both parameters were compared to GC cultured in FSH + porcine serum. Furthermore, the addition of HCG alone significantly increased P4 secretion 33- and 70-fold in GC pre-cultured with either FSH alone or FSH + LFF respectively compared with the stimulatory effect of HCG on GC pre-cultured in medium alone. These results may suggest that FSH and LFF increase the functional content of LH/HCG receptor in luteinized human GC.

Effects of human chorionic gonadotropin pretreatment on endocrine, cytological and prostaglandin F 2α receptor patterns of the bovine corpus luteum

The effects of human chorionic gonadotropin (hCG) on plasma progesterone (P) levels, size distribution of luteal cells and [ 3H]prostaglandin F 2α ([ 3H]PGF 2α) binding to luteal membranes were determined. In Experiment 1, 8 heifers were randomly assigned at synchronized oestrus to either receive 2500 IU hCG 48 h postoestrus, or remain as controls. Blood samples were taken at 6 h intervals from oestrus until Day 7, when a biopsy of luteal tissue was obtained by laparotomy. In Experiment 2, 16 heifers were assigned randomly at oestrus to one of four groups in a 2 × 2 design with hCG treatment as one factor and day of biopsy (Day 11 or Day 13) as the other. Administration of hCG was the same as in Experiment 1. Blood samples were collected at 12 h intervals from oestrus until luteal biopsy. In Experiment 1, P concentrations were lower ( P < 0.05) in hCG-treated animals than controls at 42, 66, 72, and 84 h post-hCG. In contrast, P concentrations were higher in the hCG-treated heifers than controls at 114 h post-hCG (4.47 ± 0.74 vs 3.41 ± 0.52 ng/ml; mean ± s.e.m.). In Experiment 2, P levels were higher ( P<0.05) in hCG-treated heifers than controls from 96–192 h post-hCG. The proportion of large luteal cells (>25 μm) on Day 7 was lower in the hCG-treated heifers than controls (17.8±4.7% vs 29.0 ± 3.9%; mean ± s.e.m.). The small luteal cells of control heifers on Day 7 had a greater ( P < 0.05) diameter than those of hCG-treated heifers (13.7 ± 0.2 vs 11.9 ± 0.7 μm; mean ± s.e.m.). No differences were observed in the percentage of large cells between treatments on Day 11 or Day 13. The large luteal cells of control heifers on Day 13 had a greater ( P < 0.05) diameter (50.9 ± 1.4 vs 44.5 ± 1.9 μm) than those of hCG-treated animals. Binding of [ 3H]PGF 2α to Day 7 (Experiment 1) luteal membranes was lower ( P < 0.05) in control than hCG-treated heifers (16.3 ± 6.9 vs 54.1 ± 5.3 femtomoles/mg of membrane protein; mean ± s.e.m.). Differences in [ 3H]PGF 2α binding to luteal membranes and size distribution of luteal cells were most apparent during early dioestrus (Day 7) while the stimulatory effects of hCG on P secretion did not occur until 120 h after hCG treatment.

Prolactin and gonadotrophin interactions on progesterone formation in cultured human granulosa cells.

Human granulosa cells were isolated from preovulatory follicles during cycles stimulated with HMG-HCG or clomiphene-HMG-HCG or from unstimulated cycles. The cells were cultured for 6-8 days in medium M199 containing fetal calf serum under 5% CO2 in air. Highly purified human prolactin and human chorionic gonadotrophin were added alone or in combination to the cultures, and the content of steroids in the medium was measured every second day, utilizing conventional RIA techniques. In the presence of HCG the formation of progesterone (P) increased 3-5-fold over the control level with maximal effect after 4 days. In cells derived from clomiphene-HMG-HCG stimulated cycles, prolactin per se did not influence basal P formation but reduced the stimulatory effect of HCG. This was only seen in granulosa cells from follicles greater than 20 mm in diameter. In experiments with Forskolin, an adenylate cyclase activator, P formation was stimulated and the stimulation was counteracted by the concomitant presence of prolactin, indicating that prolactin did not interfere with the LH-HCG receptor. In cells from smaller follicles, or in cells from follicles aspirated from the natural cycle prior to the endogenous LH peak, P formation was stimulated by HCG but the addition of prolactin did not reduce this stimulatory effect. The results are discussed in relation to earlier reports on prolactin effects in vitro both on laboratory animals and human material.

Effect of GABA and benzodiazepines on testicular androgen production

We have evaluated the effect of R05-4864, a selective probe to label peripheral type benzodiazepine receptor, on “in vitro” testicular androgen production. Decapsulated testes from adult rats showed a significant increase in the basal and hCG-stimulated testosterone secretion into the medium in response to 10 −5M, 10 −6M, and 10 −7M Ro5-4864. In addition, we have studied the changes in testicular GABA content at three different ages and we found its highest concentration at 31 days of age. When we evaluated the effect of GABA on “in vitro” androgen production at different stages of gonadal maturation we observed that the highest concentration of GABA (10 −6M) was able to modify the basal and hCG-stimulated androgen production from adult (60 days) and pubertal (45 days) testes. In addition, when prepubertal testes (31 days) were incubated under basal conditions, 10 −6M GABA induced a significant increment of androstanediol production, while the stimulatory effect of hCG was reduced in the presence of the same GABA concentration. The present results suggest that GABA plays a physiological role in the regulation of rat testicular androgen production depending on the stage of sexual maturation.

The effect of hCG on cyclic AMP in rabbit ciliary processes.

Experiments designed to address the role of cyclic AMP as a mediator of the eye pressure and aqueous flow reducing effects of human chorionic gonadotropin (hCG) in the rabbit have been conducted. No significant stimulatory effect of hCG on particulate preparations of adenylate cyclase from rabbit ciliary processes could be found under several different experimental conditions. Forskolin, an activator of adenylate cyclase can, in some biological systems, reveal a significant hormonal cyclic AMP response which is undetectable in the presence of hormone alone. Forskolin did potentiate the effect of isoproterenol on cyclic AMP production by intact rabbit ciliary processes. Cyclic AMP production by intact ciliary processes was not affected by hCG, either in the presence or absence of forskolin. Significant specific binding of iodinated hCG to particulate preparations of rabbit ciliary processes could not be detected. These results dispute the role of cyclic AMP as a mediator of the flow and pressure reducing effects of hCG; however, theoretical and methodological complications relating to this interpretation are discussed.