Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 µg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 µg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

The corpus luteum (CL) is a transient ovarian endocrine gland that secretes progesterone, the hormone of pregnancy. Development of an optimally functioning CL requires the creation of a dense capillary bed through growth of new blood vessels, which is an intricate process called angiogenesis. A myriad of factors regulates angiogenesis, including the angiogenic inducer protein, cellular communication network factor 1 (CCN1). Although it is highly expressed in the early-cycle bovine CL, the mechanisms of CCN1 regulation have not been fully elucidated. In the present study, we showed that CCN1 expression in steroidogenic luteal cells from the early-cycle bovine CL was induced by Ras homolog family member A (RhoA) and by thrombin, but not by luteinizing hormone (LH). To the best of our knowledge, the involvement of thrombin and its signaling partner, RhoA, in regulating CCN1 in bovine steroidogenic luteal cells has not been previously reported. These findings will inform our future work to determine how RhoA activation by thrombin leads to increased expression of CCN1.

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat.

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

Influence of omega-3 polyunsaturated fatty acids from fish oil or meal on the structure of lipid microdomains in bovine luteal cells.

Biological membranes are composed of a lipid bilayer and proteins that form lipid microdomains. This study examined the effects of fish byproducts on lipid-protein interactions within lipid microdomains of bovine luteal cells. In Exp. 1 and 2, luteal cells were prepared from corpora lutea (CL; n = 4 to 8) collected at an abattoir. Exp. 1 was conducted to optimize ultrasonication in a detergent-free protocol for isolation of lipid microdomains. A power setting of 10 to 20% was effective in isolating lipid microdomains from bulk lipid. In Exp. 2, cells were cultured in control medium or fish oil to determine influence of fish oil on distribution of lipid microdomain markers and prostaglandin F2α (FP) receptors. Cells treated with fish oil had a smaller percentage of microdomain markers and FP receptor in microdomains (P < 0.05). In Exp. 3 and 4, cells were prepared from mid-cycle CL obtained from cows supplemented with corn gluten meal (n = 4) or fish meal (n = 4). Exp. 3 examined effects of dietary supplementation on distribution of lipid microdomain markers and FP receptor and Exp. 4 on fatty acid composition within lipid microdomains. A smaller percentage of lipid microdomain markers and FP receptor was detected in microdomains of cells collected from fish meal supplemented animals (P < 0.05). In Exp. 4, a greater percentage of omega-3 polyunsaturated fatty acids was detected in bulk lipid from fish meal supplemented cows (P < 0.05). Results show that fish byproducts influence lipid-protein interactions in lipid microdomains in bovine luteal cells.

Astaxanthin increases progesterone production in cultured bovine luteal cells.

Although astaxanthin (AST) is known to be a strong antioxidant, its effects on reproductive function in domestic animals have not yet been elucidated in detail. Therefore, we investigated the effects of AST on luteal cells, which produce progesterone (P4), an important hormone for maintaining pregnancy. Luteal cells were prepared by collagenase dispersion of the corpus luteum (CL). The addition of racemic AST at a low concentration (<10 nM) to cultured bovine luteal cells increased P4 in the culture medium (P<0.05). This effect was attributed to an increase in the ability of luteal cells to produce P4 (P4/cell·DNA); however, the level of lipid peroxide (TBARS: thiobarbituric acid reactive substances) per cell did not decrease with the addition of AST, whose values were similar to that with the addition of luteinizing hormone. When optical isomers of AST (SS and RR types) were added to the culture medium, respectively, SS-AST was more effective in increasing P4 production than RR-AST. When 1 mg/kg·body weight of SS-AST derived from green algae was fed to cows for 2 weeks, its concentration in blood plasma was 10.9 nM on average, which was sufficient to expect an in vitro effect on the production of P4 in cows. These results suggested the potential of SS-AST supplements for cows to elevate luteal function.

Adverse influence of coumestrol on secretory function of bovine luteal cells in the first trimester of pregnancy.

Coumestrol is one of a few biologically active substances present in leguminous plants, which are widely used as fodder for ruminants. Depending on the doses, coumestrol acts on the reproductive processes as an estrogen-like factor or antiestrogen to evoke a decrease in ovulation frequency, elongation of estrous cycle duration. The aim of the current investigations was to study the influence of coumestrol on secretory function of luteal cells obtained from first trimester of pregnant cows. Luteal cells (2.5 × 10(5) /mL) from 3rd to 5th, 6th to 8th, and 9th to 12th week of pregnancy were preincubated for 24 h and incubated with coumestrol (1 × 10(-6) M) for successive 48 h and the medium concentrations of progesterone (P4), oxytocin (OT), prostaglandin (PG) E2 and F2α were determined. Moreover, the expression of mRNA for neurophysin-I/oxytocin (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating mono-oxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. Coumestrol did not affect P4 secretion but increased the secretion of OT from the cells collected at all stages of gestation studied. Hence, the ratio of P4 to OT was markedly decreased. Simultaneously, coumestrol increased the expression of NP-I/OT mRNA during 9th to 12th weeks of pregnancy, and mRNA for PGA during 3rd to 5th and 9th to 12th weeks of gestation. Furthermore, coumestrol decreased PGE2 secretion from luteal cells in all studied stages of pregnancy, while it affected PGF2α metabolite (PGFM) concentration only from week 3 to 5 of pregnancy. Obtained results suggest that coumestrol impairs secretory function of the corpus luteum (CL) and this way it can affect the maintenance of pregnancy in the cow.

Expression of functional melatonin MT(1) receptors in equine luteal cells: in vitro effects of melatonin on progesterone secretion.

In the present study, we analysed the molecular mechanism(s) by which melatonin directly affects ovarian function in the mare. In Experiment 1, follicles and corpora lutea (CL) were collected from slaughterhouse ovaries and analysed for melatonin (MT(1)) receptor mRNA and protein. In Experiment 2, CL were collected from slaughterhouse ovaries and cultured in Dulbecco's modified Eagle's medium-F12 medium (control medium) supplemented with 50 ng mL(-1) equine chorionic gonadotrophin (eCG), 1 nM-1 µM melatonin, 1 µM forskolin or 1 µM luzindole. Explants were cultured for 3 h in the presence of these drugs. Conditioned media were analysed for progesterone production; luteal cells were analysed for cholesterol side-chain cleavage enzyme (P450scc), a steroidogenic enzyme that converts cholesterol into pregnenolone. Both MT(1) receptor mRNA and protein were expressed in follicles and CL. Melatonin inhibited both the eCG- and forskolin-stimulated production of progesterone, as well as the forskolin-stimulated expression of P450scc, in equine luteal cells and the effect was dose-dependent. The inhibitory effect of melatonin was blocked by luzindole, a non-selective melatonin MT(1) and MT(2) receptor antagonist. The data support the presence of functional melatonin receptors in luteal cells and a regulatory role for melatonin in the endocrine function of the equine CL.

The adverse effect of phytoestrogens on the synthesis and secretion of ovarian oxytocin in cattle.

The current investigations were undertaken to study the mechanism of the adverse effect of phytoestrogens on the function of bovine granulosa (follicles >1< cm in diameter) and luteal cells from day 1-5, 6-10, 11-15, 16-19 of the oestrous cycle. The cells were incubated with genistein, daidzein or coumestrol (each at the dose of 1 × 10(-6) m). The viability and secretion of estradiol (E2), progesterone (P4) and oxytocin (OT) were measured after 72 h of incubation. Moreover, the expression of mRNA for neurophysin-I/OT (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating monooxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. None of the phytoestrogens used affected the viability of cells except for coumestrol. The increased secretion of E2 and P4 was only obtained by coumestrol (p<0.05) from granulosa cells from follicles <1cm in diameter and decreased from luteal cells on days 11-15 of the oestrous cycle, respectively. All three phytoestrogens stimulated (p<0.05) OT secretion from granulosa and luteal cells in all stages of the oestrous cycle and the expression of NP-I/OT mRNA in the both types of cells. The expression of mRNA for PGA was stimulated (p<0.05) by daidzein and coumestrol in granulosa cells, and by genistein and coumestrol in luteal cells. In conclusion, our results demonstrate that these phytoestrogens can impair the ovary function in cattle by adversely affecting the synthesis of OT in follicles and in corpus luteum. However, their influence on the ovarian steroids secretion was less evident.

Estrogens and androgens affect human luteal cell function.

OBJECTIVE: To evaluate estrogens (Es)--E2, estrone (E1), and estriol--and androgens--T and androstendione (A)-effect on P, prostaglandin (PG) F2α, PGE2, and vascular endothelial growth factor (VEGF) release and on VEGF expression in human luteal cells. To elucidate whether androgens effects were direct or mediated by their conversion in Es, an aromatase inhibitor was used. Finally, the luteal effect of the non-aromatizable dihydrotestosterone was evaluated. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENT(S): Corpora lutea (CLs) were obtained from 36 normally menstruating patients in the midluteal phase of the menstrual cycle. INTERVENTION(S): The human luteal cells were isolated from CLs and primary cultures were established. MAIN OUTCOME MEASURE(S): P and PG release were assayed by enzyme immunoassay; VEGF secretion by ELISA; VEGF messenger RNA (mRNA) expression by real-time polymerase chain reaction (PCR). RESULT(S): P and PGF2α secretion were decreased by Es and androgens. The VEGF release was increased by Es and androgens, whereas VEGF mRNA expression was not. The aromatase inhibitor counteracted T and A luteal effects. CONCLUSION(S): Both Es and androgens could participate in the regulation of human luteal function. The effect of T and A seems to be mediated by their conversion to Es, whereas for dihydrotestosterone, both direct androgenic and indirect estrogenic luteal effects could coexist.

Effects of phytoestrogen daidzein and estradiol on steroidogenesis and expression of estrogen receptors in porcine luteinized granulosa cells from large follicles.

The aims of the study were to compare the in vitro effects of daidzein or 17beta-estradiol (E(2)) on: 1) progesterone (P(4)) secretion by luteinized granulosa cells harvested from large porcine follicles, as well as 2) estrogen receptor alpha and beta (ERalpha and ERbeta) mRNA and protein expression in the cells. In addition, the effect of daidzein on E(2) secretion and viability of the granulosa cells was examined. We found that basal and gonadotropin-stimulated P(4) secretion were inhibited in granulosa cells cultured in the presence of daidzein either for 24 or 48 hours. In contrast to daidzein, E(2) reduced P(4) secretion only during 24-hour cell cultures increasing it during longer cultures. Daidzein did not affect E(2) secretion by granulosa cells. The expression of ERalpha and ERbeta mRNA, as well as ERbeta protein, was up-regulated by daidzein but unaffected by E(2). To conclude, the soy estrogen daidzein acts directly on the porcine ovary to decrease progesterone production and to increase expression of ERbeta mRNA and protein. Daidzein actions in porcine luteinized granulosa cells differ from those of estradiol and it may suggest disadvantageous effects of the phytoestrogen on reproductive processes in females.

Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells.

The cytochrome P450 aromatase (Cyp19) gene encodes an enzyme of crucial importance in the synthesis of estradiol. Estradiol is luteotropic in the rat. In this species, luteal Cyp19 expression increases progressively during pregnancy and falls before parturition. The mechanisms that control these changes are unknown. Using gel shift assays, we sought to identify the promoter regions that control Cyp19 expression in the rat corpus luteum (CL). The Cyp19 promoter contains a cAMP response element-like sequence (CLS), two nuclear receptor elements half sites (NREs), a GATA binding site, a Yin Yang-1 (YY1) response element, and an activation protein 3 (AP3) binding site. Nuclear extracts were obtained from CL of rats on days 4, 15, and 23 of pregnancy and from the ovaries of immature rats treated with vehicle or a hormone that induces Cyp19 expression in the follicles. CLS was active in immature ovaries but inactive in the CL of pregnant rats, whereas binding to NREs and GATA was observed in both tissues. YY1 was inactive in all samples tested. In the CL, AP3 binding was higher on day 15 of pregnancy when compared with day 4 and day 23 but it was absent in ovaries of immature rats, whereas luteinization increased AP3 binding activity. Mutation of the AP3 site blunted the stimulation of Cyp19 promoter activity in granulosa cells. Our results indicate that CLS is active only in follicles; whereas in the CL, binding to the GATA, NRE, and AP3 sites associates with changes in Cyp19 expression, suggesting that they control Cyp19 promoter activity in luteal cells.

Prostaglandin F2alpha suppresses rat steroidogenic acute regulatory protein expression via induction of Yin Yang 1 protein and recruitment of histone deacetylase 1 protein.

Prostaglandin F2alpha (PGF2alpha) plays a pivotal role in ovarian luteolysis by inhibiting the expression of steroidogenic acute regulatory (StAR) protein, leading to a decrease in intracellular cholesterol transport and luteal steroid production. Previously we have demonstrated that the transcription factor Yin Yang 1 (YY1) bound to three regions in the StAR promoter in vitro and repressed promoter activity. This study further defined the YY1-mediated PGF2alpha effect on the inhibition of StAR protein expression through YY1 interaction with a single region in the StAR promoter in vivo. PGF2alpha consistently suppressed StAR mRNA and protein expression in cultured luteal cells in a dose-dependent manner. PGF2alpha also enhanced YY1 protein expression and binding to its cis-element in a time-dependent pattern that preceded the decline in StAR protein levels. The StAR promoter region bound by YY1 was also associated with histone deacetylase 1 (HDAC1). PGF2alpha treatment promoted HDAC1 binding to and suppressed the histone H3 acetylation in this region. On the contrary, YY1 knockdown decreased HDAC1 binding, increased histone H3 acetylation, enhanced StAR protein expression, and negated PGF2alpha effect on StAR protein expression. Luciferase assays showed that YY1 overexpression inhibited StAR promoter activity and the addition of a HDAC inhibitor, trichostatin A, abrogated the effect of YY1. Trichostatin A-treated luteal cells displayed increased StAR protein expression. These data indicate that PGF2alpha enhances a direct YY1/StAR promoter interaction and the recruitment of HDAC1 to the promoter, thereby preventing transcriptional activation of the StAR gene.

The pesticide heptachlor affects steroid hormone secretion in isolated follicular and luteal cells of rat.

Heptachlor, a chlorinated hydrocarbon pesticide, suppresses the production of progesterone and estradiol in the female rat in vivo or in isolated ovaries in vitro. In this study the effect of heptachlor on steroid hormone production by isolated rat luteal and follicular cells, in the presence of two precursor hormones was investigated. Ovaries were isolated from anesthetized mature normocyclic virgin rats (3 to 4 months old), under sterile conditions. Corpora lutea and follicles were microscopically dissected out and separately enzymatically dispersed with collagenase at 37 degrees C. Viable cells collected after centrifugation were used at a concentration of approximately 2.5 x 10(5) cells/10 mL. Both luteal and follicular cell preparations were separately incubated overnight (15 h) at 37 degrees C in the presence of pregnenolone (P5) and androstenedione (A4) at a concentration of 6.0 nmol/L each, and heptachlor at either 0.12 microg/mL (low dose) or 1.20 microg/mL (high dose) (test cells) or in the absence of heptachlor (control cells). At the end of the incubations, progesterone and estradiol 17beta levels were analyzed in the incubation media. The results indicate that heptachlor significantly suppressed the production of both progesterone and estradiol in both cell types in a dose related manner even in the presence of A4 and P5 as precursor hormones (P<0.05).

[Effects on secretory function of rat gonad by Erxian decoction and its disassembled prescriptions].

OBJECTIVE: To explain functions, differences and coordination of three divided combinations of the "Erxian decoction", the famous traditional Chinese formula, on the effective sites of gonad gland at the cell level. METHOD: The effects of Erxian decoction and its main disassembled prescriptions, "Kidney Warming", "Yin Nourishing" and "Chong-ren Adjusting", on the level of testosterone (T) progesterone (P) estradiol (E2), respectively secreted by the primary culture Leydig cell, luteal cell and granulosa cell, were measured by radioimmunoassay. RESULT: (1) Erxian decoction could stimulate the T secretion while its three main disassembled prescriptions would seem no individual promoting effect on the secretion of T. (2) Erxian decoction and the "Kidney Warming" had the stimulating effect on P secretion, and the action of the whole formula being better than that of the "Kidney Warming". (3) Erxian decoction and its main disassembled prescriptions had the stimulating effect on E2 secretion, especially the whole formula. CONCLUSION: Erxian decoction can stimulate the secretion of T of the Leydig cell, P of luteal cell and E2 of granulosa cell. It can be seen that the effect of the whole formula is better than that of its main disassembled prescriptions.

Influence of progesterone, pregnenolone and 17beta-hydroxyprogesterone on the function of bovine luteal cells treated with luteinizing hormone, noradrenaline and prostaglandin E2.

The aim of the present study was to investigate the influence of progesterone (P4), its precursor (pregnenolone; P5) and metabolite (17beta-hydroksyprogesterone; 17betaOHP4) on secretory function of bovine luteal cells on days 6-10 of the estrous cycle and on intracellular Ca2+ mobilization. The luteal cells were pre-incubated for 24 h and after change of medium they were incubated for 30 min with P5 and 17betaOHP4 (10(-5) each). Next, the medium was supplemented with LH (100 ng/ml), noradrenaline (NA; 10(-5) M) and prostaglandin (PG)E2 (10(-6) M), the cells were incubated for further 4 h and the medium was collected for P4 determination. Another set of luteal cells (5x10(4)/well) was incubated with P4, P5 and 17betaOHP4 at the dose of 10(-5) M each for 30 min and intracellular Ca2+ mobilization was measured every 5 s three times before and for 60 s after cells stimulation with LH, NA and PGE2. Metabolite of P4 did not affect the stimulatory effect of LH, PGE2 and NA on P4 secretion to the medium. Whereas all used steroids reduced calcium release from small but not from large luteal cells. It is suggested that steroids could temporary impair effect of luteotropins on the luteal cells via non-genomic way.

Upregulation of steroidogenic enzymes and ovarian 17beta-estradiol in human granulosa-lutein cells by Cordyceps sinensis mycelium.

There is increasing evidence that 17beta-estradiol (E2) directly influences the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. Although Cordyceps sinensis (CS) mycelium, a Chinese herbal medicine, is believed to enhance libido and fertility in both sexes, the mechanism of its effect in women has not been determined. The aim of the present study was to evaluate the effects of CS on steroidogenic enzyme expression and E2 biosynthesis in human granulosa-lutein cells (GLC). We found that CS induced E2 production by GLC in a dose- and time-dependent manner and that a 3-h treatment with CS induced increased levels of mRNAs coding for the P450 side chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and aromatase. Western blot analysis demonstrated that, after treatment with CS for 3 h, protein levels of steroidogenic acute regulatory protein (StAR) and aromatase were upregulated while P450scc and 3beta-HSD levels showed no substantial change. New protein synthesis was required for CS-induced E2 production because it was abrogated by cycloheximide pretreatment. Addition of 22(R)-hydroxycholesterol, thus bypassing the need for StAR protein, did not induce as much E2 production as CS treatment, indicating that upregulation of StAR protein was not the only factor contributing to CS-induced steroidogenesis. Cotreatment of GLCs with CS and aminoglutethimide, an aromatase inhibitor, completely abolished CS-induced E2 production. In conclusion, treatment of GLCs with CS results in increased E2 production due, at least in part, to increased StAR and aromatase expression. These data may help in the development of treatment regimens to improve the success rate of in vitro fertilization.

Exogenous steroid substrate modifies the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estradiol production of human luteinized granulosa cells in vitro.

The in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid metabolism in human luteinized granulosa cells (hLGC) have been summarized as a decreased estradiol (E(2)) production without altering either E(2) metabolism or cytochrome P450 aromatase activity. In the present study, hLGC were used to analyze the fate of different substrates for cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450(c17)) in the presence or absence of TCDD. Human LGCs were plated directly on plastic culture dishes in medium supplemented with 2 IU/ml of hCG. TCDD (10 nM) or its solvent was added directly to the cells at the time of medium change, every 48 h for 8 days. The objective of the experiment was to test the hypothesis that exogenous steroid, substrate for P450(c17), would reduce the TCDD effects on E(2) synthesis. With dehydroepiandrosterone (DHEA) (a P450(c17) product), a dose-related increase in E(2) production was observed and the effect of TCDD on lowering E(2) production disappeared. In contrast, with increasing doses, up to 10 micro M, of pregnenolone (P(5)), no change in E(2) production was observed. However, 17alpha-hydroxypregnenolone (17P(5)) at 10 micro M produced a modest but significant increase in the E(2) production. Treatments with P(5) and 17P(5) did not alter the effect of TCDD on E(2) production. Radiolabeled substrate utilization by hLGC suggests that the principal metabolic pathway for Delta5 substrates is the conversion to a Delta4 product probably by a very active 3beta-hydroxysteroid dehydrogenase. We conclude that estrogen production by hLGC is limited at the level of lyase activity. Thus, these data suggest that the most likely target for the TCDD-induced inhibition of estrogen synthesis by hLGC is the 17,20-lyase activity of the P450(c17) enzyme complex.

Tokishakuyakusan directly attenuates PACAP's luteolytic action on luteal function in the rat ovary.

We investigated the potential direct effects of Tokishakuyakusan (TS) on progestin [progesterone and 20alpha-hydroxyprogesterone (20alpha-OH-P)] and cyclic adenosine-3',5'-monophosphate (cAMP) production in cultured rat luteal cells. In addition, we examined whether TS regulates the inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP), a newly found peptide, on luteinizing hormone (LH)-stimulated progesterone production. TS significantly stimulated progesterone, but not 20alpha-OH-P, production and cAMP accumulation through 24 hours of culture. PACAP-38 significantly elevated progesterone, 20alpha-OH-P and cAMP levels at all concentrations studied. On the other hand, PACAP-38 inhibited the production of progesterone and the accumulation of cAMP enhanced by LH, while the ratio of progesterone to 20alpha-OH-P was significantly decreased by PACAP-38 + LH. Concomitant treatment with TS and PACAP-38 + LH increased the ratio of progesterone to 20alpha-OH-P more than with PACAP-38 + LH. The present data have demonstrated that TS stimulates progesterone production in rat luteal cells, reconfirming our previous evidence that TS stimulates luteal steroidogenesis. The data further suggest that TS tends to attenuate PACAP's inhibition of LH-stimulated progesterone production, suggesting a luteotrophic effect within the corpus luteum.

Isocupressic acid blocks progesterone production from bovine luteal cells.

The needles of ponderosa pine (Pinus ponderosa Laws.) were reported to induce abortions when fed to late-term pregnant beef cows in North America. An in vivo study of pregnant cows suggested that isocupressic acid (IA) was the main abortifacient isolated from needles and bark of the pine. However, the mechanism of abortifacient activity of IA is not clear yet. In a pregnant cow, the corpus luteum of the ovary helps the maintenance of pregnancy by its progesterone production. This study involved the IA extracted from the root of the Taiwan cypress (Juniperus formosana) and used a frozen-thawed bovine luteal cell culture system to investigate the action of IA on progesterone production. Thawed bovine luteal cells (1 x 10(5) cells/ml/well) in M199 medium were cultured in 24-well culture plates at 37 degrees C in a 5% CO2 incubator. Ten ml of tested drugs, IA at 1 to 1000 ng/ml and/or ovine luteinizing hormone (oLH) at 1 to 100 ng/microl or 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) with 0.1-10 mM, were added into each well. After 4 hours of incubation, the media were harvested and assayed for progesterone by an enzyme immunoassay. Progesterone production from cells was the indicator used to evaluate the action of IA. All tested doses of IA significantly inhibited progesterone production in both basal and oLH stimulating conditions. Also those dosages inhibited cyclic adenosine-3',5'- monophosphate (cAMP) stimulation, suggesting a post-cAMP mechanism is involved in the IA action. We concluded that IA can induce pregnant cows to abort partly through blocking luteal function and may be identified as a new abortifacient chemical.

Expression and regulation of P2U-purinergic receptor in human granulosa-luteal cells.

The P2U purinoceptor (P2UR) has been identified pharmacologically in the ovary. However, the expression and regulation of the P2UR messenger RNA (mRNA) in human ovarian cells are still poorly characterized. The present study was designed to examine the expression and regulation of the P2UR in human granulosa-luteal cells (hGLCs) by RT-PCR and Northern blot analysis. A PCR product corresponding to the expected 599-bp P2UR complementary DNA was obtained from hGLCs. Molecular cloning and sequencing of the PCR product revealed an identical sequence to the reported P2UR complementary DNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGLCs using Northern blot analysis. The expression of the P2UR mRNA was down-regulated by human CG in a dose- and time-dependent manner. Treatment with 8-bromo-cAMP and forskolin also attenuated P2UR mRNA levels. Calcium signaling following the activation of the P2UR in single hGLCs was studied using microspectrofluorimetry. It revealed that, like ATP, uridine triphosphate (UTP) also induced cytosolic calcium mobilization in a dose-dependent manner. These results demonstrate for the first time that the P2UR mRNA is expressed in hGLCs and that P2UR mRNA is regulated by human CG, cAMP, and forskolin. The P2UR expressed in hGLCs functional because activation of the P2UR by ATP or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cell level. These findings further support a potential role of this neurotransmitter receptor in the human ovary.

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.