LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED₅₀: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.     

Disruption of the TIMP-1 gene product is associated with accelerated endometrial gland formation during early postnatal uterine development

Postnatal uterine development is marked by periods of tissue remodeling. The objective of the present study was to examine the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a regulator of tissue remodeling events, during postnatal uterine development and to assess the phenotypic consequences of disruption of the TIMP-1 gene product during this time period. To accomplish this goal, wild-type and TIMP-1 null mice were sacrificed at Postnatal Days (PNDs) 5, 10, 15, 20, and 25 and uterine morphology, TIMP expression and matrix metalloproteinase (MMP) activity were assessed. In wild-type mice, TIMP-1 mRNA steady-state levels were highest at PND 5, after which expression decreased. TIMP-2 and TIMP-3 expression in wild-type mice showed no significant changes from PND 5 to 25. In TIMP-1 null mice, TIMP-2 and TIMP-3 expression patterns were similar to those in wild-type counterparts with the exception that, at PND 10, TIMP-2 and TIMP-3 expression was significantly lower in the null mice. Endometrial gland number and uterine histology were similar between genotypes at PNDs 5 and 10, but at PNDs 15 and 20, endometrial glands were more abundant in TIMP-1 null mice. Associated with the increased gland density in the null mice was an increase in total MMP activity above the levels expressed in wild-type mice. In summary, disruption of the TIMP-1 gene product is associated with reduced TIMP-2 and TIMP-3 steady-state mRNA levels, elevated MMP activity, and accelerated endometrial gland formation. We conclude that, during early postnatal uterine development, TIMP-1 may be critical for proper endometrial gland development.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Phosphodiesterase regulation is critical for the differentiation and pattern of gene expression in granulosa cells of the ovarian follicle

Feedback regulations are integral components of the cAMP signaling required for most cellular processes, including gene expression and cell differentiation. Here, we provide evidence that one of these feedback regulations involving the cyclic nucleotide phosphodiesterase PDE4D plays a critical role in cAMP signaling during the differentiation of granulosa cells of the ovarian follicle. Gonadotropins induce PDE4D mRNA and increase the cAMP hydrolyzing activity in granulosa cells, demonstrating that a feedback regulation of cAMP is operating in granulosa cells in vivo. Inactivation of the PDE4D by homologous recombination is associated with an altered pattern of cAMP accumulation induced by the gonadotropin LH/human chorionic gonadotropin (hCG), impaired female fertility, and a markedly decreased ovulation rate. In spite of a disruption of the cAMP response, LH/hCG induced P450 side chain cleavage expression and steroidogenesis in a manner similar to wild-type controls. Morphological examination of the ovary of PDE4D-/- mice indicated luteinization of antral follicles with entrapped oocytes. Consistent with the morphological finding of unruptured follicles, LH/hCG induction of genes involved in ovulation, including cyclooxygenase-2, progesterone receptor, and the downstream genes, is markedly decreased in the PDE4D-/- ovaries. These data demonstrate that PDE4D regulation plays a critical role in gonadotropin mechanism of action and suggest that the intensity and duration of the cAMP signal defines the pattern of gene expression during the differentiation of granulosa cells.     

Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor

The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Herein we document that epidermal growth factor (EGF)-like factors amphiregulin (Areg), epiregulin (Ereg), and betacellulin (Btc) are induced in cumulus oocyte complexes (COCs) by autocrine and paracrine mechanisms that involve the actions of prostaglandins (PGs) and progesterone receptor (PGR). Areg and Ereg mRNA and protein levels were reduced significantly in COCs and ovaries collected from prostaglandin synthase 2 (Ptgs2) null mice and Pgr null (PRKO) mice at 4 h and 8 h after human chorionic gonadotropin, respectively. In cultured COCs, FSH/forskolin induced Areg mRNA within 0.5 h that peaked at 4 h, a process blocked by inhibitors of p38MAPK (SB203580), MAPK kinase (MEK) 1 (PD98059), and PTGS2 (NS398) but not protein kinase A (PKA) (KT5720). Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. PGE2 reversed the inhibitory effects of AG on AREG-induced expression of Areg but not Ptgs2, placing Ptgs2 downstream of EGF-R signaling. Phorbol 12-myristate 13-acetate (PMA) and adenovirally expressed PGRA synergistically induced Areg mRNA in granulosa cells. In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). Collectively, FSH-mediated induction of Areg mRNA via p38MAPK precedes AREG induction of Ptgs2 mRNA via ERK1/2. PGs acting via PTGER2 in cumulus cells provide a secondary, autocrine pathway to regulate expression of Areg in COCs showing critical functional links between G protein-coupled receptor and growth factor receptor pathways in ovulating follicles.     

Prostaglandin E2 is a product of induced prostaglandin-endoperoxide synthase 2 and microsomal-type prostaglandin E synthase at the implantation site of the hamster

Certain uterine prostaglandins (PGs) are elevated at implantation sites and are needed to trigger the events of blastocyst implantation that include blastocyst-uterine attachment and stromal decidualization with vascular permeability changes. Several decades of investigations showed that treatment with PG synthesis inhibitors, prior to or during the time of implantation, resulted in either complete inhibition or a delay in implantation or reduction in the number of implantation sites with diminished decidual tissue. Consistent with these findings, we observed that whereas a selective PG endoperoxide synthase (Ptgs) 1 inhibitor SC-560 failed to inhibit implantation, a selective Ptgs2 inhibitor SC-236 showed significantly reduced number and size of implantation sites in progesterone-treated ovariectomized pregnant hamsters. It is known that Ptgs2 expression and Ptgs2-derived prostacyclin (PGI2) synthesis at implantation sites are needed for implantation in the mouse (a rodent that needs ovarian estrogen for implantation). However, it is unknown which Ptgs and PG synthases produce which PGs at implantation sites of the hamster (a rodent that does not need ovarian estrogen for implantation). Here we demonstrate that as blastocyst implantation proceeds, a reduction in Ptgs1 expression from uterine luminal epithelial cells and a gradual induction in Ptgs2 expression exclusively in luminal epithelial and adjacent decidual cells occurred at implantation sites of hamsters. Results also reveal that PGE2, but not PGI2, is the major PG at implantation sites where Ptgs2 and microsomal type PGE synthases but not PGI synthases are co-expressed. This elevated uterine PGE2 at implantation sites may serve to initiate or amplify physiological signals required for specific aspects of the implantation process in hamsters.     

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.     

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.     

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)₂cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

Steroidogenic capacity of residual ovarian tissue in 4-vinylcyclohexene diepoxide-treated mice

Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg(-1) day(-1)) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.