Enhancement and Desensitization of Hormone-Responsive Adenylate Cyclase in Granulosa Cells of Preantral and Antral Ovarian Follicles: Effects of Estradiol and Follicle-Stimulating Hormone*

The expression of LH receptor (LHR) mRNA was studied in fetal rat gonads using polymerase chain reaction multiplication of reverse-transcribed mRNA. A primer pair corresponding to the extracellular domain of the receptor revealed the expression of LHR mRNA in fetal ovaries and testes as early as embryonic Day 13.5, the earliest age studied. The localization of LHR mRNA was examined in the perinatal rat ovary using in situ hybridization with two antisense probes, one encoding the extracellular and the other encoding the transmembrane domains of LHR. At the age of 4 days, only the extracellular LHR probe gave specific signal over ovarian stromal and follicular cells, excluding the ova. Three days later, similar distribution of specific hybridization was observed with both probes. In the 10- and 30-day-old rat ovaries, clear expression of LHR mRNA was found to be with both probes in theca cells. Gonadotropin-stimulated production of cAMP was studied in cultures of dispersed perinatal rat ovarian cells. When 5-day-old ovarian cells were cultured for 3 days in vitro and then stimulated by either hCG (0.1 mg/L) or FSH (1 mg/L) for 6 h, cAMP production was enhanced only in cells stimulated by FSH. In a similar experiment with 7-day-old ovarian cells, cAMP production was stimulated by both FSH and hCG. Stimulation with hCG (0.1 mg/L) during the 3-day culture caused homologous desensitization of cAMP production, but stimulation with FSH (0.1 mg/L) had no such effect. The desensitization of the LHR was also investigated by treating neonatal rats in vivo with a high dose of hCG (600 IU/kg BW s.c. as a single injection on Day 7) or with a dosage of recombinant human (rec) FSH on Days 3-9 (0.3 IU s.c twice daily). Thereafter, at the age of 10 days, the ovaries were incubated either with recFSH (200 IU/L) or hCG (CR-121; 0.1 mg/L) for 1 h. Homologous desensitization of cAMP production by hCG was observed, but the FSH-mediated cAMP production was not affected. The hCG-induced steroidogenesis (progesterone and testosterone production) was not desensitized. In conclusion, these findings indicate that 1) the expression of the mRNA encoding the extracellular domain of LHR, i.e., truncated receptor, occurs in fetal rat gonads as early as embryonic Day 13.5; 2) the expression of the truncated LHR mRNA occurs uniformly in the differentiating ovarian cells before the appearance of the functional theca cell layer; 3) full-length LHR message appears in the developing ovary concomitantly with appearance of differentiated theca cells; 4) homologous desensitization of cAMP output by hCG, without steroidogenic desensitization, is present in perinatal rat ovaries; and 5) no FSH-evoked desensitization of cAMP production occurs in perinatal ovaries.

Section: Discussionsupporting

confidence: 92%

“…The missing effect of the hCG treatment on subsequent recFSH stimulation of cAMP agreed with the findings in cell culture and with earlier in vivo results [41]. The recFSH injections in vivo did not cause desensitization of cAMP in subsequent in vitro incubations with recFSH or hCG.…”

Section: Discussionsupporting

confidence: 90%

Development of Luteinizing Hormone Action in the Perinatal Rat Ovary1

Sokka

Hämäläinen

Kaipia

et al. 1996

“…In granulosa cells this positive feedback loop between estradiol and prostaglandin also seems plausible based on previous findings of PGE 2 stimulation of CYP19A1 activity in granulosa cells [51]. However, other mechanisms may also be important because previous studies have found that FSHresponsive ADCY (also known as adenylate cyclase) was increased by treatment with estradiol alone [52] and that estrogen increased CREB (known as cAMP responsive element binding protein) phosphorylation to promote CRE-dependent gene transcription [53][54][55]. Further studies are needed to dissect the molecular events leading to the synergism between FSH/cAMP and ESR in regulating CYP19A1.…”

Section: Discussionmentioning

confidence: 78%

Distinct Regulation by Steroids of Messenger RNAs for FSHR and CYP19A1 in Bovine Granulosa Cells

Luo

Wiltbank

2006

Steroidal regulation of gene expression in follicular cells is not completely defined. Granulosa cells from 5 mm bovine follicles were cultured and treated and steady-state mRNA levels determined for FSHR (follicle-stimulating hormone receptor) and CYP19A1 (aromatase). Cells were treated for 5 days with (0.1-300 ng/ml) 17beta-estradiol (E2), testosterone (T), or 5alpha-dihydrotestosterone (DHT). FSHR mRNA was increased by T and DHT but not E2. In contrast, CYP19A1 mRNA was induced by all doses of E2 but only high doses of T and DHT. Similarly, varying treatment duration (1-5 days) showed that FSHR was increased by T and DHT and CYP19A1 mRNA increased by E2 and T at all times. Synergism between steroid hormones and FSH or forskolin was also evaluated. FSH or E2 did not alter FSHR mRNA and did not enhance DHT stimulation of FSHR mRNA. In contrast, DHT alone had no effect on CYP19A1 mRNA but synergized with FSH plus E2 to increase CYP19A1 mRNA, probably due to induction of FSHR by DHT. Effects of E2 and T on CYP19A1 were blocked by ICI 182,780, indicating mediation by estrogen receptors. However, the specific androgen receptor antagonist bicalutamide did not block E2 or T effects on CYP19A1 but did block T and DHT stimulation of FSHR. Thus, FSHR is specifically regulated through androgen receptor, whereas CYP19A1 is regulated by multiple pathways, including estrogen receptors and cAMP/protein kinase A induced by FSHR activation in granulosa cells. These inter- and intracellular regulatory mechanisms may be critical for normal follicle growth and dominant follicle selection.

“…Clearly, the ability of gonadotropins to control ovarian function is dependent not only on circulating levels of the gonadotropins but on expression of appropriate receptor proteins by potential target cells in the ovary [1,2]. Both LH and FSH act through stimulatory G protein-coupled receptors and primarily transduce their signals via activation of adenylate cyclase and production of the second-messenger cAMP [3][4][5][6]. A number of studies have established the tissue-specific expression of the LH receptor in theca cells and FSH receptor in granulosa cells of developing follicles, as well as the induction of LH receptors by FSH in granulosa cells of preovulatory follicles [7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Early Growth Response Gene-1 Regulates the Expression of the Rat Luteinizing Hormone Receptor Gene1

Yoshino

Mizutani

Yamada

et al. 2002

LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between -171 and -137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between -171 and -137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.