Gonadotropin-dependent regulation of the prostaglandin E2 receptor in equine preovulatory follicles during the ovulatory process in mares.

The objectives of the study were to clone the primary structure of the prostaglandin E2 receptor subtype 2 (PTGER2) cDNA and to characterize its regulation in equine follicles during gonadotropin-induced ovulation. Results from DNA isolation indicated that the equine PTGER2 cDNA encodes a predicted 353-amino acid protein, which is highly similar (76-85%) to known mammalian homologues. The regulation of PTGER2 was studied by semi-quantitative RT-PCR/Southern blot using preparations of theca interna and mural granulosa cells isolated from equine follicles 0-39 hr post-treatment with human chorionic gonadotropin (hCG). Results indicated that a significant increase of PTGER2 mRNA occurred at 24 and 39 hr post-hCG in granulosa cells, and 30 and 33 hr post-hCG in theca cells (P < 0.05). Immunohistochemical staining and immunoblotting performed on equine follicular samples showed a corresponding increase of PTGER2 protein in both cell types after treatment with hCG. Levels of PTGER2 mRNA were also high in uterus, thymus and spleen, but moderate to low in other tested tissues. In the ovary, the expression of PTGER4 mRNA was observed and predominantly occurred in granulosa cells, with highest abundance of transcripts observed at 12 and 39 hr post-hCG. Thus, this study reports for the first time in mares that the ovulatory process is accompanied by the gonadotropin-dependent up-regulation of PTGER2 and PTGER4, which may in turn regulate PGE2-mediated preovulatory effects.

Regulation of bovine tumor necrosis factor-alpha-induced protein 6 in ovarian follicles during the ovulatory process and promoter activation in granulosa cells.

To study the regulation of bovine TNFalpha-induced protein 6 (TNFAIP6) prior to ovulation, preovulatory follicles obtained after the treatment with human chorionic gonadotropin (hCG) were used. RT-PCR analyses showed that levels of TNFAIP6 mRNA were low before hCG but significantly increased after hCG treatment in follicles. Further analyses and immunohistochemistry indicated that this increase in transcript and protein levels occurred in theca and granulosa cells. To investigate molecular mechanisms involved in TNFAIP6 transactivation, the activity of bovine TNFAIP6 promoter was studied in granulosa cell cultures. Mutant studies identified the minimal region conferring full-length promoter activity, in which activator protein-1 (AP1) and cAMP response element (CRE) elements were required for promoter activity. Overexpression of dominant-negative AP1 and activating transcription factor/cAMP response element-binding protein (CREB) inhibited forskolin-inducible promoter activity. DNA binding assays demonstrated the importance of AP1 and CRE for activity and identified JunD, FosB, Fra2, CREB1, and CREB2 as being part of the AP1 complex, and FosB, Fra2, and CREB1 for the CRE complex. Chromatin immunoprecipitation assays confirmed binding of these proteins with endogenous TNFAIP6 promoter. Treatment with forskolin, prostaglandin E2, and catalytic subunit protein kinase (cPKA) stimulated, but H89, PKA inhibitor peptide, and indomethacin inhibited, TNFAIP6 promoter activity and gene expression in granulosa cells. Collectively, this study is the first to describe that the ovulatory process in cows is associated with a gonadotropin-dependent induction of TNFAIP6 in ovarian follicles and provide the molecular basis through which AP1 and CRE sites and PKA activation played important roles in the regulation of TNFAIP6 in granulosa cells.

Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process.

The mammalian ovulatory process is accompanied by a gonadotropin-dependent increase in follicular levels of prostaglandin E2 (PGE2) and PGF2alpha, which are metabolized by 15-hydroxy prostaglandin dehydrogenase (PGDH). Little is known about ovarian PGDH regulation in non-primate species. The objectives of this study were to characterize the structure of equine PGDH and its regulation in follicles during human chorionic gonadotropin (hCG)-induced ovulation. The full-length equine PGDH was obtained by RT-PCR, 5'- and 3'-rapid amplification of cDNA ends (RACE). Its open reading frame encodes a 266-amino acid protein that is 72-95% homologous to other species. Semi-quantitative RT-PCR/Southern blot were used to study PGDH regulation in follicles isolated 0-39 h post-hCG. Results showed that PGDH mRNA expression was low in follicles obtained at 0 h, increased at 12 and 24 h (P < 0.05), and decreased at 36-h post-hCG. This induction of expression was biphasic, with elevated abundance of transcripts at 12 and 33 h post-hCG (P < 0.05) in mural granulosa and theca cells. Immunohistochemistry and immunoblotting confirmed regulated expression of PGHD protein in both cell types of preovulatory follicles after hCG. High levels of PGDH mRNA were observed in corpus luteum and other non-ovarian tissues tested, except kidney, muscle, brain, and heart. Thus, this study is the first to report the gonadotropin-dependent regulation of PGDH during ovulation in a non-primate species. PGDH induction was biphasic in theca and mural granulosa cells differing from primates in which this induction was monophasic and limited to granulosa cells, suggesting species-specific differences in follicular control of PGDH expression during ovulation.

Molecular cloning of equine 17beta-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo.

The type 1 form of 17beta-hydroxysteroid dehydrogenase (17betaHSD1) was the first isoform to be identified and is capable of converting estrone to 17beta-estradiol. This study was aimed at characterizing the molecular structure of the equine 17betaHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17betaHSD1 gene was cloned from an equine genomic library and shown to have a conserved genomic structure composed of six exons. Its cDNA sequence was also identified and coded for a 308 amino acid protein, 72 x 1-74 x 5% homologous to other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of the 17betaHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17betaHSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 h after hCG (P<0 x 05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17betaHSD1 transcript expression and downregulation (P<0 x 05). A 1412 bp fragment of the equine 17betaHSD1 proximal promoter was sequenced and shown to contain many putative transcription factor binding sites. Electromobility shift assays (EMSA) using a fragment of the proximal promoter (-230/-30) and nuclear extracts prepared from granulosa cells isolated prior to hCG (0 h post-hCG) revealed the presence of a major complex, and results from competition assays suggest that steroidogenic factor-1 (SF-1), NFkappaB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFkappaB led to the displacement of the binding nuclear proteins to the DNA probe, whereas the use of an anti-equine SF-1 antibody demonstrated the clear formation of a DNA-protein-antibody complex, confirming the potential role of these transcription factors in EMSA results. Interestingly, a notable decrease in DNA binding was observed when granulosa cell nuclear extracts isolated 30 h post-hCG were used, which paralleled the decrease in 17betaHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17betaHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17betaHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17betaHSD1 cDNA and gene.

Down-regulation of messenger ribonucleic acid encoding an importer of sulfoconjugated steroids during human chorionic gonadotropin-induced follicular luteinization in vivo.

Members of the organic anion transporting polypeptide (SLCO/OATP) superfamily are capable of importing anionic compounds across the lipid bilayer in a sodium-independent manner. Member 2B1 has been shown to transport few substrates, two of which are dihydroepiandrosterone-3-sulfate (DHEA-S) and estrone-3-sulfate. Steroid sulfatase (STS) catalyses the hydrolysis of these steroids into their unconjugated counterparts. The objective of this study was to investigate the regulation of SLCO2B1 and STS mRNAs during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine SLCO2B1 cDNA was cloned and shown to encode a 709-amino acid protein (OATP2B1) that is highly conserved when compared to mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of SLCO2B1 and STS transcripts in equine preovulatory follicles isolated between 0 and 39h after hCG treatment. Results showed high levels of SLCO2B1 mRNA expression before hCG, with a marked decrease observed in follicles obtained 24-39h post-hCG (P<0.05). Analyses of isolated granulosa and theca interna cells identified high mRNA expression in both cell types prior to hCG treatment, with granulosa cells showing a more rapid SLCO2B1 mRNA down-regulation. No significant change in STS mRNA was observed in intact follicle walls. However, when both cell types were isolated, a significant decrease in STS mRNA was observed in granulosa cells 24-39h post-hCG. Collectively, these results demonstrate that the hCG-dependent induction of follicular luteinization is accompanied by the down-regulation of SLCO2B1 and STS transcripts. Considering that OATP2B1 can import sulfoconjugated DHEA and estrogens, and that STS can remove the sulfonate moiety from these steroids, their down-regulation in luteinizing preovulatory follicles may provide an additional biochemical basis for the decrease in ovarian 17beta-estradiol biosynthesis after the LH surge.

Molecular cloning and gonadotropin-dependent regulation of equine prostaglandin F2alpha receptor in ovarian follicles during the ovulatory process in vivo.

The progressive rise in gonadotropins prior to ovulation triggers a marked increase in intrafollicular levels of prostaglandin F(2alpha)(PGF(2alpha)), which is known to interact with PGF(2alpha) receptor (FP). Little is known about the regulation of FP during ovulation. This study was undertaken to characterize the equine FP and its gonadotropin-dependent regulation in preovulatory follicles prior to ovulation. The full-length equine FP encodes a 366-amino acid protein that is 82-93% homologous to other species. Using semi-quantitative RT-PCR/Southern blot, we showed that FP mRNA expression was low in follicles obtained before hCG treatment (0h) and at 24, but increased at 12 and 36h post-hCG (P<0.05). This expression was regulated in both follicular cells, with high levels of the transcript at 33 and 36h post-hCG in granulosa cells, and at 12, 30 and 33h post-hCG in theca cells (P<0.05). Immunohistochemistry confirmed the induction of FP protein in both follicular cells after hCG, and immunoblotting revealed the increase of FP protein in preovulatory follicles 36h post-hCG. High levels of FP mRNA were detected in the corpora lutea and heart, but very low or undetectable in other tissues. This study reports for the first time the expression of FP and its up-regulation by hCG in preovulatory follicles prior to ovulation. FP regulation was occurred in different pattern than that observed in other species, suggesting a distinct and species-specific follicular control of FP expression during ovulation, and a potential involvement of PGF(2alpha), acting on granulosa and theca cells, in the ovulatory process.

Molecular characterization of equine P-selectin (CD62P) and its regulation in ovarian follicles during the ovulatory process.

Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes an 829-amino acid protein that is highly conserved when compared to the human protein (80% identity). Semiquantitative RT-PCR/Southern blot analyses were performed to study the regulation of P-selectin transcript in preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (ovulation occurs between 39 and 42 h post-hCG in this model). Results showed that levels of P-selectin mRNA remained very low or undetectable throughout the ovulatory process in extracts prepared from the granulosa cell layer. In contrast, a significant increase in P-selectin transcript was observed between 30 and 39 h post-hCG in extracts obtained from thecal layers (P < 0.05). Likewise, immunohistochemistry revealed an increase of immunoreactive P-selectin protein in the vascular endothelium present in thecal layers of follicles isolated 36 and 39 h post-hCG. Thus, the present study describes, to our knowledge for the first time, the primary structure of equine P-selectin and the regulation of P-selectin transcript and protein in follicular thecal endothelial cells before ovulation.

Cyclooxygenase-2 and its role in ovulation: a 2004 account.

The pre-ovulatory surge of gonadotrophins triggers a marked and obligatory increase in follicular prostaglandin synthesis prior to ovulation, and the cyclooxygenase (COX) enzyme is a key rate-limiting step in the biosynthesis of prostaglandins. In the early 1990s, the pre-ovulatory rise in follicular prostaglandin synthesis was shown to result from the selective induction of a novel COX isoform, now referred to as COX-2. Differences in the time-course of COX-2 induction in species with a short versus a long ovulatory process suggest that the enzyme could be a molecular determinant that sets the alarm of the mammalian ovulatory clock. Some of the fine molecular mechanisms involved in the transcriptional activation of the COX-2 gene in granulosa cells have also been elucidated. The binding of trans-activating upstream stimulatory factors (USF) to a consensus E-box cis-element in the proximal region of the promoter was shown to play a predominant role in COX-2 transcription. Studies showed that COX-2 expression could also serve as a valuable marker for follicular commitment to ovulation during hyperstimulatory cycles. This paper presents a comprehensive review of the events that led to the characterization of COX-2 in pre-ovulatory follicles, updates current concepts on the control of COX-2 expression in pre-ovulatory follicles, and addresses the consequences of COX-2 inhibition to women fertility and potential implications of COX-2 expression in ovarian cancer.

Human chorionic gonadotropin-dependent regulation of 17beta-hydroxysteroid dehydrogenase type 4 in preovulatory follicles and its potential role in follicular luteinization.

17Beta-hydroxysteroid dehydrogenase type 4 (17betaHSD4) has a unique multidomain structure, with one domain involved in 17beta-estradiol inactivation. The objective of the study was to investigate the regulation of 17betaHSD4 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine 17betaHSD4 cDNA was cloned and was shown to encode a 735-amino acid protein that is highly conserved (81-87% identity) compared with other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of 17betaHSD4 transcripts in equine preovulatory follicles isolated between 0-39 h after hCG treatment. Results showed the presence of basal 17betaHSD4 mRNA expression before hCG treatment, but an increase was observed in follicles obtained 24 h after hCG (P < 0.05). Analyses of isolated preparations of granulosa and theca interna cells identified basal mRNA expression in both layers, but granulosa cells appeared as the predominant site of follicular 17betaHSD4 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and used to study regulation of the 17betaHSD4 protein. Immunoblots showed an increase in full-length 17betaHSD4 protein in follicles 24 h after hCG (P < 0.05), in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. Collectively, these results demonstrate that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in 17betaHSD4 expression. Considering the estrogen-inactivating function of 17betaHSD4, its regulated expression in luteinizing preovulatory follicles appears as a potential complementary mechanism to reduce circulating levels of 17beta-estradiol after the LH surge.

Molecular characterization and role of bovine upstream stimulatory factor 1 and 2 in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells.

The transcriptional activation of the prostaglandin G/H synthase-2 (PGHS-2) gene in granulosa cells is required for ovulation. To directly study the ability of upstream stimulatory factor 1 (USF1) and USF2 to trans-activate the bovine PGHS-2 promoter in granulosa cells, USF1 or USF2 expression vectors were cotransfected with the PGHS-2/luciferase (LUC) chimeric construct, -149/-2PGHS-2.LUC. Results revealed that overexpression of USF1 or USF2 caused a marked and significant increase in basal and forskolin-inducible promoter activities (p<0.05), and these effects were dependent on the presence of a consensus E-box cis-element within the promoter fragment. Co-transfections with different N- and C-terminal truncated USF mutants led to significant reductions in promoter activation, as compared with full-length constructs (p<0.05), thus allowing identification of putative bovine USF functional domains. Overexpression of a USF2 truncated mutant lacking the first 220 residues (U2Delta1-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U2Delta1-220 blocked the forskolin-dependent induction of PGHS-2 mRNA in granulosa cells, whereas transfections with full-length USF2 increased PGHS-2 transcript levels. Immunoblot analyses confirmed overexpression of full-length and truncated USF proteins, and electrophoretic mobility shift assays (EMSAs) and supershift EMSAs established that the observed effects were dependent on specific interactions between USF proteins and the consensus E-box cis-element. Stimulation of cells with forskolin increased, whereas treatment of extracts with phosphatase decreased USF binding activities to the E-box. Thus, this study presents for the first time direct evidence for a role of USF proteins in the regulation of the PGHS-2 promoter in preovulatory granulosa cells.

Induction of hyaluronan synthase 2 by human chorionic gonadotropin in mural granulosa cells of equine preovulatory follicles.

In contrast to other species, the preovulatory rise in gonadotropins in mares causes a remarkable expansion of the entire granulosa cell layer in vivo, suggesting that hyaluronan (HA) synthesis may be regulated in mural granulosa cells in this species. The objectives of this study were to clone and characterize equine hyaluronan synthase 2 (HAS2) and investigate the regulation of its transcript and of HA synthesis in equine follicles during human chorionic gonadotropin (hCG)- induced ovulation. Results showed that the equine HAS2 cDNA contains a 5'-untranslated region of 436 bp, an open reading frame of 1659 bp, and a 3'-untranslated region of 707 bp. The open reading frame encodes a 552-amino acid protein that is highly conserved (98-99% identity), compared with other known mammalian homologs. The regulation of HAS2 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h after an ovulatory dose of hCG and in corpora lutea obtained on d 8 of the estrous cycle. Results from semiquantitative RT-PCR/Southern blotting analyses revealed a transient induction of HAS2 during the ovulatory process. Levels of HAS2 transcripts were undetectable in follicles before hCG treatment (0 h), increased markedly after gonadotropin treatment (P < 0.05), but returned to undetectable levels in corpora lutea. Analyses performed on isolated preparations of theca interna and granulosa cells showed that the granulosa cell layer was the predominant site of HAS2 expression. An immunohistochemical approach showed that this induction of HAS2 transcript was accompanied by a dramatic increase in HA production after hCG treatment. The isolation and characterization of a 1.8-kb fragment of genomic sequence located immediately upstream of equine HAS2, and comparison with corresponding human and mouse genomic regions identified several conserved putative cis-acting elements. Thus, this study describes the primary structure of equine HAS2, demonstrates for the first time the regulation of HAS2 in mural granulosa cells during the ovulatory process in vivo and identifies a valuable model in which to study the molecular control of HAS2 gene expression.

Molecular characterization of canine prostaglandin G/H synthase-2 and regulation in prostatic adenocarcinoma cells in vitro.

Induction of PG G/H synthase-2 (PGHS-2), a key rate-limiting enzyme in the PG biosynthetic pathway, has been implicated in prostatic adenocarcinomas in humans and dogs in vivo, but the molecular control of PGHS-2 expression in prostate cancer remains poorly understood. Using the dog model, the specific objectives of this study were to clone and characterize canine PGHS-2, and to study the regulation of its transcript, protein, and activity in a canine prostatic adenocarcinoma (CPA) cell line in vitro. The canine PGHS-2 cDNA was cloned by a combination of cDNA library screening and 5'-rapid amplification of cDNA ends, and shown to contain a 5'-untranslated region of 28 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1655 bp. The open reading frame encodes a 604-amino acid protein that is 89% identical to the human homolog. The regulation of PGHS-2 protein and PGE(2) synthesis was studied in CPA cells cultured in the absence or presence of graded doses of phorbol 12-myristate 13-acetate (PMA), TNFalpha, and lipopolysaccharides. Results from immunoblots, immunocytochemistry, and RIAs showed that PGHS-2 protein and PGE(2) were present at low levels in control cells and were significantly induced after agonist treatment (P < 0.05), with PMA being the strongest inducer. Northern blot analyses also revealed a significant increase of PGHS-2 mRNA by PMA, TNFalpha, and lipopolysaccharides treatment (P < 0.05). Agonist-dependent induction of PGHS-2 mRNA was not dependent on new protein synthesis (coincubation with cycloheximide; 10 microg/ml) but was blocked by transcription inhibitor actinomycin D (5 microg/ml), suggesting that PGHS-2 acts an immediate early-response gene in prostatic epithelial cells. Thus, this study characterizes for the first time the structure of canine PGHS-2 and provides an in vitro model to unravel the molecular basis of PGHS-2 expression in prostatic adenocarcinomas.