Carlos Stocco

Aromatase expression in the ovary: Hormonal and molecular regulation

Estrogens are synthesized by the aromatase enzyme encoded by the Cyp19a1 gene, which contains an unusually large regulatory region. In most mammals, aromatase expression is under the control of two distinct promoters a gonad- and a brain-specific promoter. In humans, this gene contains 10 tissue-specific promoters that are alternatively used in various cell types and tumors. Each promoter is regulated by a distinct set of regulatory sequences and transcription factors that bind to these specific sequences. The cAMP/PKA/CREB pathway is considered to be the primary signaling cascade through which the gonad Cyp19 promoter is regulated. Very interestingly, in rat luteal cells, the proximal promoter is not controlled in a cAMP dependent manner. Strikingly, these cells express aromatase at high levels similar to those found in preovulatory follicles, suggesting that alternative and powerful mechanisms control aromatase expression in luteal cells and that the rat corpus luteum represents an important paradigm for understanding alternative controls of the aromatase gene. Here, the molecular and cellular mechanisms controlling the expression of the aromatase gene in granulosa and luteal cells are discussed.

Prostaglandin F2α-induced Expression of 20α-Hydroxysteroid Dehydrogenase Involves the Transcription Factor NUR77

Prostaglandin F2α (PGF2α) binding to its receptor on the rat corpus luteum triggers various signal transduction pathways that lead to the activation of a steroidogenic enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), which in turn catabolizes progesterone. The molecular mechanism underlying PGF2α-induced 20α-HSD enzyme activity has not yet been explored. In this report we show, using mice lacking PGF2α receptor and pregnant rats, that PGF2α is responsible for the rapid and massive expression of the 20α-HSD gene at the end of pregnancy leading to a decrease in progesterone secretion. We also present evidence that PGF2α enhances 20α-HSD promoter activity. We have determined a region upstream of the −1590 position in the 20α-HSD promoter that confers regulation by PGF2α in ovarian primary cells. This region encompasses a unique transcription factor-binding site with a sequence of a NUR77 response element. Deletion of this motif or overexpression of a NUR77 dominant negative protein caused a complete loss of 20α-HSD promoter activation by PGF2α. NUR77 also transactivated the 20α-HSD promoter in transient transfection experiments in corpus luteum-derived cells (GG-CL). This induction required the NUR77-transactivating domain. We also show that PGF2α induces a very rapid expression of NUR77 that binds to a distal response element located at −1599/−1606 but does not interact with another proximal putative NUR77 response element located downstream in the promoter. A rapid increase in NUR77 mRNA was observed in mice corpora lutea just before parturition at a time when 20α-HSD becomes expressed. This increase in the expression of both genes was not seen in PGF2α receptor knockout mice. By using cyclosporin A and PGF2α treatment, we established that inhibition of NUR77 DNA binding in vivo prevents PGF2α induction of the 20α-HSD gene in the corpus luteum. Taken together, our results demonstrate, for the first time, that PGF2α induces in the corpus luteum the expression of the nuclear orphan receptor and transcription factor, NUR77, which in turn leads to the transcriptional stimulation of 20α-HSD, triggering the decrease in serum progesterone essential for parturition.

Tissue physiology and pathology of aromatase

Aromatase is expressed in multiple tissues, indicating a crucial role for locally produced oestrogens in the differentiation, regulation and normal function of several organs and processes. This review is an overview of the role of aromatase in different tissues under normal physiological conditions and its contribution to the development of some oestrogen-related pathologies.

Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy.

In this study, we investigated whether progesterone exerts a local action regulating the function of the corpus luteum of pregnancy in rats. The luteal activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD), involved in progesterone biosynthesis, and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), that catabolizes progesterone and reduces progesterone secretion by the corpus luteum, were evaluated after intrabursal ovarian administration of progesterone in pregnant rats that had received a luteolytic dose of prostaglandin F2alpha (PGF2alpha). Luteal 3beta-HSD activity decreased and 20alpha-HSD activity increased after PGF2alpha treatment (100 microg x 2 intraperitoneally on Day 19 of pregnancy at 12:00 p.m. and 4:00 p.m.) when compared with controls sacrificed at 8:00 p.m. on Day 20 of pregnancy. This effect of PGF2alpha on the luteal 3beta-HSD and 20alpha-HSD activities was abolished in animals that also received an intraovarian dose of progesterone (3 microg/ovary on Day 19 of pregnancy at 8:00-9:00 a.m.). In a second functional study, luteal cells obtained from 19-day pregnant rats responded to the synthetic progestin promegestone (R5020) in a dose-dependent manner, with an increase in the progesterone output. In addition, the glucocorticoid agent hydrocortisone did not affect progesterone accumulation in the same luteal cell culture. We also examined by immunocytochemistry the expression of progesterone receptors (PR) in the corpora lutea during pregnancy and demonstrated the absence of PR in this endocrine gland in all the days of pregnancy studied. In the same pregnant rats, positive staining for PR was observed in cells within the uteroplacental unit, such as cells of the decidua basalis and trophoblast giant cells of the junctional zone. In addition, positive PR staining was observed in the ovarian granulosa and theca cells of growing follicles, but not in corpora lutea of ovaries obtained from cycling rats at proestrus. In summary, this report provides further evidence of a local action of progesterone regulating luteal function in the rat despite the absence of a classic PR.

Opposite Effect of Prolactin and Prostaglandin F2α on the Expression of Luteal Genes as Revealed by Rat cDNA Expression Array

It is well established that prolactin (PRL) sustains, while prostaglandin F2α (PGF2α) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20α-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF2α. In addition to 20α-HSD, PGF2α stimulated and PRL inhibited PGF2α-receptor, phospholipase Cδ1 and TGFβ1 expression. In contrast PRL stimulated and PGF2α inhibited the LH receptor, 11β-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GSTμ2, inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF2α. PGF2α stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-β, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF2α also up-regulated the expression of the sodium channel β1,...

IGF-I Signaling Is Essential for FSH Stimulation of AKT and Steroidogenic Genes in Granulosa Cells

FSH and IGF-I synergistically stimulate gonadal steroid production; conversely, silencing the FSH or the IGF-I genes leads to infertility and hypogonadism. To determine the molecular link between these hormones, we examined the signaling cross talk downstream of their receptors. In human and rodent granulosa cells (GCs), IGF-I potentiated the stimulatory effects of FSH and cAMP on the expression of steroidogenic genes. In contrast, inhibition of IGF-I receptor (IGF-IR) activity or expression using pharmacological, genetic, or biochemical approaches prevented the FSH- and cAMP-induced expression of steroidogenic genes and estradiol production. In vivo experiments demonstrated that IGF-IR inactivation reduces the stimulation of steroidogenic genes and follicle growth by gonadotropins. FSH or IGF-I alone stimulated protein kinase B (PKB), which is also known as AKT and in combination synergistically increased AKT phosphorylation. Remarkably, blocking IGF-IR expression or activity decreased AKT basal activity and abolished AKT activation by FSH. In GCs lacking IGF-IR activity, FSH stimulation of Cyp19 expression was rescued by overexpression of constitutively active AKT. Our findings demonstrate, for the first time, that in human, mouse, and rat GCs, the well-known stimulatory effect of FSH on Cyp19 and AKT depends on IGF-I and on the expression and activation of the IGF-IR.

Loss of GATA-6 and GATA-4 in Granulosa Cells Blocks Folliculogenesis, Ovulation, and Follicle Stimulating Hormone Receptor Expression Leading to Female Infertility

Single GATA-6 (G6(gcko)), GATA-4 (G4(gcko)), and double GATA-4/6 (G4/6(gcko)) granulosa cell-specific knockout mice were generated to further investigate the role of GATA transcription factors in ovarian function in vivo. No reproductive defects were found in G6(gcko) animals. G4(gcko) animals were subfertile as indicated by the reduced number of pups per litter and the release of significantly fewer oocytes at ovulation. In marked contrast, G4/6(gcko) females fail to ovulate and are infertile. Furthermore, G4/6(gcko) females had irregular estrous cycles, which correlate with the abnormal ovarian histology found in unstimulated adult G4/6(gcko) females showing lack of follicular development and increased follicular atresia. Moreover, treatment with exogenous gonadotropins did not rescue folliculogenesis or ovulation in double-knockout G4/6(gcko) mice. In addition, ovary weight and estradiol levels were significantly reduced in G4(gcko) and G4/6(gcko) animals when compared with control and G6(gcko) mice. Aromatase, P450scc, and LH receptor expression was significantly lower in G4(gcko) and G4/6(gcko) mice when compared with control animals. Most prominently, FSH receptor (FSHR) protein was undetectable in granulosa cells of G4(gcko) and G4/6(gcko). Accordingly, gel shift and reporter assays revealed that GATA-4 binds and stimulates the activity of the FSHR promoter. These results demonstrate that GATA-4 and GATA-6 are needed for normal ovarian function. Our data are consistent with a role for GATA-4 in the regulation of the FSHR gene and provide a possible molecular mechanism to explain the fertility defects observed in animals with deficient GATA expression in the ovary.

Testosterone, not 5α-dihydrotestosterone, stimulates LRH-1 leading to FSH-independent expression of Cyp19 and P450scc in granulosa cells.

Androgens are crucial for normal folliculogenesis and female fertility as evidenced in androgen receptor-null and granulosa cell conditional knockout mice. It is thought, however, that the multiple effects of androgens in the ovary are mainly complementary to the actions of gonadotropins. Using primary rat granulosa cells, we demonstrated that in the absence of gonadotropins, testosterone (T) increases aromatase (Cyp19) and P450 side-change cleavage expression, two enzymes crucial for normal ovarian function. T can be converted into estradiol, a classical estrogen, by Cyp19 and into 5α-dihydrotestosterone, a pure androgen, by 5α-reductase. However, inhibition of Cyp19 and/or 5α-reductase did not prevent the stimulatory effects of T. In contrast, the effect of this steroid was potentiated by blocking 5α-reductase. Additionally, T, not 5α-dihydrotestosterone, stimulates liver receptor homolog-1 (LRH-1) expression, whereas the expression of steroidogenic factor-1 (SF-1) was not affected by either steroid. LRH-1 and SF-1 are transcription factors known to be involved in the regulation of Cyp19. Accordingly, small interference RNA against LRH-1 prevented Cyp19 and P450 side-change cleavage up-regulation whereas anti-SF-1 small interference RNA had no effects. Chromatin immunoprecipitation demonstrated that T stimulation of LRH-1 leads to the recruitment of LRH-1 to the native Cyp19 promoter, which was not affected by cotreatment with 5α-reductase and Cyp19 inhibitors. Finally, gel shift and supershift analysis demonstrated that the androgen receptor binds to an androgen response element located within the LRH-1 promoter. These results provide novel evidence that T has a direct effect on the expression of genes involved in granulosa cell differentiation.

Prostaglandin E2 increases cyp19 expression in rat granulosa cells: Implication of GATA-4

The expression of Cyp19, the key gene of estrogen biosynthesis, in granulosa cells (GC) is essential for follicular growth and coordination of the ovulatory process. The goal of this study was to examine the effect of PGE2 and PGF2alpha on Cyp19 expression in undifferentiated and luteinized GC (UGC and LGC). In UGC, PGE2 increased Cyp19 mRNA and Cyp19 protein levels whereas PGF2alpha had no effect. In LGC, PGF2alpha decreased Cyp19 expression whereas PGE2 had no effect. Gene-reporter experiments demonstrated that PGE2 increases Cyp19 transcription in UGC. A protein kinase A inhibitor blocked PGE2-induced increase in Cyp19 promoter activity. PGE2 increased GATA-4 binding to the Cyp19 promoter. Mutation of the GATA binding site resulted in the loss of PGE2 stimulation. This study demonstrates that PGE2 stimulates Cyp19 expression in rat GC and suggests that GATA-4 may mediate (at least in part) the stimulatory effect of PGE2.

Luteal Expression of Cytochrome P450 Side-Chain Cleavage, Steroidogenic Acute Regulatory Protein, 3β-Hydroxysteroid Dehydrogenase, and 20α-Hydroxysteroid Dehydrogenase Genes in Late Pregnant Rats: Effect of Luteinizing Hormone and RU486

Abstract A decrease in serum progesterone at the end of pregnancy is essential for the induction of parturition in rats. We have previously demonstrated that LH participates in this process through: 1) inhibiting 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and 2) stimulating progesterone catabolism by inducing 20α-hydroxysteroid dehydrogenase (20α-HSD) activity. The objective of this investigation was to determine the effect of LH and progesterone on the luteal expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), 3β-HSD, and 20α-HSD genes. Gene expression was analyzed by Northern blot analysis 24 and 48 h after administration of LH or vehicle on Day 19 of pregnancy. StAR and 3β-HSD mRNA levels were lower in LH-treated rats than in rats administered with vehicle at both time points studied. P450scc mRNA levels were unaffected by LH. The 20α-HSD mRNA levels were not different between LH and control rats 24 h after treatment; however, greater expression of 20α-HSD, with respect to controls, was observed in LH-treated rats 48 h after treatment. Luteal progesterone content dropped in LH-treated rats at both time points studied, whereas serum progesterone decreased after 48 h only. In a second set of experiments, the anti-progesterone RU486 was injected intrabursally on Day 20 of pregnancy. RU486 had no effect on 3β-HSD or P450scc expression but increased 20α-HSD mRNA levels after 8 h treatment. In conclusion, the luteolytic effect of LH is mediated by a drop in StAR and 3β-HSD expression without effect on P450scc expression. We also provide the first in vivo evidence indicating that a decrease in luteal progesterone content may be an essential step toward the induction of 20α-HSD expression at the end of pregnancy in rats.