Effects of follicle stimulating hormone on estradiol-17 beta production and P-450 aromatase (CYP19) activity and mRNA expression in brown trout vitellogenic ovarian follicles in vitro

In order to determine whether follicle stimulating hormone (FSH) regulates P-450 aromatase (P-450 arom) in salmonid fish, we investigated the in vitro effects of FSH on estradiol (E(2)) production and P-450 arom activity and expression in brown trout (Salmo trutta) vitellogenic ovarian follicles. Brown trout ovarian follicles were incubated in the presence of coho salmon FSH and the production of E(2) into the medium was measured by RIA, the activity of P-450 arom by the tritiated water release assay and the expression of P-450 arom by Northern blotting using a homologous cDNA probe obtained by RT-PCR. Results from this study indicate that the dose- and time-dependent stimulatory effects of FSH on E(2) production are dependent on new RNA and protein synthesis. The basal and FSH-stimulated E(2) production was completely blocked by fadrozole, a specific aromatase inhibitor. FSH was capable of stimulating P-450 arom activity but this stimulation was only detectable with short incubation times (30 min) since longer incubation times with FSH resulted in the inhibition of P-450 arom activity. In addition, FSH increased the steady-state P-450 arom mRNA levels. In conclusion, our results indicate, for the first time in teleost fish, that FSH stimulates the expression of P-450 arom, as well as its activity, albeit after a short-term treatment with FSH, and that FSH plays a fundamental role in the regulation of the production of E(2) in the salmonid ovary.     

Immunohistochemical localization of 17 alpha-hydroxylase/C17-20 lyase and aromatase cytochrome P-450 in the human ovary during the menstrual cycle.

Immunohistochemical localization of 17 alpha-hydroxylase/C17-20 lyase (P-450(17 alpha,lyase)) and aromatase cytochrome P-450 (P-450arom) in normal human ovaries during the menstrual cycle was studied using specific polyclonal antibodies which were raised against corresponding enzymes. In the follicular phase of matured follicles, P-450(17 alpha,lyase) was localized in theca interna cells and P-450arom in granulosa cells. P-450(17 alpha,lyase) was expressed in theca interna cells before P-450arom was expressed in granulosa cells. The corpus luteum showed immunoreactivity to both enzymes and, after menstruation, immunoreactivity decreased gradually until it could not be detected in the corpus albicans. In corpus luteum graviditatis the immunoreactivity continued to be expressed strongly. In some atretic follicles, P-450(17 alpha,lyase) and/or P-450arom continued to be expressed. In the stromal layer, P-450(17 alpha,lyase was detected in secondary interstitial cells, which originated from the theca interna of atretic follicles, and P-450arom was detected in hilar cells. Immunoreactivity to both enzymes was also detected in oocytes of developing follicles. These results are consistent with the two cell theory in the human ovary. They also suggest that androgens and oestrogens are produced not only by follicles and corpora lutea but also by stroma and oocytes.     

Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro

The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence.     

Oestrogen formation and connective tissue growth factor expression in rat granulosa cells

Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450(aromatase) (P450(arom))) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450(arom) mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450(arom) mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450(arom). Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450(arom). Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSH-stimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10(-7)-10(-5) M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450(arom) diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.     

Stimulation of salmon calcitonin on secretion of 17beta-estradiol by the ovarian follicles of common carp, Cyprinus carpio

The effects of salmon calcitonin (sCT) on the secretion of 17beta-estradiol (E(2)) were examined in female common carp, Cyprinus carpio. Vitellogenic stage fish adapted to high-Ca water were i.p. injected with vehicle, sCT, human chorionic gonadotropin (hCG), or hCG plus sCT. To determine whether ovarian follicles are equipped with CT receptors, a CT binding assay was conducted. In the in vitro experiments, vitellogenic follicles were incubated with stimulators and inhibitors. Administration of sCT increased the basal and hCG-stimulated E(2) release in vivo and in vitro. Binding characteristics of [(125)I]sCT to plasma membrane preparation of carp ovarian follicles showed saturability with high-affinity (K(d)=48.48 pmol/l and B(max)=1.2 pmol/mg protein). To clarify the mechanism of E(2) production by sCT, in vitro effect of sCT and hCG on aromatase activity (conversion of testosterone to E(2)) and cytochrome P450 aromatase (P450arom) gene expression in carp ovarian follicles were investigated. Salmon CT-stimulated both aromatase activity and P450arom gene expression in ovarian follicles of carp. sCT-stimulated E(2) release by the ovarian follicles in vitro was augmented in the presence of dibutyryl cAMP. Inhibitor of protein kinase A (PKA), SQ 22536 inhibited sCT-stimulated steroid production in a dose-dependent manner. Specific inhibitor of protein kinase C (PKC), NPC-15437 dihydrochloride had no inhibitory effects on sCT-induced E(2) release. The present study indicates that sCT binds specifically to carp ovary and stimulates E(2) production by increasing the activity of cytochrome P450 aromatase and P450arom gene expression. The results further suggest that stimulatory action of sCT on E(2) production is mediated through cAMP pathway.     

Expression of P450 aromatase protein in developing and in sex-reversed gonads of the XX/XY type of the frog Rana rugosa

Gonadal differentiation in some species of amphibians is sensitive to steroids. The phenotypic sex of XX/XY-type frogs such as Rana rugosa can be reversed from female to male by injection of testosterone into tadpoles, but little is known about the molecular mechanism of this sex reversal. To elucidate the mechanism of the sex differentiation, we examined the role of P450 aromatase (P450arom), an enzyme that converts testosterone to estrogen, during gonadal differentiation of amphibians. In this study, we first cloned a P450arom cDNA homolog of the frog R. rugosa and analyzed by RT-PCR its expression profile in developing and in female-to-male sex-reversed gonads. P450arom expression was observed in the gonad of tadpoles during ovarian differentiation and became much stronger in the developing ovary in which only immature oocytes were observed. However, its expression declined significantly in the ovary of frogs 2 months after metamorphosis, when oocytes were growing; and it was no longer seen in adult ovaries. By RT-PCR, we also examined the expression of P450arom and SF-1 (steroidogenic factor-1; the orphan nuclear receptor) in the female-to-male sex-reversed gonad. The level of P450arom mRNA was high in the ovary, but it declined rapidly after the injection of testosterone. In contrast, no change in the SF-1 (also known as Ad4BP) expression was observed. Moreover, to identify the type(s) of cells expressing P450arom protein, we performed immunostaining with an antibody against frog P450arom protein. Cells giving positive signals were observed around oocytes in the ovary of frogs 1 month after metamorphosis. They were identified as follicle cells by both light and electron microscopy. The results, taken together, indicate that P450arom protein is synthesized in follicle cells and that P450arom is very much involved in ovarian differentiation in R. rugosa.     

Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle

To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes.     

Increasing aromatase cytochrome P-450 level in human placenta during pregnancy: studied by immunohistochemistry and enzyme-linked immunosorbent assay.

We investigated the immunohistochemical localization of aromatase cytochrome P-450 (P-450arom) using a specific polyclonal antiserum (PAb R-8-2). We compared catalytic activity, as detected by the tritiated water assay, and tissue levels of P-450arom, as detected by the specific enzyme-linked immunosorbent assay, in placental samples from early pregnancy to term. Immunostaining and subsequent detection by light and electron microscopy demonstrated that P-450arom is localized in the microvilli and endoplasmic reticulum of the syncytiotrophoblasts of the chorionic villi, but is not present in the mitochondria, nuclei, or cytotrophoblasts at any time during gestation. The P-450arom concentration and aromatase activity were greater in the microsomal fraction than in the mitochondrial fraction or total homogenate at each gestational stage and increased linearly as pregnancy progressed. However, the specific activity of P-450arom was comparable among subcellular fractions in each gestational period. These results suggest that the nature and localization of P-450arom are unchangeable, and the P-450arom concentration increases during pregnancy. It appears that the aromatase detected in the mitochondrial fraction is a contamination of microsomal aromatase, and the increase in aromatase activity can be attributed to an increase in the number of P-450arom molecules rather than an increase in the catalytic ability of each molecule.     

Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis

The following study was undertaken to compare the content of aromatase cytochrome P450 (P450arom) mRNA with the content of the enzyme in rat ovarian tissues and to relate these changes with estradiol biosynthesis by follicles and corpora lutea isolated throughout pregnancy. A deoxyoligonucleotide (62 mer) probe derived from an amino acid sequence of purified human placental P450arom was used to screen a rat granulosa cell lambda gt11 cDNA expression library. Seven cDNA clones, ranging in size from 0.6-2.0 kilobases (kb), were identified and plaque purified. In vitro translation using mRNA that had been selected by hybridization to a 1.2-kb rat P450arom cDNA insert yielded an 35S-labeled translation product that bound antihuman aromatase immunoglobulin and comigrated with purified human placental aromatase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thus verifying that the clones do encode for P450arom. Using the 1.2-kb cDNA insert as a radiolabeled probe, the hormonal regulation, tissue distribution, content, and size of mRNA for P450arom were analyzed. Filter hybridization assays demonstrated that P450arom mRNA was low in small antral (SA) follicles, increased 16-fold in preovulatory (PO) follicles, and reached a peak in granulosa cells within 1 h after an ovulatory dose of hCG. In the corpus luteum of pregnancy, P450arom mRNA content was low on day 4, and increased 3-fold on days 7-11 and 10-fold on days 15-19 of gestation. P450arom mRNA then decreased on days 21 and 23, the day of parturition. Northern analyses of RNA from PO follicles and corpora lutea revealed three bands of P450arom mRNA that were 3.3, 2.6, and 1.9 kb in size. Immunoblots of soluble cell extracts of SA, PO, and luteinizing (PO plus hCG) follicles and corpora lutea of pregnancy demonstrated that aromatase enzyme was low in SA follicles, increased 1.5- to 3-fold in PO follicles, and decreased within 3-5 h after an ovulatory dose of hCG. Changes in the content of P450arom enzyme in luteal cells during pregnancy exhibited a pattern similar to that observed for P450arom mRNA. In contrast, changes in estradiol biosynthesis by follicles and corpora lutea were not directly related to the contents of P450arom mRNA and enzyme. For example, although corpora lutea isolated on days 15-21 of gestation contain the highest amount of P450arom mRNA and enzyme, these tissues did not produce the most estradiol when incubated for 5 h at 37 C in the presence of aromatizable androgen substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell-specific expression of immunoreactive cholesterol side-chain cleavage cytochrome P-450 during follicular development in the rat ovary

Using a specific antiserum against rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc), we examined the expression of this key steroidogenic enzyme during follicular development in PMSG-treated immature rats. The accumulation of the enzyme was monitored in ovary homogenates by quantitative immunodot blot assay, while expression of P-450scc in various cell types was visualized concomitantly by immunofluorescent staining of ovarian cryosections. Before PMSG treatment, no labeling of P-450scc could be observed in follicular granulosa cells. In contrast, steroidogenic cytochrome was markedly expressed in interstitial cells, part of theca interna cells, and hypertrophied theca of atretic follicles. As a result of PMSG treatment, the interstitial thecal cells promptly enriched their P-450scc content within 24 h, whereas the granulosa cells acquired the enzyme at a later time, between 30 and 48 h after hormone administration. After ovulation, many corpora lutea filled most of the ovarian volume, and the ovarian content of P-450scc was 47 times higher than that in control ovaries of untreated rats. In granulosa cell population of a single preovulatory follicle, a downward gradient of P-450scc expression was observed, starting high in the cells abutting the basal lamina and decreasing toward the cells lining the antrum. Cumulus cells failed to express P-450scc. Referring to the basal lamina, theca interna cells exhibited a reverse gradient of P-450scc expression, starting high in peripheral cells close to the theca externa layer and decreasing in cells located near the follicular basement membrane. Immunofluorescent labeling revealed a major difference between P-450scc expression in thecal cells compared to that in granulosa cells. While expression of P-450scc in granulosa cells was restricted exclusively to cells within preovulatory follicles, P-450scc labeling was observed throughout the ovary in thecal and interstitial cells associated with follicles at any phase of follicular maturation. Therefore, it may be proposed that the thecal and interstitial cells represent an all ovarian network which expresses its steroidogenic capacity at early stages of follicular maturation and thereby is able to supply androgens necessary for the follicular development.