Immunohistochemical evidence identifying the site of androgen production in the ovary of the protogynous grouper <Emphasis Type="Italic">Epinephelus merra</Emphasis>

Androgen plays an important role in the developing ovaries of female fish. However, little is known regarding either the sites of production of androgen or its functional roles. In the present study, we investigated immunohistochemically the localization of cholesterol-side-chain-cleavage (P450scc) and cytochrome P45011-hydroxylase (P45011) with antibodies P450scc and P45011 in the ovary of the female honeycomb grouper Epinephelus merra during its reproductive cycle. Clusters of strongly immunopositive cells, with 100–1000 cells in each cluster, against both P450scc and P45011, were observed throughout the annual reproductive cycle in tissue near blood vessels in the tunica ovary surrounding the outer periphery of the ovary. The ultrastructural characteristics of these cells showed that they were steroid-producing cells. In contrast, immunopositive cells against P450scc but not against P45011 were localized in the theca layer surrounding the outer periphery of oocytes. These results suggest that two distinct steroid biosynthesis sites exist in the ovary and that cells at the two sites differ functionally. The only cells that biosynthesize 11-ketotestosterone are found in clusters in the vicinity of blood vessels; they possibly play a physiological role in oocyte growth and gonadal restructuring during the sex change of individuals of this species. CREST, JST (Japan Science and Technology Corporation), and the Takeda Science Foundation provided grants supporting this work. This study was also partially supported by a grant for the 21st century COE project entitled, The Comprehensive Analyses of Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions. We also gratefully acknowledge a Japanese Government (Monbukagakusho) Scholarship.     

Immunohistochemical evidence for 11beta-hydroxylase (P45011beta) and androgen production in the gonad during sex differentiation and in adults in the protandrous anemonefish Amphiprion clarkii

To obtain basic information on the endocrine mechanisms underlying sex change in the protandrous anemonefish Amphiprion clarkii, we examined the immunolocalization of the steroidogenic enzyme cytochrome 11beta-hydroxylase (P45011beta), which is involved in 11-ketotestosterone (11-KT) production, and analyzed the ability of gonads to produce steroid hormones throughout the sex differentiation process and at the breeding stage. Immunopositive reactions against P45011beta appeared in sexually undifferentiated gonads at 30 days post hatching (dph). The number of immunopositive cells continued to increase during ovarian differentiation (from 60 to 180 dph) and throughout the formation of ambisexual gonads with both ovarian and testicular tissue until 270 dph. In the male phase, strongly immunopositive cells were observed in the cellular interstices of both testicular and ovarian tissues. P45011beta was localized only in the theca cells enclosing developed oocytes in the female phase. In-vitro 11-KT production in the gonads gradually increased with testicular differentiation (before, during, and after differentiation). Production of 11-KT in the gonads was higher in the male phase than during testicular differentiation or in the female phase. Our results suggest that androgen is involved in testicular differentiation during sex differentiation and spermatogenesis.     

Adrenal P-450scc modulates activity of P-45011 beta in liposomal and mitochondrial membranes. Implication of P-450scc in zone specificity of aldosterone biosynthesis in bovine adrenal.

Bovine adrenal P-45011 beta catalyzes the 11 beta- and 18-hydroxylation of corticosteroids as well as aldosterone synthesis. These activities of P-45011 beta were found to be modulated by another mitochondrial cytochrome P-450 species, P-450scc. The presence together of P-45011 beta and P-450scc in liposomal membranes was found to remarkably stimulate the 11 beta-hydroxylase activity of P-45011 beta and also stimulate the cholesterol desmolase activity of P-450scc. The stimulative effect of P-450scc on 11 beta-hydroxylase activity diminished by the addition of protein-free liposomes to proteoliposomes containing P-45011 beta and P-450scc, thus showing P-450scc to interact with P-45011 beta in the same membranes. Kinetic analysis of this effect indicated the formation of an equimolar complex between P-45011 beta and P-450scc on liposomal membranes. P-45011 beta in the complex had not only stimulated activity for 11 beta- and 18-hydroxylation of 11-deoxycorticosterone but also suppressed activity for production of 18-hydroxycorticosterone and aldosterone. When the inner mitochondrial membranes of zona fasciculata-reticularis from bovine adrenal were treated with anti-P-450scc IgG, aldosterone formation was stimulated to a greater extent than that of zona glomerulosa. This indicates the aldosterone synthesizing activity of P-45011 beta in the zona fasciculata-reticularis to be suppressed by interaction with P-450scc. The zone-specific aldosterone synthesis of P-45011 beta in bovine adrenal may possibly be induced by differences in interactions with P-450scc of mitochondrial membranes in each zone.     

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation in the rainbow trout, Oncorhynchus mykiss.

In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.     

Gonadal restructuring and correlative steroid hormone profiles during natural sex change in protogynous honeycomb grouper (Epinephelus merra)

The honeycomb grouper shows protogynous hermaphroditism. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are largely unknown. In the present study, we investigated changes in the gonadal structures and levels of serum sex steroid hormones during female to male sex change in the honeycomb grouper. On the basis of histological changes, entire process of sex change was assigned into four developmental phases: female, early transition (ET), late transition (LT), and male phase. At the female phase, the oocytes of several developmental stages were observed including gonial germ cells in the periphery of ovigerous lamellae. At the beginning of ET phase, perinucleolar and previtellogenic oocytes began degenerating, followed by proliferation of spermatogonia toward the center of lamella. The LT phase was characterized by further degeneration of oocytes and rapid proliferation of spermatogenic germ cells throughout the gonad. At the male phase, no ovarian cells were observed and testis had germ cells undergoing active spermatogenesis. Serum levels of estradiol-17beta (E2) were high in females in the breeding season, but low in the non-breeding female, transitional and male phase, and those of 11-ketotestosterone (11-KT) and testosterone (T) were low in females and gradually increased in the transitional and male phase. The present results suggest that low serum E2 levels and degeneration of oocytes accompanied by concomitant increase in the 11-KT levels and proliferation of spermatogenic germ cells are probably the events mediating protogynous sex change in the honeycomb grouper.     

Adrenal cortical 11 beta-hydroxylase and side-chain cleavage enzymes. Requirement for the A- or B-pyridyl ring in metyrapone for inhibition

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.     

Electron paramagnetic resonance studies of the purified cytochrome P-450scc and P-45011beta from bovine adrenocortical mitochondria.

Electron paramagnetic resonance studies have been carried out on two species of cytochrome P-450 (P-450scc and P-45011beta) purified from bovine adrenocortical mitochondria. The g values of the steroid-bound cytochromes in the high spin form were determined at 4.2 degrees K to be 8.07, 3.60 and 1.70 for P-450scc and 8.00, 3.65 and 1.71 for P-45011beta. The E/D values were estimated to be 0.103 for P-450scc and 0.099 for P-45011beta. Either high spin P-450 was converted into the low spin form by the treatment with an NADPH dependent electron donating system and subsequent gel filtration in order to remove the steroid. The g values of the low spin ferric cytochromes were 2.423, 2.247 and 1.914 for P-450scc and 2.430, 2.251 and 1.919 for P-45011beta at 77 degrees K. The values for magnitude of delta/gamma, magnitude of V/gamma and k were 5.69, 5.21 and 1.11 for P-450scc and 5.94, 5.38 and 1.16 for P-45011beta. These studies indicate that there are some differences in the ferric heme environment between P-450scc and P-45011beta.     

Stoichiometry of mitochondrial cytochromes P-450, adrenodoxin and adrenodoxin reductase in adrenal cortex and corpus luteum. Implications for membrane organization and gene regulation

We have estimated the concentrations of cytochromes P-450scc and P-45011 beta and the electron-transfer proteins adrenodoxin reductase and adrenodoxin in the adrenal cortex and corpus luteum using specific antibodies against these enzymes. While in the adrenal cortex the concentrations of these enzymes are relatively constant in different animals and show no significant sex differences, in corpora lutea they vary considerably and can increase at least up to fifty-fold over the levels found in the ovary. The average relative concentrations of adrenodoxin reductase, adrenodoxin and P-450 are 1:3:8 in the adrenal cortex (which has two cytochromes P-450, P-450scc and P-450(11) beta, in equal concentrations) and 1:2.5:3 in the corpus luteum (which has only P-450scc). We further present evidence that the levels of cytochrome c oxidase also show a degree of correlation with the levels of the mitochondrial steroidogenic enzymes.     

Mitochondrial P-450 activities in aldosteronoma tissues

Adrenal P-450 activities were measured by an in vitro reconstitution system from tissues obtained from human aldosteronomata, and the results compared with those of the normal adrenal tissues from patients with Grawitz's tumor. The P-45011 beta activity was significantly increased in adenoma tissue (55.6 +/- 5.3 vs 9.0 +/- 6.2 nmol corticosterone/mg of protein/min in the control tissues, P less than 0.01). P-450scc activity in adrenal adenomata was 13.4 +/- 2.0 nmol pregnenolone/mg of protein/min, significantly higher than control (P less than 0.05). The present results suggest that increased mitochondrial P-450(11 beta) activities may be characteristic of aldosterone-producing adenomata.     

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.     

Flash photolysis studies on the CO complexes of ferrous cytochrome P-450scc and cytochrome P-45011 beta. Effects of steroid binding on the photochemical and ligand binding properties

Upon irradiation by a light flash (100-J), the carbon monoxide complex of cytochrome P-450scc was fully photodissociated in both the presence and absence of cholesterol, while less than 20% of the CO complex was photodissociable with those of deoxycorticosterone-bound and -free forms of cytochrome P-45011 beta. When the quantum yield of the reaction was measured for each photodissociable portion, the values were 0.5 and 1.0 for the substrate-free and -bound forms of cytochrome P-450scc, and 0.03 and 0.8 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. Thus, CO complexes of these enzymes become more photosensitive upon binding with the specific substrates. Steroid binding also affected kinetic constants of reactions between the ferrous enzymes and CO. The rate constants for the CO recombination at 15 degrees C were 2.7 X 10(6) and 2.3 X 10(5) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-450scc, and were 7.0 X 10(5) and 5.4 X 10(3) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. The rate constants for the CO dissociation also decreased upon the steroid bindings. The products of the enzyme reactions, pregnenolone and corticosterone, had similar effects on the kinetic constants. From these findings, we postulate that the binding of a steroid to the substrate site of each enzyme alters the bonding character of CO with the heme-iron, thereby affecting both photochemical and kinetic properties of the CO complex. The nature of the photoindissociable portion of the CO complex of cytochrome P-45011 beta is also discussed.     

Efferent duct differentiation during female-to-male sex change in honeycomb grouper Epinephelus merra

Efferent duct (ED) differentiation was examined histologically during female-to-male sex change in the honeycomb grouper Epinephelus merra . During natural sex change, ED differentiation began with the appearance of slit-like structures between the stromal tissue and the tunica ovary and small oval-shaped spaces within the wall of the ovarian cavity, accompanied by oocyte degeneration and the initiation of spermatogenesis, i.e. the early transitional phase. In the late transitional phase, ED structure formation expanded and further ED differentiation occurred, including the rapid multiplication of spermatogonial germ cells. In sex-changed males, the slit-like structures increased in size, fused with each other and finally formed a well-developed ED. The oval-shaped spaces also increased in size and fused to form an ED. In contrast, during artificial sex change, induced by aromatase inhibitor (AI, 1 mg kg⁻¹), ED differentiation in E. merra was first observed as the appearance of slit-like structures and small oval-shaped spaces in the restructuring gonads in the third week after AI treatment. These were accompanied by oocyte degeneration and the proliferation of gonial germ cells into spermatogonia. In the fifth week, the rapid multiplication of spermatogonial germ cells, increases in 11-ketotestosterone (11-KT), and further differentiation of EDs were observed. Sex-changed males had testes containing sperm in the completely differentiated EDs; the significantly highest levels of 11-KT were observed in the sixth week. Simultaneous increases in 11-KT and initiation of ED differentiation were observed, suggesting a role of 11-KT in ED differentiation during sex change. There were no basic differences in the mechanisms of natural and artificially induced ED differentiation. Two types of structure led to the formation of EDs in two different areas of the newly formed testis during sex change.     

Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes.

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.     

Gonadal structure and P450scc and 3β-HSD immunoreactivity in the gobiid fish Trimma okinawae during bidirectional sex change

The process of sex change in the gobiid fish Trimma okinawae was investigated by gonad histology and immunohistochemistry of two steroidogenic enzymes, P450 cholesterol-side-chain-cleavage (P450scc) and 3-hydroxysteroid dehydrogenase (3-HSD). Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. Females changed sex to male within 7 days, reverting again to female over an 11-day period. In each sexual phase of the females, the 2nd (2DF-M) and 4th (4DF-M) day after the initiation of sex change to male, the males, and 2nd (2DM-F), 4th (4DM-F), and 6th (6DM-F) days after the initiation of reversion from male to female, histological observations were made. In the ovary during the female, 2DF-M, 4DF-M, and 6DM-F phases, both vitellogenic and previtellogenic oocytes were present, but only previtellogenic oocytes were found in the other phases. The testis contained sperm in all phases, but sperm ducts were not visible in the female phase. In the ovary, P450scc immunoreactivity of interstitial cells was strongly or moderately detected, although weak in the male phase. In contrast, P450scc immunoreactivity in thecal cells was found in all but the male and 2DM-F phases. 3-HSD immunoreactive interstitial cells were detected in all phases, but only weakly so in the male and 2DM-F phases. 3-HSD immunoreactive thecal cells were observed in all stages without the male and 2DM-F and 4DM-F phases. In the testis, moderate P450scc and 3-HSD immunoreactivity was regularly found in the Leydig cells in all the phases. These results suggest that functional steroids including testosterone are produced in any sexual phases.     

Sex inversion of sexually immature honeycomb grouper (Epinephelus merra) by aromatase inhibitor

Previous studies have shown that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that the treatments with aromatase inhibitor (AI) cause estrogen depletion and complete sex inversion of pre-spawning females into functional males. In the present study, we examined whether AI causes sex inversion of sexually immature females. Female honeycomb groupers were implanted with various doses of Fadrozole (0, 100, 500 and 1000 microg/fish) in the non-breeding season, and resultant changes in the gonadal structures and the plasma levels of sex steroid hormones (estradiol-17 beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) were examined three months after implantation. Vehicle-implanted groups did not change sex, while 100 and 500 microg AI-implanted groups had turned into transitionals with intersex gonad. In contrast, the highest dose receiving group exhibited both transitional and male phases. Transitional phase gonad had atretic oocytes and spermatogenic germ cells at the late stages of spermatogenesis, while male phase testis contained spermatozoa accumulated in the seminiferous tubules. All males released sperm upon slight pressure on the abdomen. In the AI-implanted fish, plasma levels of E2 decreased in a dose-dependent manner, while the levels of 11-KT were high in the highest dose receiving group. Present results suggest that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that treatments with AI potentially inhibits endogenous E2 production in vivo, causing oocyte degeneration and subsequently the sex inversion from female to male. The Fadrozole could be an important tool for manipulating the sex of hermaphrodite fishes.     

Hydroxylation of steroids in a dienzyme system consisting of cytochrome P-450 and immobilized adrenodoxin

Experimental systems for the hydroxylation of steroids (11-deoxycorticosterone and cholesterol) with reduced electron transfer chain, in which flavoprotein was omitted, were investigated. Incubation of chemically reduced immobilized adrenodoxin either with cytochrome P-45011 beta or cytochrome P-450scc in the presence of substrate of hydroxylation and oxygen yields the specific reaction products, corticosterone or pregnenolone. The catalytic activity of the experimental dienzyme systems proves the possibility of the steroid hydroxylation mechanism based exclusively on dissociation and reassociation of the electron transporting protein complexes.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.     

Intrafollicular concentrations of steroids and steroidogenic enzymes in relation to follicular development in the mare

The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare. Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulatory protein (StAR), P450-side chain cleavage (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase, and aromatase were assessed by semiquantitative Western blot and densitometry. Follicular fluid was assayed for cholesterol concentrations by colorimetric assay and for progesterone, testosterone, and estradiol-17beta concentrations by RIA. Intrafollicular concentrations of progesterone and estradiol-17beta significantly increased in the dominant follicle during growth. After injection of gonadotropin, follicular maturation was characterized by a decrease in estradiol-17beta concentrations and a further increase in progesterone concentrations. Granulosa cells from dominant follicles had increased levels of StAR, P450(scc), 3betaHSD, and aromatase during growth, but decreased levels during maturation. Levels of StAR, P450(scc), 3betaHSD, and aromatase, as well as progesterone and estradiol-17beta, were lower in granulosa cells from subordinate than from dominant follicles. We did not observe a relationship between the steroidogenic activity of follicles and the capacity of their enclosed oocytes to complete meiosis in vitro.