Influence of experimental cryptorchidism on cholesterol side-chain cleavage enzyme and delta5-3beta-hydroxysteroid dehydrogenase activities in rat testes.

Testicular cholesterol side-chain cleavage enzyme (CSCCE) and delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD) activities were assessed 12 hours and 2, 4, 8, 16, and 32 days after surgical induction of bilateral cryptorchidism in adult rats. Within 12 hours after surgery CSCCE activity (expressed as dpm of isocaproic acid-14C formed from cholesterol-26-14C/3 hours/testis) was significantly reduced (P less than 0.01) in cryptorchid testes to approximately 55% of sham-operated control values and remained depressed at less than 50% of control activities 2, 4, 16, and 32 days after surgery. Cryptorchid testis delta5-3beta-HSD activity (measured by a pregnenolone substrate-depletion assay and expressed as mumoles of products/30 minutes/testis) did not differ from controls (P greater than 0.05) 1/2, 2, or 4 days after translocation of testes to the abdominal cavity. By day 8 of cryptorchidism, however, delta5-3beta-HSD activity was reduced to 60% of control values (P less than 0.05) and continued to decline to approximately 30% of controls during the remainder of the experimental period. These observed alterations in enzyme activities suggest an impairment in the ability of cryptorchid rat testes to synthesize androgens and further indicate that testicular CSCCE is more acutely sensitive to the cryptorchid milieu than delta5-3beta-HSD.     

Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles

The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles. Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM). Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration. Northern blot analysis revealed a single band approximately 2.1 kb in size. Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels. Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH. Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar. In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles. These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion. This last observation indicates that control at sites other than SCC mRNA can affect progesterone production.     

Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro

We determined 1) whether the previously observed induction of estradiol secretion in bovine granulosa cells cultured in serum-free conditions is associated with an increase in cytochrome P450 aromatase (P450(arom)) mRNA abundance and 2) whether P450(arom) mRNA levels are responsive to FSH in vitro. Granulosa cells from small (2-4-mm) follicles were cultured in serum-free medium. Estradiol secretion increased with time in culture and was correlated with increased P450(arom) mRNA abundance. Progesterone secretion also increased with time in culture, but P450 cholesterol side-chain cleavage (P450(scc)) mRNA abundance did not. FSH stimulated estradiol secretion and P450(arom) mRNA abundance; the effect was quadratic for both estradiol and P450(arom) mRNA. Estradiol secretion and P450(arom) mRNA levels were correlated. FSH stimulated progesterone secretion and P450(scc) mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450(arom) mRNA levels but not progesterone or P450(scc) mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450(arom) mRNA abundance responsiveness to FSH and insulin, whereas P450(scc) mRNA abundance and progesterone secretion were responsive to FSH but not insulin.     

Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4),delta(5) isomerase compared to normal dominant follicles

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, cytochrome P450 aromatase, 3 beta-hydroxysteroid dehydrogenase Delta(4),Delta(5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.     

Localization of immunoreactive testosterone and 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in cynomolgus monkey (Macaca fascicularis) testes during postnatal development.

The age-related expression of testosterone and 3beta-HSD in the testes of cynomolgus monkeys was detected using light-microscopic immunocytochemistry. Intense deposits of immunoreactive testosterone were labeled in parts of Leydig cells in neonatal, late infantile, pubertal, and adult testes, and only a few Leydig cells in early infantile testes. The immunoreactive 3beta-HSD was labeled in parts of Leydig cells and in all Sertoli cells in neonatal, late infantile, pubertal, and adult testes, whereas only a few Leydig cells, but no Sertoli cells, were labeled in early infantile testes. The fluctuations of testosterone and 3beta-HSD expression in testes correlated well with those already observed plasma testosterone levels during postnatal development in cynomolgus monkeys.     

Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3beta-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.     

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc)-regulated steroidogenesis as an organ-specific molecular and cellular target for endocrine disrupting chemicals in fish

Biologically active steroids are synthesised de novo in specialised cells of several organs, including the adrenal gland, testis, ovary, brain, placenta and adipose tissue. Regardless of organ or tissue, the rate-limiting step in steroid hormone synthesis is the movement of cholesterol across the mitochondrial membrane (i.e. from the outer to the inner membrane) mediated by the steroidogenic acute regulatory (StAR) protein. Subsequent conversion of cholesterol to pregnenolone by cytochrome P450 side-chain cleavage (P450scc) represents the initiation of steroidogenesis. Chemically mediated disruption of StAR and P450scc expression may represent the first step in the sequence of related event cascades underlying xenoestrogen-induced toxicity and transmittable disturbances to the whole organism level. This may include, but is not limited to, alterations in sexual differentiation, growth, reproduction, development and metabolism. Despite the integral role of StAR and P450scc in acute steroidogenesis, and popular demand from regulatory agencies, bioassays for evaluating the effect of endocrine-disrupting chemicals have the potential to overlook chemicals that may modulate estrogenic responses through mechanisms that do not involve direct binding to estrogen receptors (ERs). In addition to their effect as direct ER agonists, the effects of endocrine disruptors may be evaluated and interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of juvenile, male and female individuals. Knowledge of these effects is scarce, indicating that relatively little is known about the mechanisms or mode-of-action of chemical alterations to steroidogenesis and their potential toxicity for wildlife species. In addition, analytical methods for the complete adaptation of these responses as biomarkers of response and effect are yet to be properly validated.     

Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.