Steroid metabolism in granulosa and theca interna cells from preovulatory follicles of domestic hen (Gallus domesticus)

The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P4, but we also observed low amounts of 5β-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5β-androstan-3,17-dione (5β-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5β-dione, whereas T was only transformed into A4. On the other hand, P5 metabolism by theca interna cells yielded A4 as the main product, also P4, 17α-OHP4, 17α-OHP5, 5β-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5β-dione. A4 was mainly transformed into 5β-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3β-hydroxysteroid dehydrogenase/5-4 isomerase (3β-HSD from P5 and DHEA), 17β-hydroxysteroid dehydrogenase (17β-HSD from T) and 5β-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3β-HSD (from P5 and DHEA), 17β-HSD (from T) and 5β-reductase (from P4, DHEA and A4). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.     

Ovarian steroidogenesis during follicular maturation in the domestic fowl (Gallus domesticus)

The steroidogenic potential of various physiological compartments within the ovary of the hen were examined using in vitro systems. Three-hour incubations of individual whole small follicles (less than 1 mm-1 cm) or 100,000 collagenase-dispersed theca cells of the five largest ovarian follicles (F1-F5) were conducted in 1 ml of Medium 199 at 37 degrees C in the presence and absence of luteinizing hormone (LH) (0.39, 0.78, 1.56, 3.13 and 6.25 ng), progesterone (5 ng), and dehydroepiandrosterone (DHEA, 5 ng). Steroid output was measured by radioimmunoassay of incubation media. Progesterone was not produced by small follicles although they are a major source of DHEA and estradiol and a significant source of androstenedione. Output of DHEA, androstenedione and estradiol was highly stimulated by LH. The substrate for androstenedione and estradiol in small follicles is probably DHEA. Output of DHEA and androstenedione in theca cells of F2-F5 was stimulated by LH in a dose-related manner. A dose-response relationship between estradiol output and the concentration of LH in media was not apparent in theca cells from F2-F5. Steroidogenesis in theca tissue of large follicles occurs predominantly via the delta 4 pathway. The ability of these theca cells to metabolize progesterone to androstenedione is lost between 36 and 12 h before ovulation. Their ability to metabolize DHEA to androstenedione is still present 12 h before ovulation. Aromatase activity is significantly reduced between 36 and 12 h before ovulation. These data indicate that both large and small follicles can be stimulated by LH. The small follicles are the major source of estrogen. As the large yolky follicles mature, steroidogenesis shifts from the delta 5 to the delta 4 pathway. By 12 h before ovulation, the F1 follicle has lost the ability to convert progesterone to androstenedione. The inability of the largest ovarian follicle to convert progesterone to androstenedione contributes at least in part to the preovulatory increase in the plasma concentration of progesterone that generates the preovulatory LH surge by positive feedback.     

Androgen-induced changes in rat ovarian granulosa cells in vitro

Ovarian granulosa cells collected from small antral follicles from immature rats were cultured in McCoy's 5A medium, for 1-6 days in the presence of delta 4-androstenedione, testosterone, dihydrotestosterone, and dehydroepiandrosterone (10(-5) M and 10(-7) M). Granulosa cells examined by electron microscopy demonstrated many lipid droplets, mitochondria with tubular cristae and profiles of smooth endoplasmic reticulum, all suggestive of active metabolism in the cell. Cells cultured in androstenedione, testosterone, dihydrotestosterone and dehydroepiandrosterone produced estrogen and progesterone as measured by radioimmunoassay. By day 4, cells cultured in androgen had almost completely degenerated. The control cells acquired none of the aforementioned characteristics and survived up to beyond 6 days, at which time the experiments were terminated. This study supports the hypothesis that high concentrations of androgens in cultured granulosa cells contribute to their degeneration through altered structure, which is associated with functional change.     

Steroid production from 17alpha-hydroxypregnenolone and dehydroepiandrosterone by human granulosa cells in vitro.

Granulosa cells were aspirated 3--4 h before the expected time of ovulation from 10 follicles of 4 patients treated with gonadotrophins: 4 of the follicles were immediately preovulatory. The granulosa cells were cultured for 10 h with 17alpha-hydroxypregnenolone or dehydroepiandrosterone and samples of medium removed at 3 and 10 h were assayed for 6 steroids. Granulosa cells were unable to synthesize androgens from endogenous substrate or undertake conversions via the delta5 pathway, but cells from all follicles were capable of aromatizing exogenous androgens to oestrogens although this capability was reduced in cells from follicles beginning to luteinize. Granulosa cells from preovulatory follicles synthesized more progesterone from endogenous substrate than cells from follicles which had not begun to luteinize. The results provide further support for the two-cell theory of oestrogen biosynthesis whereby granulosa cells aromatize androgens which are synthesized by the thecal cells in vivo.     

The source of follicular androgens in the hamster follicle

The comparative ability of granulosa cells and theca of the hamster preovulatory follicle to produce androgens in vitro from endogenous and exogenous substrates was assessed. The results indicate that theca are the major source of follicular androstenedione, but that the granulosa cells may be the major source of follicular testosterone. Theca and granulosa cells accumulate comparable amounts of dihydrotestosterone from exogenous androstenedione and testosterone and both may be a significant source of follicular DHT. LH stimulates the conversion of progesterone and 17 alpha-OH progesterone to androstenedione, testosterone and DHT in theca. LH does not stimulate the conversion of androstenedione to testosterone or DHT, and that of testosterone to DHT in either granulosa cells or theca. FSH, in granulosa cells but not in theca, stimulates the conversion of adrostenedione to testosterone but it has no effect in DHT accumulation from exogenous testosterone.     

In vitro metabolism of androgens by mammalian cauda epididymal spermatozoa.

The in vitro metabolism of [3H] testosterone (17beta-hydroxy-4-androsten-3-one), [3H] androstenedione (4-androstene-3,17-dione) and [3H] dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) by cauda epididymal spermatozoa from the rat, rabbit, hamster, guinea-pig and ram, varied between species. There were differences in the androgens utilized, the extent of their conversion and the identities of the metabolites formed. Of the steroid substrates tested rat spermatozoa metabolized testosterone preferentially while spermatozoa from guinea-pig transformed [3H] dehydroepiandrosterone (DHEA) almost exclusively. Rabbit spermatozoa converted all three [3H] androgens while hamster sperm utilized [3H] testosterone and [3H] DHEA. Spermatozoa collected from rams killed at the abattoir metabolized both [3H] androstenedione and [3H] DHEA but this capacity was dramatically reduced in spermatozoa collected from rams subjected to short-term anaesthesea. The results are discussed in relation to the possible direct roles of androgens in sperm physiology.     

Pharmacokinetics of oral dehydroepiandrosterone (DHEA) in the ovariectomised cynomolgus monkey

Humans and primates are unique in having adrenals that secrete large amounts of DHEA and DHEA-S in the circulation. These steroids act as precursors of active androgens and estrogen's in a long series of peripheral target intracrine tissues. The marked decline of serum DHEA and DHEA-S concentrations with age in men and women has been incriminated in the development of various pathologies. This study provides detailed information on the effect of a single 50mg oral dose of DHEA on circulating estrogen's as well as androgens and their metabolites over 10h in adult ovariectomised (OVX) Cynomolgus monkeys. Serum DHEA, DHEA-S, testosterone (Testo) and androstenedione (4-dione) concentrations increased rapidly with a maximal value at approximately 1h after DHEA administration followed by a 60-80% decrease during the next 2-6h. An important sulfatation of DHEA occurs through first hepatic pass, thus, leading to a marked increase in serum DHEA-S. Serum androst-5-ene-3beta,17beta-diol and androsterone glucuronide (ADT-G) levels remained elevated on a plateau for 6h. Androstan-3alpha,17beta-diol-glucuronide, estradiol and estrone levels remained unchanged. The present data indicate the predominant transformation of the adrenal precursor DHEA into active androgens in peripheral tissues and support the importance of measurement of circulating glucuronide derivatives as index of peripheral or intracrine androgen formation and action.     

Androgen inhibition of FSH-stimulated progesterone production by granulosa cells of prepubertal pigs

Androgens have been reported to stimulate progesterone production by granulosa cells of several species, and to act synergistically with FSH in stimulation of progesterone accumulation by rat granulosa cells. Studies were undertaken to examine the effect of androgens on FSH-stimulated progesterone production in culture by granulosa cells derived from prepubertal pig ovaries. When included in 24-h culture with FSH, both androstenedione and testosterone caused a reduction in progesterone accumulation, but dihydrotestosterone and androsterone did not. Granulosa cells were cultured for 24 h with FSH and [14C]progesterone with or without testosterone; testosterone did not affect the rate of overall metabolism of [14C]progesterone and it was therefore concluded that testosterone inhibited progesterone synthesis, rather than enhancing its catabolism. 17 beta-Estradiol also inhibited FSH-stimulated progesterone accumulation. To determine whether the action of testosterone was mediated by conversion to estradiol, granulosa cells were cultured with FSH and testosterone with or without an aromatase inhibitor (4-acetoxy-androstenedione). The aromatase inhibitor failed to prevent the testosterone-induced reduction in progesterone accumulation, although it markedly inhibited estradiol accumulation. These results indicate that theca-derived androgens can inhibit FSH-stimulated progesterone production by granulosa cells in the prepubertal pig, independently of estradiol.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Influence of oral dehydroepiandrosterone (DHEA) on urinary steroid metabolites in males and females

Oral dehydroepiandrosterone (DHEA) replacement therapy may have a multitude of potential beneficial effects and exerts its action mainly via peripheral bioconversion to androgens (and estrogens). A daily dose of 50-mg DHEA has been shown by us and others to restore low endogenous serum DHEA concentrations to normal youthful levels followed by an increase in circulating androgens and estrogens. As the hepatic first-pass effect may lead to a non physiological metabolism of DHEA after oral ingestion we studied the influence of two single DHEA doses (50 and 100 mg) on the excretion of steroid metabolites in 14 elderly males [age 58.8+/-5.1 years (mean +/- SEM)] with endogenous DHEAS levels <1500 ng/ml and in 9 healthy females (age 23.3+/-4.1 years) with transient suppression of endogenous DHEA secretion induced by dexamethasone (dex) pretreatment (4x0.5 mg/day/4 days). Urinary steroid profiles in the elderly males were compared to the steroid patterns found in 15 healthy young men (age 28.9+/-5.1 years). In the females the results were compared to their individual baseline excretion without dex pretreatment. Urinary steroid determinations were carried out by semiautomatic capillary gas-liquid chromatography. In both genders DHEA administration induced significant increases in urinary DHEA (females: baseline vs. 50 mg vs. 100 mg: 361+/-131 vs. 510+/-264 vs. 1541+/-587 microg/day; males: placebo vs. 50 mg vs. 100 mg: 434+/-154 vs. 1174+/-309 vs. 4751+/-1059 microg/day) as well as in the major DHEA metabolites androsterone (A) and etiocholanolone (Et). Fifty mg DHEA led to an excretion of DHEA and its metabolites only slightly above baseline levels found in young females and in young men, respectively, whereas 100 mg induced clearly supraphysiological values. After 50 mg DHEA the ratios of urinary DHEA metabolites (A/DHEA, Et/DHEA) were not significantly different between elderly males vs. young male volunteers and young healthy females versus their individual baseline levels. In conclusion, an oral dose of 30 to 50 mg DHEA restores a physiological urinary steroid profile in subjects with DHEA deficiency without evidence for a relevant hepatic first-pass effect on urinary metabolites.     

Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle

Isolated hamster granulosa cells and theca from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroid substrates. The purpose of the experiments was to determine, in theca, the relative activities of the delta 5 and delta 4 pathways under controlled conditions, and to compare the ability of granulosa cells and theca to form progesterone from exogenous pregnenolone. The results of the experiments show that the delta 5 pathway in theca predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from delta 5 to delta 4 as the major metabolic pathway. Progesterone formation from exogenous pregnenolone is 7 to 10 times greater in unstimulated granulosa cells than in theca. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theca. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone.     

Involvement of insulin and growth hormone (GH) during follicular development in the bovine ovary

Insulin and growth hormone (GH) play critical roles in the process of follicular development and maturation. However, the involvement of insulin receptor (IR) and GH receptor (GHR) during follicular development is not well understood. The aim of this study was to investigate the expression of IR and GHR mRNAs in the granulosa cells (GCs) and theca tissues (TCs) of the follicle at different developmental stages (preovulatory dominant follicles, POFs; estrogen-active dominant follicles, EADs; estrogen-inactive dominant follicles, EIDs; and small follicles, SFs), and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of IR and GHR genes in cultured bovine GCs. Although the concentration of insulin in follicular fluid (FF) was constant at all developmental stages, the GH concentration in FF was significantly increased in the EAD and POF compared with the EID. IR mRNA in GCs and TCs was significantly increased in the POF compared with other follicles. Regarding GHR expression, significant increases of mRNA expression were observed in GCs of EAD compared to those of SF, EID and POF. GHR mRNA in TCs was significantly decreased in the SF compared with other follicles. In cultured GCs, FSH, but not E2, stimulated the expression of IR and GHR genes. Our results suggest that the increase in the expression of GHR may be a turning point for follicles to enter the ovulatory phase during final follicular development and that the insulin system may support the maturation of preovulatory follicles.     

Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens (Gallus domesticus)

Blocking testosterone action with immunization or with a specific antagonist blocks the preovulatory surge of progesterone and ovulation in laying hens. Thus, testosterone may stimulate progesterone production in a paracrine fashion within the ovary. To test this hypothesis, we evaluated the effects of testosterone and its interaction with LH on the production of progesterone by granulosa cells in culture. Hen granulosa cells obtained from preovulatory follicles were cultured in 96 well plates. The effects of testosterone (0-100ng/ml) and/or LH (0-100ng/ml) were evaluated. LH-stimulated progesterone production in a dose response manner up to 10ng/ml (p<0.01). Testosterone, up to 10ng/ml, increased progesterone production in a dose response manner in the absence of LH and at all doses of LH up to 1ng/ml (p<0.001). However, at supraphysiological concentrations of LH (10 and 100ng/ml) there was no further increase in progesterone production caused by testosterone (p>0.05). Finally, the addition of 2-hydroxyflutamide (0-1000mug/ml) to hen granulosa cells cultured with 10ng/ml of testosterone reduced progesterone production in a dose response manner (p<0.001). In conclusion, testosterone stimulates progesterone production in preovulatory follicle granulosa cells and interacts with physiological concentrations of LH to increase progesterone production. In addition, testosterone stimulation on granulosa cells is specific since the testosterone antagonist decreased testosterone stimulatory action.     

Identifying androsterone (ADT) as a cognate substrate for human dehydroepiandrosterone sulfotransferase (DHEA-ST) important for steroid homeostasis: structure of the enzyme-ADT complex

In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 A resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.     

In vitro metabolism of C19 steroids in human endometrium

In order to quantitate the extent of intracellular metabolic conversions of C19 steroids in human endometrium, specimens of proliferative and secretory tissue were superfused at a constant rate with several pairs of labeled compounds at low concentrations. About 16% of dehydroepiandrosterone sulfate interacting with endometrial cells was converted to dehydroepiandrosterone and about 3% of this compound was converted to androstenedione. Androstenedione was reversibly reduced to testosterone and the extent of this conversion was shown to be several-fold higher in secretory than in proliferative tissue. About 1% of testosterone entering the cells was reduced to 5 alpha-dihydrotestosterone. These results demonstrate that the conversion of the main circulating C19 steroids in women, i.e. dehydroepiandrosterone sulfate and androstenedione, to 5 alpha-dihydrotestosterone, the compound considered to be the true intracellular androgen, is very small. In contrast, formation of testosterone from androstenedione is extensive and increases during the luteal phase under the influence of progesterone, a hormone known to stimulate the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrium.     

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.     

Aromatase activity in ovarian follicles of the golden hamster

The aromatizing ability of recombined granulosa and thecal cells in culture, isolated from hamsters 72-78 h and 96-102 h after PMSG-stimulation, was assessed by the addition to the culture medium of androstenedione, testosterone, dehydroepiandrosterone (DHEA) or 5 alpha-dihydrotestosterone (DHT), and measuring the output of oestradiol 4 h later. The cells from all follicles taken after 96-102 h had a reduced oestradiol output compared to those isolated after 72-78 h (P less than 0.02). Recombined cells from the unluteinized follicles at 96-102 h (Group I) showed similar oestradiol output in the presence of androstenedione, testosterone and DHEA to the cells from follicles taken at 72-78 h. However, the recombined cells from the luteinized follicles (Group II) showed a reduced output of oestradiol in the presence of androstenedione, testosterone and DHEA when compared to the recombined cells from the previous period cultured with the corresponding C19 steroid. The results show that a reduced oestradiol output can be caused by (1) the reduced availability of aromatizable substrate and (2) a reduced potential aromatase activity.     

The effect of ACTH therapy on serum dehydroepiandrosterone, androstenedione, testosterone and 5 alpha-dihydrotestosterone in infants

Serum concentrations of dehydroepiandrosterone (DHEA), androstenedione, testosterone, 5 alpha-dihydrotestosterone and cortisol were measured in 10 infants (age 5-22 months) before, during and after 6-weeks of ACTH therapy for infantile spasms. During therapy, their mean DHEA concentrations increased 2.3-fold, androstenedione 12.3-fold, testosterone 2.7-fold, 5 alpha-dihydrotesterone 2.5-fold and cortisol 2.9-fold compared to pre-therapy values. Serum dehydroepiandrosterone sulphate (DHEA-S) concentrations were also increased during ACTH therapy above the normal prepubertal range. Three days after the cessation of ACTH treatment, all androgens had returned to the pre-therapy level. We conclude: At least in pharmacologic doses ACTH alone stimulates adrenal androgen secretion in infants, excluding the necessity of a separate adrenal androgen stimulating hormone.     

Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken

Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400–760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.     

Metabolism of pregnenolone by human breast cancer. Evidence for 17 alpha-hydroxylase and 17,20-lyase.

The metabolism of 7-(3)H-pregnenolone was studied in vitro using 16 human breast carcinomas. All mammary tumors transformed pregnenolone to progesterone. All estrogen receptor poor tumors and 4 out of 8 estrogen receptor rich tumors converted pregnenolone to 17-hydroxypregnenolone. Five estrogen receptor poor tumors showed the presence of 17,20-lyase as evidenced by formation of dehydroepiandrosterone and androstenedione. In two estrogen receptor poor tumors, conversions of pregnenolone to progesterone, 17-hydroxy pregnenolone, dehydroepiandrosterone, androstenedione and finally to estradiol was documented, providing a hypothetical pathway for steroid metabolism in human breast cancer. The conversion of pregnenolone to 17-hydroxypregnenolone was significantly less in receptor rich tumors and was totally absent in 4 receptor rich tumors with estrogen receptors of over 45 fmol/mg protein.