Testosterone and testosterone precursors in the spermatic vein and in the testicular tissue of old men. Reduced oxygen supply may explain the relative increase of testicular progesterone and 17 alpha-hydroxyprogesterone content and production in old age

Testosterone and the testosterone precursors pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, androstenedione, androstenediol and dehydroepiandrosterone were measured in the spermatic vein plasma and in the testicular tissue of young and old men. Testosterone and its precursors decreased in the testicular tissue of old men. However, progesterone and 17 alpha-hydroxyprogesterone increased in relation to testosterone in the testicular tissue and in the spermatic vein of old men. It is assumed that these age-dependent changes are caused by an impaired oxygen supply of the ageing testes. This hypothesis is supported by the observation that the same changes in steroid pattern seen in old age can be observed under reduced oxygen supply in in vitro incubation experiments with testicular tissue.     

The molecular basis of isolated 17,20 lyase deficiency

Human P450c17 catalyzes the 17alpha-hydroxylation of pregnenolone to 17OH pregnenolone and of progesterone to 17alpha-OH progesterone; the same P450c17 enzyme also catalyzes 17,20 lyase activity on the same active site, converting 17OH-pregnenolone to DHEA. Rodent and porcine P450c17 also catalyze 17,20 lyase activity with delta4 substrates, converting 17OH-progesterone to delta4 androstenedione, but human P450c17 catalyzes this reaction very inefficiently, so that virtually all human C19 sex steroids are made via 17OH pregnenolone and DHEA. P450c17 is encoded by a single gene and a single species of mRNA. Many mutations of this gene have been described, but until recently all of these either entirely eliminated both 17alpha-hydroxylase and 17,20 lyase activity, or affected each activity equivalently. We have identified and characterized the first patients with P450c17 mutations that selectively ablate 17,20 lyase activity while retaining 17alpha-hydroxylase activity. Through a combination of enyzmologic experiments in transfected mammalian cells and in genetically manipulated yeast, plus a computer model of human P450c17, we have proven that the responsible mutations, R347H and R358Q lie in the redox-partner binding site of P450c17. This site, through which P450c17 interacts with P450 oxidoreductase to receive the electrons needed for catalysis, can be allosterically influenced by cytochrome b5. These two mutations have contributed substantially to our understanding of the mechanisms by which 17alpha-hydroxylase and 17,20 lyase activities are regulated independently, and thus have contributed to the study of regulated 17,20 lyase activity in adrenarche, aging, and the polycystic ovary syndrome.     

The inhibition of androstenedione production in mature thecal cells from the ovary of the domestic hen (Gallus domesticus): evidence for the involvement of progestins

In the 24 hours before ovulation, there is an abrupt decline in the ability of theca cells from the largest chicken preovulatory follicle to produce androstenedione from all substrates except dehydroepiandrosterone. In this study, we tested the hypothesis that progesterone from granulosa cells might inhibit andostenedione production by the adjacent theca cells. Physiological concentrations of progesterone inhibited andostenedione production by dispersed thecal cells from the substrate 17 alpha-hydroxyprogesterone but not dehydroepiandrosterone in both a dose- and time-dependent manner. In contrast, the metabolites of progesterone, 17 alpha-hydroxyprogesterone, and androstenedione at a high concentration (100 nM) failed to produce such an inhibitory effect. In addition, this inhibitory effect of progesterone was reversed by the protein synthesis inhibitor cycloheximide. The results of this study seem to suggest that progesterone acts indirectly through its nuclear receptor to induce the synthesis of a protein that possibly inhibits C17,20 lyase activity and/or C17,20 lyase gene expression.     

Biochemical differences between rat and human cytochrome P450c17 support the different steroidogenic needs of these two species

Microsomal 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes both the 17alpha-hydroxylase reaction required to produce cortisol, the major glucocorticoid in many animals, and the 17, 20-lyase activity required for the production of androgens in all animals. In rodents such as rat, which utilize corticosterone as the major glucocorticoid, P450c17 is expressed predominantly in the gonads, and is absent in the adrenal. In other species including humans, P450c17 is expressed in both adrenal and gonads and participates in both glucocorticoid and androgen production. Rat and human forms of P450c17 are 69% identical at the amino acid level. Based on the differences in physiological roles between P450c17 in these two species, it could be predicted that major differences would be observed in their hydroxylase activities. Contrary to this hypothesis, using partially purified, recombinant human and rat P450c17, we found that the most significant differences lie in their lyase activities. Lyase activities demonstrate that the rat enzyme favors Delta4 (progesterone) substrates while the human enzyme favors Delta5 (pregnenolone) substrates. This substrate preference is also observed in the ability of steroids to decrease uncoupled H2O2 production and to increase stability during turnover. Cytochrome b5, a microsomal electron-transfer protein, enhances lyase activities of rat and human P450c17. However, the most dramatic stimulatory effect is on the human HO-PROG lyase activity. This enhancement of activities is not associated with electron transfer. These differences in biochemical properties between the two forms of P450c17 indicate that human P450c17 has evolved as an enzyme system that limits androgen production to the gonads where a favorable b5:P450c17 ratio exists. Even though orthologous forms of P450c17 are capable of catalyzing the same enzymatic activities, specific physiological requirements of each species ensure biochemical differences between these enzymes.     

A simple method for the assay of eight steroids in small volumes of plasma

A simple method is described for the simultaneous radioligand assay of four delta5-3beta-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17alpha-hydroxypregnenolone, dehydroepiandrosterone and 5-androsterone-3beta, 17beta-diol), and their four delta4-3keto products (progesterone, 17alpha-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid in extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17, 20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.     

Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography

Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.     

Potent and selective aromatase inhibitor: in vitro and in vivo studies with s-triazine derivative SEF19

We found a potent aromatase inhibitor through the screening of agents for estrogen-dependent breast cancer. SEF19 (2-(imidazol-1-yl)-4,6-dimorphorino-1,3,5-triazine) decreased 50% of human placental aromatase activity in vitro at the concentration of 5.3 nM. In order to clarify the selectivity of SEF19 for enzyme inhibition, we determined the effect of SEF19 on the activities of four steroidogenic cytochrome P450 enzymes in porcine adrenal gland, P450SCC(side-chain cleavage of cholesterol), P450(11 beta) (11 beta-hydroxylase), P450(17 alpha)(17 alpha-hydroxylase/C17,20 lyase) and P450C21 (21-hydroxylase). SEF19 failed to inhibit the activities of porcine adrenal P450SCC, P450(17 alpha) and P450C21 up to the concentration of 100 microM and showed some inhibition on P450(11 beta) activity at 100 microM, while SEF19 completely nullified the aromatase activity at 1 microM. We also determined the potency of SEF19 for the suppression of aromatase activity in vivo. SEF19 suppressed dose-dependently the uterine hypertrophy of immature rats caused by administration of androstenedione (30 mg/kg, s.c.). The ED50 of SEF19 for the suppression of uterine hypertrophy was 0.8 mumol/kg. These results suggest that SEF19 may serve as a potent and selective agent for the treatment of estrogen-dependent breast cancer.     

17 alpha-Hydroxyprogesterone responses to leuprolide and serum androgens in obese women with and without polycystic ovary syndrome offer dietary weight loss

Insulin resistance and increased ovarian cytochrome P450c17 alpha activity (i.e. increased 17 alpha-hydroxylase and, to a lesser extent, increased 17,20-lyase) are both features of the polycystic ovary syndrome (PCOS). Evidence suggests that hyperinsulinemia may stimulate ovarian P450c17 alpha activity in obese women with PCOS. We hypothesized that weight loss would decrease serum insulin and P450c17 alpha activity in PCOS. Therefore, we measured serum steroid concentrations and 17 alpha-hydroxyprogesterone responses to leuprolide administration and performed oral glucose tolerance tests before and after 8 weeks of a hypocaloric diet in 12 obese women with PCOS (PCOS group) and 11 obese women with normal menses (control group). Serum insulin decreased in both groups. In the PCOS group, basal serum 17 alpha-hydroxyprogesterone decreased from 4.2 +/- 0.6 to 3.0 +/- 0.5 nmol/L (P < 0.05), and leuprolide-stimulated peak serum 17 alpha-hydroxyprogesterone decreased from 14.9 +/- 2.6 to 8.9 +/- 0.8 nmol/L (P < 0.025). Serum testosterone decreased from 2.47 +/- 0.52 to 1.56 +/- 0.33 nmol/L (P < 0.05), and free testosterone decreased from 9.03 +/- 1.39 to 5.95 +/- 0.50 pmol/L (P < 0.02). None of these values changed in the control group. Serum sex hormone-binding globulin increased by 4.5- and 3-fold in the PCOS (P < 0.003) and control (P < 0.007) groups, respectively. We conclude that dietary weight loss decreases ovarian P450c17 alpha activity and reduces serum free testosterone concentrations in obese women with PCOS, but not in obese ovulatory women. The changes in women with PCOS may be related to a reduction in serum insulin.     

Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor beta 1

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factor beta 1 (TGF beta 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTHR), cytochrome P-450scc (cholesterol side-chain cleavage), P450 17 alpha (17 alpha-hydroxylase/17-20 lyase) and 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTHR and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTHR, P450scc and P450 17 alpha mRNA levels. In contrast, TGF beta 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGF beta 1 had no effect on ACTHR and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17 alpha both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3 beta-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGF beta may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.     

Expression of steroid sulfatase during embryogenesis

Neurosteroids are steroids that are synthesized de novo in the brain from cholesterol and, in general, mediate their effects through ion-gated channel receptors such as gamma-aminobutyric acidA (GABA[A]) and N-methyl-D-aspartate receptors rather than through classical nuclear steroid hormone receptors. Steroid hormones are known to exist not only as free compounds, but also as sulfated derivatives. Pharmacological studies indicate that unconjugated and sulfated steroids, such as pregnenolone and pregnenolone sulfate, may have opposite effects on GABA(A) receptors. Thus, pregnenolone acts as a potent positive allosteric modulator of gamma-aminobutyric acid action at GABA(A )receptors, whereas pregnenolone sulfate acts as a potent negative modulator. Recent experiments also suggest that dehydroepiandrosterone and dehydroepiandrosterone sulfate may have distinct effects on growth of neurites from embryonic neocortical neurons in vitro. Thus, regulation of steroid sulfation may have profound behavioral and morphological effects on the nervous system. We, therefore, studied the developmental expression of the enzyme steroid sulfatase (STS), which converts sulfated steroids to free steroids. By in situ hybridization, STS messenger RNA was expressed in the embryonic mouse cortex, hindbrain, and thalamus during the last third of gestation. The sites of expression of STS were similar to those of P450c17, suggesting that these two enzymes may have concerted actions in similar functional processes.     

Ovarian enzymatic divergence in patients with polycystic ovary syndrome excreting urinary pregnanetriolone

When ovarian mitochondria from patients with polycystic ovary syndrome (POS) were incubated with [7-3H]17alpha-hydroxypregnenolone and [4-14C]-17alpha-hydroxyprogesterone, 11beta-hydroxylated metabolites were obtained. The mitochondria, prepared from pooled, frozen, polycystic ovarian tissue of 5 patients, converted [7-3H]17alpha-hydroxypregnenolone to 3beta, 11beta, 17alpha--trihydroxy-5-pregnen-20-one (yield 0.065%) and to 3beta, 17alpha-dihydroxy-5-pregnene-11,20-dione (0.22%), while [4-14C]17alpha-hydroxyprogesterone was converted to 21-deoxycortisol (0.1%). Incubation of mitochondria, prepared from 4 pooled samples of frozen, normal ovarian tissue, yielded no evidence of 11beta-hydroxylation of either of the substrates. Mitochondria obtained from fresh, polycystic ovarian tissue of a single patient with POS converted [7-3H]17alpha-hydroxypregnenolone to 3beta,17alpha-dihydroxy-5-pregnene-11,20-dione (2.1%) and [4-14C]17alpha-hydroxyprogesterone to 21-deoxycortisol (0.1%). When the same mitochondrial preparation was incubated simultaneously with [7-3H]17alpha-hydroxypregnenolone and [4-14C]11-deoxycortisol, it converted 17alpha-hydroxypregnenolone to 3beta,17alpha-dihydroxy-5-pregnene-11,20-dione (1.9%), but no 11beta-hydroxylated derivatives of 11-deoxycortisol were found. These results demonstrate that ovaries of patients with POS contain an 11beta-hydroxylase active towards C-21-deoxysteroids but inert to C-21-hydroxysteroids such as 11-deoxycortisol.     

Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.     

Dehydroepiandrosterone 7alpha- and 7beta-hydroxylation in mouse brain microsomes. Effects of cytochrome P450 inhibitors and structure-specific inhibition by steroid hormones

Presently, several works question the effects of dehydroepiandrosterone (DHEA) reported in vivo and designate its 7-hydroxylated metabolites as native antiglucocorticoids and potent mediators in the triggering of immune response. Among mouse tissues and organs, and second to liver, the largest production of 7alpha-and 7beta-hydroxylated derivatives of DHEA takes place in brain microsomes. To contribute to identification of cytochromes P450 (CYPs) responsible for 7alpha- and 7beta-hydroxy-DHEA production, effects of CYP inhibitors and of several steroid hormones on DHEA 7-hydroxylation were examined. Using mouse brain microsomes as a source of enzyme, we report now that strong and smaller inhibitions of DHEA 7alpha-hydroxylation were obtained with ketoconazole and alpha-naphthoflavone, respectively, and that neither changed DHEA 7beta-hydroxylation. Metyrapone and antipyrine also inhibited 7alpha-hydroxylation, but by contrast, significantly increased 7beta-hydroxylation of DHEA. This indicated that at least, two different CYPs were responsible for 7alpha- and 7beta-hydroxylation of DHEA. Steroids sharing a 3beta-hydroxylated structure with DHEA, namely pregnenolone, 5-androstene-3beta,17beta-diol and 3beta-hydroxy-5alpha-androstan-17-one, were strong inhibitors of DHEA 7alpha-hydroxylation (non-competitive inhibition with pregnenolone, Ki=2.0 +/- 0.3 microM). In contrast, 7beta-hydroxylation yields were not decreased by the 3beta-hydroxysteroids tested. Moderate inhibition of 7alpha- and 7beta-hydroxylation was obtained with 3-oxosteroids, namely testosterone, progesterone, corticosterone and 4-androsten-3,17-dione. Taken together, these data indicate specific inhibition patterns of DHEA 7alpha- and 7beta-hydroxylation by CYP inhibitors and steroid hormones in mouse brain microsomes and may be used as criteria necessary for identification of the responsible CYP species.     

Variation of dorsal horn cell dendritic spread with map scale

OBJECTIVE: To examine the hypothesis that, in polycystic ovary syndrome (PCOS), ovarian steroids induce adrenal enzyme dysfunction or adrenal androgen hyperresponsiveness to ACTH. DESIGN: Prospective controlled clinical study. SETTING: Reproductive endocrinology unit of an academic medical center. PATIENTS: Twelve women with PCOS who had adrenal androgen excess were compared with five weight-matched ovulatory women. In half of the women with PCOS, prestudy screening was suggestive of mild 3 beta-hydroxysteroid dehydrogenase (HSD) deficiency. INTERVENTIONS: Basal and adrenal dynamic blood sampling before and after GnRH agonist (GnRH-a) administration for 6 months. MAIN OUTCOME MEASURES: Basal E2 and androgen levels as well as dexamethasone-suppressed, ACTH-stimulated 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and androgen levels before and after ovarian suppression. RESULTS: Although none of the subjects with PCOS proved to have mild 3 beta-HSD deficiency, the majority of them (58%) met the criteria for 17,20 lyase hyperactivity before and after GnRH-a therapy. As a group, the remaining subjects with PCOS exhibited an elevated DHEAS response to ACTH before GnRH-a treatment, which may have normalized after GnRH-a treatment. CONCLUSION: Adrenal androgen excess in PCOS may be heterogeneous in etiology, whereas 17,20 lyase hyperactivity appears to be an intrinsic adrenal disorder, adrenal androgen hyperresponsiveness to ACTH may be ovarian induced. Reliance on historical controls may lead to overdiagnosis of mild 3 beta-HSD deficiency.     

Purification and characterization of constituent androstenedione 15 alpha-hydroxylase (cytochrome P450(15 alpha AD)) from mouse liver. Sex- and tissue-dependent expression

Hepatic microsomal androstenedione 15 alpha-hydroxylase (i.e.cytochrome P450(15)alpha AD was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15 alpha-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450(15)alpha AD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7 alpha- and 15 alpha-hydroxylation of androstenedione, representing, respectively, > 5% and > 93% of the total metabolites. Polyclonal antibodies against cytochrome P450(15)alpha AD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7 alpha- and 15 alpha-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450(15)alpha AD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450(15)alpha AD was 53,000, and the protein appeared as a single band or sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450(15)alpha AD is a member of the murine cytochrome P450 2c subfamily.     

A new method for the determination of steroidal hormones in biological material (adrenal tissue) (author's transl)

A rapid method is described for the determination of some steroidal hormones in adrenal tissue. The following steroids were measured: pregnenolone, progesterone, deoxycorticosterone, corticosterone, aldosterone, 17-alpha-OH-pregnenolone, deoxycortisol, cortisol, cortisone, 17-alpha-OH-progesterone, dehydroepiandrosterone, androstendione, and testosterone. After extraction of the steroids the purification steps were performed by thin layer chromatography. Gas chromatography was used for further separation and quantitative analysis of underivatized steroids. The GC-analysis of steroids without any derivatisation makes this procedure comparatively simple and exact. Recovery of the steroid content of the tissue ranged from 30% to 70%. This method described herewith has several advantages, and allows the analysis of two tissues at the same time for a large number of adrenal steroids within two weeks.     

The influence of prostaglandin F-2alpha on pregnenolone metabolism by the autotransplanted ovary of the ewe

Eight ewes each with an autotransplanted ovary received infusions of tritium-labelled pregnenolone (41 muCi/hr) for 8 hr into the artery supplying the ovary, together with prostaglandin (PG) F-2alpha (30 mug/hr) for 3 hr beginning 2 hr after the start of the pregnenolone infusion. All animals exhibited oestrus 2-3 days after the start of the experiment. During the PGF-2alpha infusion blood flow through the ovaries was increased by 13%, but subsequently returned to pre-infusion levels. Secretion rates of endogenous progesterone and 20alpha-hydroxypregn-4-en-3-one dropped rapidly 5 hr after the PGF-2alpha infusion had started from values of 250 mug/hr and 25 mug/hr to values below 60 mug/hr and 8 mug/hr, respectively. At this time the conversion of radioactive pregnenolone to progesterone was reduced by 50% of its initial value, but the secretion of endogenous pregnenolone and the formation of radioactive metabolites other than progesterone were not diminished. In 4 control animals, which received pregnenolone only, no changes in ovarian blood flow, steroid secretion rates, or in the conversion of labelled pregnenolone were observed. These results suggest a possible involvement of PGF-2alpha in the regulation of progesterone biosynthesis by an action on the 3beta-hydroxysteroid oxidoreductase-delta5(-4) isomerase enzyme system.     

The effect of opioid antagonists in local regulation of testicular response to acute stress in adult rats

The present study examined the effects of naloxone (N) and naltrexone-methobromide (NMB; an opioid receptor antagonist that does not cross the blood-brain barrier) on testicular steroidogenesis during acute immobilization stress (IMO; 2 h) in adult rats. Unstressed rats as well as IMO rats were treated by unilateral intratesticular injection of N (20 micrograms/testis), NMB (36 micrograms/testis), or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats serum T levels were significantly reduced, while serum luteinizing hormone levels were not affected. N and NMB normalized serum T levels in IMO rats and had no effects in controls. In IMO rats the activities of 3 beta-hydroxysteroid dehydrogenase (HSD) and P450(17 alpha, lyase) were significantly reduced, while the activity of 17 beta-HSD was not affected. N and NMB antagonized the inhibitory effect of IMO on 3 beta-HSD and P450(17 alpha, lyase) but did not alter enzyme activity in freely moving rats. Acute IMO decreased basal and human chorionic gonadotropin-stimulated androgen production by hemitestis preparation, but N (10(-4) M) added directly to the incubation medium blocked the decrease and had no effect on testes from freely moving control rats. These results support the conclusion that endogenous opioid peptides are potentially important paracrine regulators of testicular steroidogenesis under stress conditions.