Changes in steroid pattern following acute and chronic adrenocorticotropin administration in premature adrenarche

Biochemically adrenarche is characterized by increased production of 5-ene steroids, in particular Dehydroepiandrosterone (DHA) and its sulphate (DHA-S). It is still not clear if ACTH is responsible for this adrenal steroid production. The aim of the present study was to evaluate the effect of acute and chronic ACTH administration, without dexamethasone pretreatment, on hormonal patterns in 20 patients (5 males aged between 6 8/12 and 7 10/12 years and 15 females aged between 5 9/12 and 7 6/12 years) with idiopathic premature adrenarche. Pregnenolone (5P), DHA, DHA-S, 17-hydroxyprogesterone (17-OHP), androstenedione (A), 11-deoxycortisol (S) and cortisol (F) have been determined by Radioimmunoassay. The results of the hormonal evaluation (means +/- standard error) showed high plasma levels of DHA [329.2 +/- 41.7 ng/100 ml (dl)] and DHA-S (169.1 +/- 54 micrograms/dl) and slightly increased levels of 5P (74.4 +/- 7.1 ng/dl), of A (45.4 +/- 4.6 ng/dl) and 17-OHP (69.3 +/- 11.3 ng/dl) in comparison to those of controls, thus indicating a decrease in 3 beta-hydroxysteroid dehydrogenase activity and an increase in 17-20-lyase and 17-hydroxylase activities, characteristic for adrenarche. Acute and chronic ACTH stimulation did not amplify the characteristic basal hormonal pattern, but they induced a shift of adrenal steroid metabolism to 4-ene pathway, suggesting that the basal hormonal pattern in premature adrenarche may be independent or, at least, not exclusively dependent on ACTH control.     

The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

Effects of dehydroepiandrosterone and 16 alpha-hydroxydehydroepiandrosterone on the reduction of glucose to glucitol by the human placenta.

The metabolism of glucose by subcellular preparations of human full term placentae has been investigated. It has been shown that in the presence of NADPH two transformation products can be detected of which one has been identified as glucitol. The effects of dehydroepiandrosterone and 16alpha-hydroxydehydroepiandrosterone on the reduction of glucose to glucitol have also been studied. It has been found that at a concentration of DHA 1.2 X 10(-4)M, the reduction of glucose is strongly inhibited (35-51%), while at a concentration of DHA 5.8 X 10(-6)M this reaction is stimulated by 13 +/- 2.3%. 16alpha-hydroxyepiandrosterone at concentrations ranging from 1.2 X 10(-4)M to 3 X 10(-6)M inhibits the formation of glucitol from 63% to 9%.     

Metabolism of adrenal androgens by human endometrium and adrenal cortex

The enzyme 17 beta-hydroxysteroid dehydrogenase (17OHSD) was studied in human endometrium and adrenal cortex with respect to the metabolism of 5-androstene-3 beta,17 beta-diol (androstenediol) and dehydroepiandrosterone (DHA). The aim was to provide further information concerning the origin and biological significance of these androgens in endometrium, particularly the increased concentrations of the secretory phase and to compare the characteristics of the enzyme in the two tissues. In both endometrium and adrenal cortex the metabolism of androstenediol to DHA was linear with time and increasing enzyme concentration. The preferred cofactor was NAD and the apparent Km values were 3.4 +/- 0.2 (SD) microM (n = 3) for endometrium and 30.5 +/- 6.1 microM (n = 3) for adrenal cortex. In endometrium DHA was not metabolised to androstenediol in the presence of either NADH or NADPH whereas in the adrenal cortex both cofactors were utilised. However, the concentration of NADH required to achieve maximum enzyme activity was 10-fold higher (1 mM) than for NADPH (0.1 mM) and maximum activity with NADH was only 30% of that using NADPH. The apparent Km was 125 microM DHA (n = 2). The study indicates that androstenediol in endometrium does not arise from DHA metabolism but that its presence could be due to a binding protein particularly during the secretory phase. Our findings also suggest that the enzyme of endometrium differs from that of the adrenal cortex and that the kinetic properties may be related to the physiological requirements of the two tissues.     

The effects of androgens and cortisol on the in vivo metabolism of oestradiol

We have examined the effect of co-administration of dehydroepiandrosterone sulphate, 5-androstenediol or cortisol on the metabolic clearance rate of oestradiol (MCR-E2) and conversion of oestradiol to oestrone (CRE2E1). Previous studies have shown that these androgens influence the metabolism of oestradiol in vitro while cortisol alters the distribution of oestradiol in plasma. The MCR-E2 and CRE2E1 were measured after 2.5 and 5 h of [3H]oestradiol infusion with co-infusion of androgen or cortisol starting after 2.5 h of tracer infusion. For one subject who did not receive co-infusion of another steroid no significant change in MCR-E2 or CRE2E1 occurred over the 5-h period. For other subjects, however, the MCR-E2 decreased by 18 +/- 7% (mean +/- SD) while the CRE2E1 increased by 45 +/- 12%. It is possible that these results are due to: changes in the distribution of oestradiol in plasma; differences in the metabolism of oestradiol bound to albumin or SHBG, or an effect of androgens or cortisol on the uptake of [3H]oestradiol by the liver.     

Modulation of phospholipase A2 activity in human endometrium and amniotic membrane by steroid hormones

Phospholipase A2 (PLA2) activity was measured in endometrium and amnion by a double isotope ratio technique using 1-palmitoyl-2-oleoyl phosphatidylcholine as substrate in the presence and absence of a range of unconjugated steroids and steroid sulphates (0.2–6.4 × 10⁻⁴ M). In the presence of 0.1% Triton, PLA2 activity was inhibited by the majority of steroids tested, pregnenolone sulphate being the most effective (12.9 ± 3.0% control activity) while oestradiol sulphate, oestrone and testosterone had only a minimal or no effect (99.1 ± 19.0, 85.4 ± 4.4 and 104.2 ± 16.3% control respectively). In the absence of Triton, the inhibitory effect of the free steroids was reduced or absent but oestradiol sulphate and testosterone sulphate stimulated activity by 2–13 and 1.5–3 times respectively. The effect was dose related, linear with time and independent of the stage of the menstrual cycle. Inhibition by pregnenolone sulphate, dehydroepiandrosterone (DMA sulphate and oestrone sulphate was maintained in the absence of Triton (24.9 ± 3.8, 67.1 ± 10.1 and 87.2 ± 13.8% control respectively). In amnion all 5 steroid sulphates caused a marked stimulation of PLA2 activity in both the presence and absence of Triton. The effect was greatest without Triton and at 6.4 × 10⁻⁴ M, testosterone, pregnenolone, oestrone, DHA and oestradiol sulphates increased PLA2 activity 20, 15, 12, 10 and 6-fold respectively. These findings indicate a direct action of steroid sulphates on PLA2 activity in endometrium and amnion.     

Inhibition of steroid sulphatase activity by steroidal methylthiophosphonates: Potential therapeutic agents in breast cancer

The hydrolysis of steroid sulphates, by steroid sulphatase, is an important source of oestrogenic steroids (oestrone, oestradiol and 5-androstene-3β,17β-diol) which are found in tumours. In the present study, we have examined the effect of dehydroepiandrosterone-3-O-methylthiophosphonate (DHA-3-MTP), pregnenolone-3-O-methylthiophosphonate (pregnenolone-3-MTP) and cholesterol-3-O-methylthiophosphonate (cholesterol-3-MTP) on the inhibition of oestrone sulphatase as well as DHA sulphatase activities in intact MCF-7 breast cancer cells and in placental microsomes. All three methylthiophosphonates significantly (P< 0.01) inhibited the hydrolysis of oestrone sulphate (E₁ S) in intact MCF-7 cells (31–85% inhibition at 1 μM and 53–97% inhibition at 10 μM). Significant inhibition of DHA sulphatase was also achieved. At a concentration of 50 μM, all three compounds inhibited the hydrolysis of dehydroepiandrosterone sulphate (DHAS) by > 95%. Using human placental microsomes, the K_m and V_max of E₁S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding K_i values for DHA-3-MTP, pregnenolone-3-MTP and cholesterol-3-MTP were found to be 4.5, 1.4 and 6.2 μM, respectively. Such inhibitors which are resistant to metabolism may have considerable potential as therapeutic agents and may have additional advantage over aromatase inhibitors in also reducing tumour concentrations of the oestrogenic steroid, 5-androstene-3β,17β-diol, by inhibiting the hydrolysis of DHAS.     

Endometrial tissue and plasma concentrations of 5-androstene-3 beta, 17 beta-diol during the menstrual cycle in normal premenopausal and perimenopausal women

A radioimmunoassay for 5-androstene-3 beta, 17 beta-diol (ADIOL) in human endometrium and plasma is described. The recognised criteria of reliability have been fulfilled. Plasma and endometrial tissue concentrations of ADIOL were determined in samples obtained from normal premenopausal and perimenopausal women (average ages 37 and 48 years respectively) at different phases of the menstrual cycle. In perimenopausal women plasma concentrations of ADIOL did not vary throughout the cycle (proliferative phase: 411 +/- 95 (SEM) pg/ml; secretory phase: 462 +/- 28.5 (SEM) pg/ml). For the premenopausal group the pattern was similar (proliferative phase: 568.4 +/- 56.9 (SEM) pg/ml; secretory phase: 663.1 +/- 64.7 (SEM) pg/ml) although a significant difference (P less than 0.05) was noted between late proliferative and late secretory phase levels in these women. A different pattern was observed for endometrial tissue concentrations of ADIOL. In both groups of women a significant (3-4-fold) increase occurred during the secretory phase. There was no apparent relationship between plasma and tissue concentrations of ADIOL either during the proliferative or the secretory phase. There was, however, an age associated decrease for both tissue and plasma ADIOL. Theories are proposed to account for the increase in ADIOL concentration during the luteal phase.     

Production of monoclonal antibodies to dehydroepiandrosterone-sulphate after immunization of mouse with dehydroepiandrosterone-bovine serum albumin conjugate

Monoclonal antibodies with a much higher specificity for DHA-S than for DHA were obtained from a BALB/c mouse immunized with a non-sulphated DHA-7CMO-BSA antigen. An improved fusion technique using PEG containing 10% DMSO instead of PEG alone increased the number of positive hybridomas. One of the five monoclonal antibodies obtained, showed a high affinity for DHA-S (Ka = 10(10) M-1) and very low cross-reactions with androsterone (0.62%) and androsterone sulphate (0.83%) which made it potentially useful for direct quantitation of DHA-S in human serum.     

Effects of DHA-S on placental 3 beta-hydroxysteroid dehydrogenase activity, progesterone and 20 alpha-dihydroprogesterone concentrations in placenta and serum

To study the effects of dehydroepiandrosterone sulfate (DHA-S) on placental steroid metabolism and maternal steroidal profiles at term, the following in vivo and in vitro experiments were performed. Two hundred mg of DHA-S was given to five pregnant women 30 minutes prior to delivery. After delivery, the placenta was collected and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and sulfatase activity was determined by measuring the rate of conversion of pregnenolone to progesterone and DHA-S to DHA. The amount of C21-delta 4-steroid in the placental tissue was measured by gas chromatography mass spectrometry (GC-MS) and compared with the control groups. The maternal serum concentration of several steroids was also measured by GC-MS before and after the administration of DHA-S. 3 beta-HSD activity in the placentae from the mothers who received DHA-S before delivery was significantly lower than in the controls. On the other hand, no significant change was observed in the activity of sulfatase. The serum concentration of progesterone (P) and 20 alpha-dihydro-P (20-P) before DHA-S loading decreased following the administration whereas estradiol (E), DHA, and androstenedione (A) levels increased. To study the direct effect of DHA-S and its related steroids on placental 3 beta-HSD activity, placental tissue samples were incubated with pregnenolone in vitro. Several other steroids were added simultaneously into the medium. It was observed that placental 3 beta-HSD activity was directly inhibited by DHA-S. These results indicate that DHA-S inhibits 3 beta-HSD activity in the placenta and subsequently causes a reduction in P and 20-P.     

A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was covalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 microliter serum. The method is precise, accurate and specific and can detect 19.5 pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.     

Olfactory responses to steroids in an African mouth-brooding cichlid, Haplochromis burtoni(Günther)

Underwater electro‐olfactogram (EOG) recordings involving 150 steroids and eight prostaglandins were used to determine which of these potential odorants are detected by the olfactory organ of an African cichlid, Haplochromis burtoni. In initial EOG tests at 10^?9 M, H. burtoni did not respond to unconjugated steroids or prostaglandins, but did respond to 17 conjugated steroids, 11 of which (17β‐oestradiol‐17β‐glucuronide; 17β‐oestradiol‐3‐sulphate; 17β‐oestradiol‐3,17β‐disulphate; epiandrosteron‐3β‐sulphate; etiocholanolone‐3α‐glucuronide; testosterone‐17β‐sulphate; dehydroepiandrosterone‐3β‐sulphate; 5α‐pregnan‐3β‐ol‐20‐one‐3β‐sulphate; 5β‐pregnan‐3α,17‐diol‐20‐one‐3α‐glucuronide; 5β‐pregnan‐3α,17,21‐triol‐11,20‐dione‐3α‐glucuronide; pregnenolone‐3β‐sulphate) were selected for EOG concentration‐response, cross‐adaptation and binary mixture tests. The EOG detection thresholds ranged from 10^?11 to 10^?9 M in all but one instance (female threshold to pregnenolone‐3β‐sulphate; 10^?8 M), and males and females exhibited only minor differences in EOG threshold or response magnitude. Results of EOG cross‐adaptation tests, which were supported by results of binary mixture tests, indicated that the response to the 11 steroid conjugates is mediated by five putative olfactory receptor mechanisms characterized by specificity for conjugate position and type: 3‐sulphate, 17‐sulphate, 3,17‐disulphate, 3‐glucuronide, 17‐glucuronide. Although there is no evidence that H. burtoni releases, or exhibits biological response to, the steroids shown to be detected in this study, the present results are suggestive of a complex pheromone system utilizing steroid conjugates.     

Steroidogenic activity of hACTH and related peptides on the human neocortex and fetal adrenal cortex in organ culture.

Explants prepared from the neocortex and the fetal zone of the human fetal adrenal (gestational age 13 to 18weeks) were maintained under conditions of organ culture for 7 to 9 days during which time they were exposed to hACTH and various related peptides. Corticotrophic activity was monitored by the daily release of dehydroepiandrosterone sulfate (3beta-hydroxy-5-androsten-17-one, 3-sulfate; DHA-S) and cortisol as quantified by radioimmunoassay, hACTH (2.2 x 10(-9) - 2.2 x 10(-8)M) was the most active in sustaining steroidogenesis by both neocortical and fetocortical cells. alpha-MSH possessed similar properties but not at concentrations lower than 10(-6)M, whereas CLIP (4.4 x 10(-9) - 1.1 x 10(-7)M), the 18-39 C-terminal moiety of ACTH, was devoid of activity. Corticotrophic activity with respect to fetocortical explants appeared to be that of maintenance of function best illustrated by dehydroepiandrosterone sulfate biosynthesis, while enhancement of steroidogenesis was observed in the neocortex as manifested by cortisol release. Although not eliminating the possible existence of a specific fetal corticotrophin related to ACTH1-39, the data indicate that hACTH is capable of regulating steroidogenesis in the fetal zone which is primarily geared to the formation of dehydroepiandrosterone sulfate.     

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.     

Plasma sex hormone concentrations during the reproductive cycle in the male lizard, Podarcis s. sicula

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard.     

Pharmacological actions of 17 beta-oestradiol on articular cartilage chondrocytes and chondrosarcoma chondrocytes in the absence of oestrogen receptors

(1) Pharmacological concentrations (greater than 10(-5) M) of 17 beta-oestradiol inhibited 35S-labelled proteoglycan synthesis in bovine articular cartilage explant cultures. They also inhibited 35S-labelled proteoglycan synthesis and 3H-labelled protein synthesis in cell cultures of chondrocytes from bovine articular cartilage and Swarm rat chondrosarcoma. Maximal inhibition was about 30-50%. Physiological concentrations (10(-9)-10(-8) M) of oestradiol had no effect on the synthesis of either protein or proteoglycan. (2) The inhibitory action of high concentrations of oestradiol on these biosynthetic pathways is not common to all steroids since 10(-4) M cortisol had no effect on articular chondrocyte cell cultures. 10(-4) M testosterone had a similar action to oestradiol. (3) Neither physiological nor pharmacological concentrations of 17 beta-oestradiol had any effect on 35S-labelled proteoglycan turnover in the cartilage explant system. (4) 10(-5) M oestradiol inhibited cell division in cultures of articular chondrocytes which had entered the log growth phase. 10(-7) M oestradiol had no effect on articular chondrocyte growth. (5) In male rats implanted with silastic capsules releasing 17 beta-oestradiol, increase in body weight was retarded by about 25% over a period of 6 weeks, compared to control rats. Rat chondrosarcoma grew to the same size in oestrogen-treated rats as it did in controls. (6) Oestrogen receptors could not be detected in freshly isolated bovine articular chondrocytes or in rat chondrosarcoma. (7) In conclusion, neither the mitotic rate of articular chondrocytes nor their proteoglycan metabolism is under the direct physiological control of oestradiol. Growth and biosynthetic activity of the rat chondrosarcoma chondrocytes are independent of either direct control by the hormone or control effected by oestradiol regulation of a second hormone or growth factor.     

Adrenal androgen concentrations in breast tumours and in normal breast tissue. The relationship to oestradiol metabolism

Concentrations of ADIOL, DHA and DHAS were measured in human breast tumours and normal tissue from the same breast and related to 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity in these tissues. ADIOL and DHA were significantly higher in tumour tissue compared to normal tissue from the same breast (paired t-test: P less than 0.05 and P less than 0.01 respectively) whereas the difference between concentrations of DHAS in normal tissue and tumour tissue was not significant. There was a positive correlation between ADIOL and DHA in both tissues (P less than 0.001) but for DHAS the relationship was only significant in normal tissue (ADIOL:DHAS, P less than 0.001; DHA:DHAS, P less than 0.002). An increase in 17 beta-HSD activity was associated with an increase in DHAS concentrations in both normal and tumour tissue (P less than 0.01 and P less than 0.001 respectively) and with an increase in DHA concentrations in normal tissue (P less than 0.05). These results might be explained by an impairment in the balance between sulphatase and sulphotransferase activity in breast tumours.     

Metabolism of [3H]oestradiol in vivo by normal breast and tumour tissue in postmenopausal women

Tumour and normal breast tissue was obtained from postmenopausal breast cancer patients following [3H]oestradiol infusion (50 mu Ci over a 12 h period). The fraction of radioactivity present as oestradiol or oestrone was measured and the results expressed both as the ratio of oestradiol-oestrone and as the percentage oestrogen present as oestrone, and the findings compared with in vitro measurements of 17 beta-hydroxysteroid dehydrogenase activity. Concentrations of 5-androstene-3 beta, 17 beta-diol, dehydroepiandrosterone and its sulphate and testosterone were measured and related to oestradiol metabolism. The study demonstrated that tumour tissue is less able to metabolise oestradiol to oestrone than is normal breast tissue and indicated that the ability of the tissue to detoxify oestradiol may be dependent on cofactor availability. The results also supported the possibility that increased tissue concentrations of adrenal androgens inhibit oestradiol and thus increase tissue exposure to oestradiol.     

Effects of dihydrotestosterone treatment on adrenal gland function and morphology in adult female guinea-pigs

The effect of chronic treatment of female guinea-pigs with dihydrotestosterone (DHT) on growth and function of the adrenal gland and, in particular, on the reticular zone is described. Two groups of 6 young adult, female guinea-pigs were treated with DHT (1 mg/kg dissolved in peanut oil and injected s.c.) for 30 and 60 days. Two other groups of animals, treated only with oil, were used as controls. At the end of treatment, animals were killed and adrenal glands were quickly removed. Plasma levels of pregnenolone, dehydroepiandrosterone (DHA) and its sulfate (DHA-S), 17 alpha-hydroxyprogesterone, androstenedione, testosterone, estradiol, 11-deoxycortisol, androstenedione, DHT and 3 alpha-androstanediol were determined by R.I.A. following celite microcolumn chromatography. Animals treated for 30 days showed only elevated DHT and 3 alpha-androstanediol plasma levels, whereas animals treated for 60 days also showed increased values of pregnenolone (251 +/- 62 vs 193 +/- 51 ng/dl; P less than 0.05), DHA-S (12,046 +/- 4110 vs 2780 +/- 888 ng/dl; P less than 0.001) and slightly increased values of DHA (110 +/- 31 vs 86.5 +/- 55.4). In the 30-day-treated animals no histological changes were observed, but in the 60-day-treated group the total size as well as cell volumes of the zona reticularis were significantly increased. Normal estrous cycles were observed in the 30-day-treated animals whereas the 60-day-treated animals showed a progressive acyclicity during the second month of treatment. These results indicate that in guinea-pigs, prolonged treatment with DHT induces a growth of the zona reticularis of the adrenal gland associated with increased levels of 5-ene steroids, particularly DHA-S. The mechanisms inducing these modifications are probably mediated by a DHT effect at the hypothalamic-pituitary level. A direct effect of DHT on the zona reticularis, however, cannot be excluded.     

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.