Robert K. Tcholakian

Direct measurement of androgen receptors in cultured Sertoli cells.

Cytoplasmic and nuclear forms of macromolecules with the properties of androgen receptors have been demonstrated by direct labeling techniques in cultured Sertoli cells. The cytoplasmic form was excluded from Sephadex G-200 and could be distinguished from androgen binding protein (ABP) on the basis of size, heat stability, relative electrophoretic mobility, and binding complex dissociation rate. When cultured Sertoli cells were incubated with 3H-testosterone, a time- and temperature-dependent accumulation of label into the nuclear fraction was observed, 46% of which crystallized as authentic testosterone. Specific binding was saturable with an apparent association constant of 0.4nM-1. Approximately 30% of the nuclear bound hormone was extracted within 1 h by 0.4M KCl and 34% of this was associated with macromolecular species as measured by gel filtration. Unlabeled androgens and to some degree progestogens competed with 3H-testosterone for binding sites. These data constitute direct evidence that Sertoli cells contain androgen receptors.

Effect of Prednisone on Plasma Testosterone Levels and on Duration of Phases of the Menstrual Cycle in Hyperandrogenic Women

A group of 106 women of reproductive age with laboratory and clinical evidence of hyperandrogenism was treated with prednisone. The daily dosage varied between 7.5 and 10 mg. Ovulatory activity was assessed prior to and during therapy by basal body temperature and observation of changes in the cervical os and cervical mucus. Plasma testosterone levels were significantly suppressed by prednisone therapy. This was associated with initiation of ovulatory activity in 5 of 14 (35.7%) amenorrheic patients and 10 of 11 (90.9%) anovulatory patients. In 81 ovulatory patients, prednisone therapy resulted in statistically significant shortening of the follicular phase and lengthening of the luteal phase of the menstrual cycle. The mean length of the menstrual cycle was unchanged. Significant correlations between percentage suppression of plasma testosterone and shortening of the follicular phase or lengthening of the luteal phase were observed. Suppression of plasma testosterone by prednisone was maximal after 2 months of treatment, while the effect on the phases of the menstrual cycle was progressive with duration of treatment. The effects of prednisone at daily dosages of 7.5 or 10 mg were not significantly different. These results suggest that prednisone therapy in hyperandrogenic women exerts an effect on both phases of the menstrual cycle, possibly related to suppression of plasma testosterone levels.

Unusual Anti-Reproductive Properties of the Analog [d-Leu6,Des-Gly- N H 2 10 ]-Luteinizing Hormone-Releasing Hormone Ethylamide in Male Rats *

Nanogram quantities of a luteinizing hormone-releasing hormone (LHRH) agonist, [D-Leu6,des-Gly-NH2(10)]-LHRH ethylamide, were injected into immature male rats twice daily for 40 days. Treatment with the analog significantly increased serum luteinizing hormone and follicle-stimulating hormone levels, but suppressed accessory sex organ weights. Plasma and testicular concentrations of testosterone were also significantly decreased. The agonist did not alter body, pituitary, or testis weights. Spermatogenesis and the morphology or density of the Leydig cells also appeared not to be affected. A possible direct effect of the analog on the testes is discussed.

In vitro steroid metabolic studies in human testes I: Effects of estrogen on progesterone metabolism

A technique of incubation of testicular tissue in vitro with radiolabeled precursors was applied in the investigation of the steroid biosynthesis by testes of four young men after long-term, high-dose estrogen treatment. A positive correlation between plasma and testicular steroid levels, and in vitro capacity of the testes to metabolize progesterone was demonstrated. Estrogen administration produced a very significant inhibition of plasma and testicular levels of testosterone. The in vitro synthesis of testosterone from progesterone was very severely impaired; especially 17alpha-hydroxylation of progesterone. 20alpha-hydroxysteroid-dehydrogenase activity was found to be increased after estrogen treatment, both in vivo and in vitro. These findings suggest that testicular 17alpha-hydroxylase activity (and possibly also 17-20 lyase activity) is either under gonadotropin regulation, or is directly suppressed by estrogen. This could result by decreased enzyme synthesis, direct enzyme inhibition or affectation of the cofactors or cytochromes necessary for the enzymatic activity. 20alpha-reduction of C21-steroids would represent an alternative pathway for their catabolism, not regulated by gonadotropin or not affected by estrogen, that would be significant in situations with reduced 17alpha-hydroxylase activity.

The Relation Between Plasma Testosterone Levels and The Lengths of Phases of the Menstrual Cycle

Plasma testosterone levels were measured in 331 women of reproductive age. The incidence of clinical signs of hyperandrogenism (hirsutism, acne) was recorded. Ovulatory activity was evaluated clinically by basal body temperature and frequent observation of changes in the appearance of the cervical os and cervical mucus. Plasma testosterone levels were abnormally elevated in patients with clinical signs of hyperandrogenism. The highest mean testosterone levels were noted in the group of hyperandrogenic women with amenorrhea. Significant prolongation of the follicular phase and shortening of the luteal phase were demonstrated to be associated with clinical signs of hyperandrogenism and elevated plasma testosterone levels. Statistically significant correlations between plasma testosterone levels and duration of phases of the menstrual cycle were observed. Testosterone levels were directly related to the length of the follicular phase and inversely related to the length of the luteal phase. A significant inverse correlation between the lengths of the two phases of the menstrual cycle was also demonstrated. These results suggest an association between hyperandrogenism and prolongation of the follicular phase and shortening of the luteal phase of the menstrual cycle, possibly related to the high incidence of infertility and menstrual irregularity reported for hyperandrogenic women.

Effects of various doses of testosterone propionate on intratesticular and plasma testosterone levels and maintenance of spermatogenesis in adult hypophysectomized rats

Adult hypophysectomized rats were maintained on different regimens of testosterone propionate (TP) treatment for 27 days (0.2, 0.4, 0.6 and 1 mg/day) and autopsied 16 hours after the last injection. Blood samples were taken, sex organs were weighed and one testis from each animal was fixed in Bouins fluid for histologic analysis. The other testis and blood were used for testosterone (T) determinations. Both testicular and plasma T were below detectable levels in hypophysectomized control rats. The plasma T level showed a dose response relationship with increasing dose of TP but such was not the case for intratesticular T concentrations. Qualitative and quantitative evaluation of testis sections showed that spermatogenesis was incomplete in rats receiving 0.2 mg TP/day characterized by the absence of step 15 to 19 spermatids, degeneration of some pachytene spermatocytes and a significantly lower yield of B type spermatogonia. Analysis of testis sections from animals treated with 0.4 to 1 mg TP/day showed complete maintenance and maturation of pachytene spermatocytes, meiosis and spermiogenesis. However, even with the highest dose of TP (1 mg/day) the total yield of B type spermatogonia was only about 58% of the intact controls. It is concluded that at least 0.4 mg/day of exogenous TP is essential for qualitative maintenance of spermatogenesis in hypophysectomized rats with an intratesticular T concentration of 17 to 18 ng/gm testis.

Testosterone levels in female partners of infertile couples: Relationship between androgen levels in the woman, the male factor, and the incidence of pregnancy

Plasma testosterone levels were measured in the female partners of 146 consecutive infertile couples. The incidence of hyperandrogenism in the woman was correlated with ovarian function, incidence of pregnancy, male factor, and response of plasma testosterone levels to prednisone treatment. Over 70 per cent of the patients had pretreatment testosterone levels above 40 ng. per 100 ml. while after a minimum of two months of therapy approximately 80 per cent had levels below 40 ng. per 100 ml. High levels of plasma testosterone were associated with significant prolongation of the follicular phase of the cycle and increased incidence of amenorrhea or anovulation. An over-all pregnancy rate of 50.4 per cent resulted from the treatment. A direct relationship between pregnancy rates and sperm density as well as between pregnancy rates and degree of suppression of plasma testosterone after therapy was observed. These results demonstrate a high incidence of hyperandrogenism in female partners of infertile couples. The effectiveness of glucocorticoid treatment appears to be related to suppression of excessive androgen levels. The data also suggest that infertility is a relative state related to the fertility potential of each member of the couple. Improvement of the fertility potential of either member may result in conception.

In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH.

Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-17 beta. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures; testosterone (unconverted substrate), androstenedione, dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha, 17 beta-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-17 beta-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol--17 beta failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-17 beta added as recovery tracer did. The data demonstrate that : 1) cultures of Sertoli cells from immature rats have 5 alpha-reductase, 3 alpha- and 17 beta-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-17 beta even in the presence of FSH.

Effect of Estradiol Benzoate on the Pituitary‐Gonadal Axis in the Intact Male Rat

Erwachsene männliche Ratten wurden 28 Tage lang täglich mit 50 μg Oestradiolbenzoat subcutan behandelt. Die Gewichte der Sexualorgane und der Hypophyse zeigten die für eine Oestrogenzufuhr klassischen Veränderungen. Sowohl die Plasma‐ als auch die Hoden‐Testosteronwerte gingen innerhalb von 24 Stunden nach der ersten Oestrogeninjektion auf fast Spurenwerte zurück und blieben dort während der Experimentierperiode. Die Hypophysen‐LH‐Werte und deren Konzentration zeigten einen progressiven Abfall; demgegenüber blieben die Plasma‐LH‐Werte während des Experimentes unverändert. Dieses Ergebnis legt es nahe, daß bei der gesunden männlichen Ratte das Oestrogen die Hoden‐Testosteron‐Produktion direkt beeinflußt. Die Beobachtung gibt Veranlassung für die Notwendigkeit einer Überprüfung der gegenwärtigen Vorstellungen über die Hoden‐Hypophyse‐feedback‐Mechanismen.

Steroidogenesis in testicular cells

Abstract The current state of knowledge concerning steroid biosynthesis and metabolism in isolated testicular cells is discussed. Data demonstrating conversion of progesterone to testosterone and 5α-reduced androgens in cultures of pure Sertoli cells derived from adult rat testes is presented. The constant ratio of total progesterone metabolism to total C-19 steroids observed in these studies suggests that the rate of androgen formation in Sertoli cells depends primarily upon the activities of the various steroidogenic enzymes. The data also suggest that the principal site of conversion of progesterone to androgens in the seminiferous tubules is the Sertoli cell and that the principal site of 5α-reductase activity in the testis is also the Sertoli cell. The Sertoli cell was also shown to be able to convert [ 14 C]-testosterone to steroids with Chromatographic characteristics of oestrogens which were reactive in estradiol radioimmunoassay and their production was stimulated by FSH. However, they failed to crystallize to constant specific activity or constant ratio, while authentic [ 3 H]-oestradiol 17-β used as tracer did crystallize. Thus, our studies failed to demonstrate conversion of testosterone to oestradiol-17β by cultured Sertoli cells. The identity of the steroids formed is currently under investigation.