Effect of Long Term Treatment with Gonadotrophic Hormones on In Vitro Metabolism of [3H]Progesterone in Human Testicular Tissue

Gonadotrophic hormones appear to influence the preferred pathways for intratesticular androgen biosynthesis. Individuals with low levels of gonadotrophin in the peripheral circulation, as e. g. prepubertal boys and a hypophysectomized adult male, have previously been shown to metabolize [³H]progesterone in vitro mainly to 20α-dihydroprogesterone (20α-DH-P) and less to 17α-hydroxyprogesterone (17α-OH-P), i. e. an "immature metabolic pattern" (Berg et al. 1976; Kjessler & Berg 1976b).
With physiologically increasing amounts of circulating gonadotrophins the preferred metabolic pathway in vitro shifts in favour of 17α-OH-P, i. e. a "mature metabolic pattern". Such an alteration in the preferred in vitro metabolic pattern of [³H]progesterone has also been obtained by exogenous administration of gonadotrophins to a 47, XYY-male with an initially immature metabolic pattern (Berg & Kjessler 1976).
We have sequentially analysed the metabolism of [³H]progesterone in vitro in testicular incubates derived from a chromosomally normal male before and after 16 weeks of substitution therapy with gonadotrophic hormones.
The patient originally displayed an "immature" pattern of progesterone metabolites, i. e. the ratio 20α-DH-P/17é-OH-P was 3.04. After treatment with human menopausal gonadotrophins and hCG, the metabolic activity in total had increased, and the ratio 20é-DH-P/17é-OH-P had switched to 0.39.
These results confirm and extend the concept that gonadotrophic hormones may have a regulatory function on androgen biosynthesis by stimulating the oxidative metabolic pathway from progesterone to biologically active androgens via 17α-hydroxyprogesterone.     

In Vitro Metabolism of [3H]Progesterone in Testicular Tissue Derived from Seven Males Treated with Oestrogens

Oestrogens administered to adult males are known to suppress the synthesis of androgens in the testicles. A main effect of this treatment on testicular steroid metabolism in vitro seems to be a significant reduction of 17α-hydroxylase activity. A concomitant increase in the 20α-hydroxysteroid dehydrogenase activity was, however, observed in four young oestrogen-treated transsexual males (Rodriques et al. 1976).
We have analysed the metabolism of [³H]progesterone in vitro in testicular tissue incubates derived from six males on current oestrogen treatment and from one male 19 weeks after cessation of oestrogen therapy.
The metabolism in general of [³H]progesterone in vitro was observed to be decreased in all males on oestrogen treatment as compared to control subjects. The metabolite 20α-dihydroprogesterone was found to constitute between 85–91 per cent of all metabolites formed in the patients under treatment.
On the contrary, the metabolic pattern observed in the previously treated male, appeared to be fully consistent with the patterns observed in non-treated control subjects. Thus, the main alterations in intratesticular progesterone-metabolism caused by exogenous administration of oestrogens, at least for 23 consecutive weeks, seem to be completely reversible.     

Testicular Steroid Metabolism In Vitro and Spermatogenesis in a Male with an XYY-Karyotype

Males with a 47, XYY-chromosomal constitution have been reported to generally suffer from an impaired gametogenesis, and the reason for this spermatogenetic failure is unknown.
The concentration of gonadotrophic hormones and testosterone in the peripheral circulation of XYY-males have not been found to differ significantly from normal control subjects, but so far there is a lack of information on the intratesticular steroid conversion patterns in such individuals.
We have repeately analysed the metabolism of [³H] progesterone in vitro in incubated testicular biopsy specimens derived from an XYY-male before and after gonadotrophic substitution therapy. In addition hormone levels in peripheral blood samples, gametic output and testicular histopathology were investigated.
The present patient was a 34-year-old healthy, but infertile male with a 47.XYY-karyotype. His gonads were reduced in volume (6–8 ml) and displayed a heterogenous morphological picture. The appearance of the tubules varied from normal to complete destitution of germinal cells. The Leydig cell apparatus appeared normal. Serum levels of FSH, ICSH (LH) and testosterone were repeatedly within normal limits. The seminal fluid contained less than 10 mill. sperm per ml.
Incubated testicular biopsy specimens metabolized [³H] progesterone in vitro mainly to 20α-dihydroprogesterone (20α-DH-P) and to a less extent to 17α-hydroxyprogesterone (17α-OH-P). Thus, the ratio 20α-DH-P:17α-OH-P was found to be remarkably high, i.e. an immature pattern often observed in prepubertal testicular tissue (Berg et al. 1976). By exogenous administration of gonadotrophic hormones to the present patient the steroid metabolic pattern in vitro changed in favour of an increased production of 17α-OH-P, concomitantly with an observed rise in gametic output.     

Human testicular LH receptors: correlations with circulating gonadotrophins and testicular steroid secretion

Testicular androgen production is regulated by circulating LH, the effect of which is mediated by its testicular membrane receptors. In the present study, testicular [¹²⁵I]hCG binding in man was investigated and found to be characterized by saturability and high affinity (mean K_D in testes of 21 patients with prostatic carcinoma = 1.64 times 10^-10 M), and stimulated testosterone production in dispersed interstitial cells in vitro. The concentrations of the LH receptors correlated with serum FSH concentrations (r = 0.52, P < 0.05) but not with circulating LH levels. Neither had they any correlation with intratesticular testosterone, 5α-dihydrotestosterone, androstenedione, progesterone, 17-hydroxyprogesterone or pregnenolone concentrations, which may be due to the fact that LH receptors are confined to Leydig cells, whereas steroids may be unevenly distributed in the different cells of the testis. In contrast, the concentrations of the LH receptor displayed positive correlations with the concentrations of testosterone (r = 0.81, P < 0.001), androstenedione (r = 0.54, P <0.05), 17-hydroxyprogesterone (r = 0.76, P <0.001) and progesterone (r = 0.60, P < 0.05) in the spermatic vein serum of the patients. Our data suggest that the Leydig cells mainly responsible for steroid secretion into the blood are under gonadotrophic control exerted via their receptors, whereas Leydig cell function is not rapidly reflected in steroid concentrations within the testis itself.     

Effect of Clomiphene Citrate on Testicular Steroid Metabolism in Man

Administration of clomiphene citrate to males with normal peripheral testosterone levels increased the serum levels of FSH. LH, testosterone and low polar oestrogens and also the urinary excretion of LH, low polar oestrogens, dehydroepiandrosterone, androsterone and actiocholanolone. No significant change was noted for the urinary excretion of 17-oxogenic steroids. Addition of clomiphene citrate (10^-4 to 2 times 10^-3 M) to incubations of [7^-3H, 21^-14C] progesterone with human testicular biopsy specimens in vitro caused a slight inhibition in the formation of 17α-hydroxyproge-sterone and a slightly increased formation of 20α-hydroxy-4-pregnen-3-one but no measurable changes in the C₁₉ steroid formation. The results support the hypothesis that there is selective action of clomiphene citrate upon the adrenal C₁₉ steroid biosynthesis in the human male, combined with an indirect effect upon the testes via the hypothalamic-pituitary system. A direct effect on the gonadal steroidogenesis can, however, not be ruled out.     

Effect of Chronic Neutralization of Endogenous FSH on Testicular Function in the Adult Male Bonnet Monkey – Assessment Using Biochemical Parameters

The effect of long-term passive immunization with a specific homologous antiserum to ovine FSH on testicular function in the adult bonnet monkey was studied. Testicular function was assessed by studying biochemical parameters such as hyaluronidase activity and the rate of incorporation of [³H]thymidine into DNA of testis in vitro .
The above parameters were determined in one group of monkeys at 210 days and in another group at 130 days after initiation of antiserum treatment. The testis of both groups of monkeys showed a marked reduction in the rate of [³H]thymidine incorporation into testicular DNA as well as in hyaluronidase activity compared to the corresponding control animals. Further, on withdrawal of antiserum treatment in one set of monkeys, a clear recovery was observed in these parameters, thus demonstrating that the earlier observed effects were specifically due to lack of FSH.
The results clearly indicate that chronic lack of FSH affects testicular function in the adult monkey and it is suggested that FSH could have a positive role in spermatogenesis in the adult sub-human primate.     

In vitro metabolism of progesterone by the human undescended testis

Testicular biopsy specimens from 28 boys with undescended testis, and from 6 men operated post-pubertally for undescended testis, were incubated in vitro with [³H]progesterone (P). Significant steroid metabolic activity was demonstrated in all biopsies. Before puberty the total conversion rate was low and only a small amount of 17α-hydroxy-progesterone (17α-OH-P) was formed. The amount of newly formed 20α-dihydro-progesterone (20α-DH-P) was relatively constant regardless of increasing maturity. After puberty the total conversion rate was higher. More 17α-OH-P was synthesized, and the ratio between formed 20α-DH-P and 17α-OH-P decreased significantly. The position of the undescended testis did not appear to influence progesterone metabolism. In no case could we demonstrate deficient steroidogenesis. In one 17-year old boy the 20α-DH-P/17α-OH-P ratio was lower in the undescended testis (0.9) than in the scrotal testis (4.0) suggesting increased steroidogenesis per mg tissue in the malpositioned testis. The indication from the present study, that even grossly displaced testes have a relatively undisturbed steroidogenic capability, suggests that the reason for impaired descent may not simply reflect disturbed androgen synthesis, but must involve other mechanisms.     

In Vitro Steroidogenesis and Hormone Levels in Pooled Human Vas Deferens

The ability of human vas deferens to utilize labelled pregnenolone and testosterone for the synthesis of androgens was studied and correlated with the endogenous hormone levels present in the pooled tissue. In vitro incubation of vas tissue with [4-¹⁴C]-pregnenolone resulted in accumulation of more radioactivity in dehydroepiandrosterone and testosterone compared to that observed in 4-androstene-3,17-dione and progesterone. Labelled [¹⁴C]-testosterone was mainly converted to 4-androstene-3,17-dione and 5α-dihydrotestosterone, the counts being more in the former than in the latter steroid. The endogenous levels of progesterone, testosterone and 4-androstene-3,17-dione in the vas tissue were higher than the levels of 5α-dihydrotestosterone and dehydroepiandrosterone. Thus, the vas deferens appears to have all the enzymes necessary for the synthesis of testosterone and the major metabolite of testosterone was not 5α-dihydrotestosterone as in other male accessory sex organs.     

Effect of Low Doses of Alpha Chlorohydrin on the Dehydrogenases and Oxidases of Rat Testis. A Histochemical Study

Effekt einer low-dose Behandlung mit α-Chlorohydrin auf die Dehydrogenase und Oxidasen im Rattenhoden — Eine histochemische Untersuchung
In dieser histochemischen Untersuchung wurden folgende Enzyme nach low-dose Behandlung mit α-Chlorohydrin (6,5 mg/kg/9 Tage) in den verschiedenen Zellarten des Rattenhodens untersucht: Isocitratdehydrogenase, Succinatdehydrogenase, Malatdehydrogenase, Glutamatdehydrogenase, Δ⁵- 3α-Hydroxysteroiddehydrogenase, NAD- und NADP-Diaphorase und Monoaminoxidase. Die Anwendung von α-Chlorohydrin führt zu einer Verminderung oder dem Verschwinden all dieser Enzyme bis auf NADP-Diaphorase und Δ⁵ -3α-HSD. Diese Veränderungen der Enzymaktivitäten im Rattenhoden zeigen deutlich, daß nach einer low-dose Behandlung mit α-Chlorohydrin lange bevor histologische Defekte auftreten, der Zitronensäurezyklus und Aminosäurestoffwechsel im Hoden geschädigt werden. Die Steroidbiosynthese bleibt jedoch unbeeinträchtigt, wie die gleichbleibenden Aktivitäten der Δ⁵ -3α-HSD und NADP-Diaphorase bewiesen.     

Arachidonic acid metabolites as intratesticular factors controlling androgen production

The effect of inhibitors and products of arachidonic acid metabolism on rat testicular steroidogenesis has been investigated. In the presence of indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (NDGA) (inhibitor of lipoxygenase), the activity of 3P-hydroxy steroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) were both inhibited. The LH-stimulated increase in secretion of testosterone and progesterone was also inhibited by indomethacin and NDGA. On the other hand, vitamin E (anti-oxidant and inhibitor of lipoxygenase), stimulated the activity of both 3β-HSD and 17β-HSD and enhanced LH-stimulated androgen production. The metabolites of lipoxygenase (15-HPETE, 15-HETE, 5-HPETE and 5-HETE) and cyclooxygenase (PGF_2α) pathways stimulated 3β-HSD and 17β-HSD activity and enhanced the secretion of progesterone and testosterone. It is concluded that arachidonic acid matabolites are intratesticular factors which can regulate LH-stimulated testicular steroidogenesis.     

Progestin Receptors in the Anterior Pituitary and Hypothalamus of Male Rats

Properties of progestin receptors in the pituitary and hypothalamic cytosol fraction of male rats were studied using the highly potent progestin (³H) R 5020 (17,20-dimethyl-19-nor-pregna-4,9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of (³H) R 5020 is associated with components migrating at 7 S and 4 S. The isoelectric point of the binding components were 5.8. The binding of (³H) R 5020 is highly specific since it is easily displaced by unlabelled R 5020 and progesterone. The affinity of unlabelled testosterone, 17β-estradiol and cyproterone acetate was much lower, whereas unlabelled cortisol had no affinity for the progestin receptor. The dissociation constant of pituitary R 5020 binding is approximately 1 times 10^-9 M.
Pretreatment of the animals with 17β-estradiol-valerate greatly increased the progestin receptor levels in the tissues.     

Heterogeneity in end-terminal sugars of rabbit and rat androgen binding protein (ABP)

The interaction between rabbit and rat androgen binding protein (ABP) and rabbit serum testosterone binding globulin (TeBG) with concanavalin A (Con A) was studied using affinity chromatography on Con A-Sepharose 4 B columns. When partly purified rat ABP, equilibrated with [³H]5α-dihydro-testosterone [³H]DHT was applied to Con A-Sepharose columns, approximately 50% of the ABP was retained by the column, whereas the remaining was eluted with the break-through protein fraction. A similar picture was found using partly purified rabbit ABP, or crude rabbit rete testis fluid. These studies indicate that both rat and rabbit ABP are glycoproteins, showing heterogeneity in their end-terminal sugars. When partly purified rabbit TeBG was examined by Con A-Sepharose affinity chromatography, the TeBG was completely retained by the column. The different elution patterns between rabbit ABP and rabbit TeBG indicate that these proteins, although showing identical physico-chemical and immunological properties (Weddington et al. 1975a, b). possess differences in their carbohydrate content.     

Histochemical Demonstration of Δ5,3β-hydroxysteroid Dehydrogenase Activity in Cultured Leydig Cells under the Influence of Gonadotropic Hormones and Testosterone

The influence of gonadotropic hormones or testosterone upon activity of Δ⁵, 3β-hydroxysteroid dehydrogenase (Δ⁵,3β-OH-SDH) within cultured Leydig cells was histochemically investigated.
The following amounts of hormones were added into the culture medium: LH – 10, 100, 500, and 1000 ng/ml, FSH – 100 ng/ml, Testosterone (T) – 150 ng/ml of culture medium. Doses 10 and 100 ng LH stimulated the activity of the histochemically investigated enzyme, while 500 ng decreased enzyme activity and 1000 ng failed to support culture at all. FSH did not exert any influence on enzyme activity of cultured cells while testosterone decreased its activity beginning on day 6 in culture.     

Determination of Δ5 3β-hydroxysteroid dehydrogenase activity in intact isolated rat Leydig cells

A method for the determination of Δ⁵ 3β-hydroxysteroid dehydrogenaseisomerase (3β-HSD) activity in intact isolated Leydig cells was established. This method utilizes the conversion of [7-³H]dehydroepiandrosterone (1.04 μmole) to androstenedione and expresses the activity of the enzyme as μmoles of androstenedione produced/μg DNA/h. The reaction is limited to 0.5-4 μg DNA of Leydig cells/ml (equivalent to 0.1-0.8 million of Levdig cells/ml) and to 1 h of incubation at 34°C. The 3β-HSD activity of 44 suspensions of Leydig cells isolated from adult rats was found to be 1.13 pL 0.03 (SE) μmoles/μg DNA/h. This new method for direct measurement of 3β-HSD activity in intact Leydig cells was found to be rapid, easy to perform and highly reproducible.     

Autoradiographic Study of the [3H] Carnitine Distribution in Rat Epididymis

The distribution of [³H] carnitine in the epididymis has been studied by autoradiography 93 hours after injection of [³H]deoxycarnitine into intact, hemicastrated or hypophysectomized rats. In the intact animals, radioactivity is concentrated in the lumen of the caput and corpus with only a small amount observed in the caudal portion of the epididymis. In the hemicastrated rat, devoid of spermatozoa, the amount of radioactivity in the lumen of the caput and the cauda portions are lower than in the intact rats but still greater compared to the epididymal cells. In hypophysectomized rats the luminal labelling is much reduced. In contrast to the epididymis, the amount of radioactivity is low and evenly distributed in the heart from intact, hemicastrated and hypophysectomized rats and in the pituitary from intact and hemicastrated animals. The great reduction in luminal labelling after hypophysectomy emphasizes the hormonal dependence of the carnitine concentrating mechanism. These findings indicate that carnitine initially is concentrated in the caput and corpus portion of the epididymis. This intraluminal carnitine is subsequently transported into the caudal segment.
The evidence strongly supports the existence of a hormonal dependent carnitine concentrating mechanism localized on the luminal side of the epithelium in epididymis.     

Direct Effects of Medroxyprogesterone Acetate on Testes: Possible Mechanisms Examined by Testicular Perfusion

The uptake of ³H-testosterone by the nuclear androgen receptor of rat testis was studied using a perfusion system which was adapted for the simultaneous perfusion of 8 testes. Following perfusion with ³H-testosterone, the major nuclear steroid in the testis of hypophysectomized rats was testosterone rather than one of its 5α-metabolites. The accumulation of ³H-testosterone in testicular nuclei was saturable, inhibited by perfusion with excess testosterone or 4 progestins (including medroxyprogesterone acetate, MPA) which are known to bind to the androgen receptor. Saturation of testicular androgen receptors with ³H-MPA could not be demonstrated due to the high non-specific binding of this non-polar steroid. However, specific binding of ³H-MPA was demonstrated by fractionation of salt-extractable testicular nuclear receptor on sucrose gradients.
Perfused rat testes were also used to examine the direct effects of MPA on testosterone secretion. When testes were perfused with MPA, 20 μg/ml (but not 1 μg/ml) both basal and hCG induced testosterone secretion were inhibited 30 % and 60 %, respectively. In conclusion, MPA could exert a direct effect on testis via the androgen receptor as it docs in other androgen responsive tissues. Another direct effect of this progestin on the testis could be by inhibiting testosterone secretion. 7his response requires high levels of MPA in the perfusion medium suggesting that this might be a pharmacological effect of this pregestin.     

Effects of Prostaglandins and Indomethacin on the Response of Rat Testis to LH in vivo

In adult male rats, bilateral intratesticular administration of 20 μg PGA₁ or 20 μg indomethacin significantly reduced the acute stimulation of plasma testosterone levels by concomitantly injected LH, while treatment with the same dose of PGF_2α was without effect. After six days of treatment with indomethacin, plasma testosterone levels were reduced to approximately 1/3 of the control levels.
Intratesticular administration of 10 μg LH per testis per day produced, after six days, a precipitous decline in plasma testosterone levels and a decrease in the weight of the testes. This was probably due to desensitization of Leydig cells by these high doses of gonadotropin. Administration of PGF_2α together with LH caused a further decrease in testicular weight and a significant reduction in the weights of the prostate and the seminal vesicles.     

Regional Differences in Steroidogenesis and Hormone Levels in the Epididymis and Vas Deferens of Adult Rats

In vivo and in vitro studies with different parts of the epididymis and vas deferens were carried out to determine their inherent capacity to synthesize steroids and to correlate with the endogenous levels with or without the administration of hCG.
Incubation with ¹⁴C-labelled pregnenolone and testosterone demonstrated that caput epididymidis was more active than other parts in synthesizing testosterone from ¹⁴C-pregnenolone and in converting labelled testosterone to 5α-dihydrotestosterone (DHT). The cauda epididymidis and vas deferens accumulated more radioactivity in progesterone and dehydroepiandrosterone (DHEA) than the caput epididymidis.
The levels of DHT, testosterone and 4-androstene-3,17-dione in the caput epididymidis were reduced after ligation of ipselateral efferent ductules indicating the testicular origin of these steroids. The cauda epididymidis and vas deferens had higher levels of progesterone as compared to the other regions of the epididymis, which were decreased after the ligation. Intravenous injection of hCG increased the levels of oestradiol-17β in all tissues and markedly in the cauda epididymidis and vas deferens. The high levels of progesterone and oestradiol-17β present in these organs may be of importance in maintaining fertilizing ability of spermatozoa stored in the cauda epididymidis and vas deferens and their transport.     

Effects of retinol on glycoprotein synthesis by Sertoli cells in culture: dolichyl phosphomannose synthase activation

Sertoli cells were isolated from Wistar rats aged 19 days and cultured for 48 h. The addition of retinol (10 μM) to the culture medium significantly stimulated the incorporation of [2–³H]mannose into lipid-linked oligosaccharide and into cellular and secreted glycoproteins. Incorporation of [U- 14C] leucine into proteins and of [5, 6–³ H] uridine into RNA was unaffected by retinol treatment. Incubation of microsomal fractions of retinol-treated cells showed an increase in mannose incorporation into dolichyl phosphomannose, into dolichyl pyrophosphoryl oligosaccharide and into proteins. Chromatographic analysis of the fraction soluble in chloroform/methanol (2: 1 v/v) did not show the presence of retinyl phosphomannose either in control or in retinol-treated cells. When the formation of dolichyl phosphomannose was studied in microsomes isolated from control cells and from cells treated with 10 μM retinol for 48 h in the presence of exogenous dolichyl phosphate, the results showed that the retinol effect was due to stimulation of dolichyl phosphomannose synthase.     

Effect of continual light deprivation and alpha-2u-globulin replacement therapy on serum concentration of gonadotropins and testicular activity in rats

Summary. Prolonged darkness caused a fall in testicular 17 β-hydroxysteroid dehydrogenase (17β-HSD) activity and diminished spermatogenesis, serum levels of gonadotropins, testosterone and α_2u-globulin.
Administration of α_2u-globulin at a dose of 1.5 mg rat⁻¹ per day for 7 days after 68 days of light deprivation, reversed the 17β-HSD activity and serum levels of gonadotropins, testosterone and α_2u-globulin, while spermatogenesis was restored to normal.
The animals kept in prolonged darkness for 68 days and then received saline (7 days in light-dark cycle, 14 L: 10 D), showed no significant changes of testicular activity, serum levels of gonadotropins, testosterone and α_2u-globulin, when compared with dark-exposed animals (68 days) receiving rabbit serum (7 days in light-dark cycle, 14 L: 10 D). These results suggest that α_2u-globulin plays an important role in testicular function in dark-exposed rats by inducing gonadotropins and testosterone secretion.