Testicular 3 alpha-hydroxysteroid oxidoreductase activity in the developing male rat.

In the testes, 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone, DHT) is converted to 5α-androstane-3α,17β-diol (3α-diol) by the enzyme 3α-hydroxysteroid oxidoreductase (3α-HSO). This steroid has been shown to possess biological activity in the male rat. The secretion of 3α-diol is much greater in the prepubertal animal than in the adult. This study is designed to quantitate the activity of 3α-HSO in the cytosol fraction of testes from male rats throughout sexual development. Following homogenizatlon of whole testes, the 105,000 × g supernatant or cytosol fraction was incubated with ³H DHT and varying concentrations of unlabelled DHT in the presence of 0.25μm NADPH. The incubation was carried out at 34°C for 10 min at a pH of 7.4. The K_m of 3α-HSO in testicular cytosol was calculated to be 1.25μM. The specific activity of testicular cytosol 3α-HSO, expressed as pmoles of 3α-diol converted from DHT per min per mg testicular cytosol protein, was high in young rats from 10 to 22 days of age, and was followed by a decline between day 22 and 37, with activity remaining low throughout adulthood. Total testicular cytosol activity of 3α-HSO, expressed as nmoles of 3α-diol converted from DHT per min per pair of testes, gradually increased from day 10 to day 60 and remained high in the adult rat. In the post-pubertal period, a possible lack of available substrate, DHT, or possible endogenous testicular regulatory mechanisms acting on 3α-HSO activity might account for the actual decrease in 3α-diol concentration in the blood and testes of mature rats.     

Selective inhibition of the 5 alpha-reductase of the rat epididymis.

The effect of several synthetic steroids belonging either to the 4-aza-3-oxo-steroid family or to androstene and androstane derivatives was investigated "in vitro" on the epididymal as well as prostatic 5 alpha-reductase activity. For this purpose rat caput epididymis and prostate were incubated with the different steroidal compounds at molar concentrations of 10(-7), 10(-6), and 10(-5) in the presence of labelled testosterone as substrate. The steroids 4-MA (17 beta, N,N-diethyl-carbamoyl-4-aza-5 alpha-androstan-3-one) and 4-OH-A (4-hydroxy-androstenedione), already known to be effective 5 alpha-reductase inhibitors at the level of the prostate, have been used as reference molecules. The 5 alpha-reductase activity was evaluated by measuring pg of dihydrotestosterone (DHT) formed in 2 h of incubation by mg of tissue. The steroids A, B, C, F, G and I inhibit the formation of DHT in the rat epididymis although to different extents; they are also equally effective on the formation of DHT in the rat prostate. The steroids D, E, H and L are devoid of any inhibitory property on the formation of DHT in both the rat epididymis and prostate. The most interesting results were obtained with compound M which exhibits a dose-dependent and significant inhibitory effect on the formation of DHT in the epididymis, but it is inactive at the level of the prostate. These findings suggest that it is possible (a) to selectively interfere with the 5 alpha-reductase of the epididymis without affecting that present in the prostate, and (b) consequently to envisage new ways to regulate male fertility.     

Does inhibin have intrinsic prolactin suppressing activity?

Evidence to demonstrate suppressive effect of inhibin on prolactin has been presented. The inhibin preparations derived from human testicular tissue, human seminal plasma and porcine follicular fluid were tested and all the three preparations were found to exhibit prolactin suppressing activity.     

Biological characteristics of a factor suppressing follicle stimulating hormone (inhibin) from ovine testes

Inhibin (follicle stimulating hormone suppressing factor) isolated from ovine testes has been characterized for its biological activity using a variety of tests. The bioassay used — inhibition of the human chorionic gonadotropin induced increment in the mouse uterine weight-demonstrates that there is a significant increment in specific activity (approx. 300-fold) with the progress of purification. Eventhough the final product has not been obtained in a homogenous state it has been possible to show that(a) [¹²⁵I]-labelled inhibin is preferentially taken up and retained by the pituitary, pretreatment of rats with testosterone facilitating this uptake;(b) it is able to suppress specifically the levels of follicle stimulating hormone in castrated as well as immature intact rats and (c) treatment of immature male rats with inhibin preparation for ten days results in impairment of testicular function as judged by³H-thymidine incorporation into testicular DNA and testicular hyaluronidase activity.     

Negative feedback regulation of the secretion and actions of gonadotropin-releasing hormone in males

This minireview considers the state of knowledge regarding the interactions of testicular hormones to regulate the secretion and actions of GnRH in males, with special focus on research conducted in rams and male rhesus monkeys. In these two species, LH secretion is under the negative feedback regulation of testicular steroids that act predominantly within the central nervous system to suppress GnRH secretion. The extent to which these actions of testicular steroids result from the direct actions of testosterone or its primary metabolites, estradiol or dihydrotestosterone, is unclear. Because GnRH neurons do not contain steroid receptors, the testicular steroids must influence GnRH neurons via afferent neurons, which are largely undefined. The feedback regulation of FSH is controlled by inhibin acting directly at the pituitary gland. In male rhesus monkeys, the feedback regulation of FSH secretion is accounted for totally by the physiologically relevant form of inhibin, which appears to be inhibin B. In rams, the feedback regulation of FSH secretion involves the actions of inhibin and testosterone and interactions between these hormones, but the physiologically relevant form of inhibin has not been determined. The mechanisms of action for inhibin are not known.     

Effects of inhibin on the in vitro synthesis of desoxyribonucleic acid in the rat testis

Three preparations of inhibin extracted from ram rete testis fluid (RTF) and from human seminal plasma (HSP) reduce tritiated thymidine incorporation into testicular desoxyribonucleic acids (DNA) in vitro. Effect of low molecular inhibin from RTF is dose-dependent. Castrated ram serum does not modify testicular DNA synthesis in vitro. Besides their suppressive action on follicle stimulating hormone (FSH) secretion in vivo and in vitro, these inhibin preparation display a direct inhibiting effect on testicular DNA synthesis and, thus, on mitotic activity. Identity between inhibin and testicular chalones are discussed.     

Short term treatments with prolactin, HMG or testosterone fail to affect testicular inhibin content in rats

To investigate the effect of prolactin (PRL) on testicular function, especially on spermatogenesis, testicular inhibin content in male rats treated with PRL was compared with those treated with HMG and testosterone. Mature Wistar male rats were given 10 or 50 IU of ovine PRL, 10 IU of HMG and 5 mg of testosterone, i. m. for 5 consecutive days and testes were removed for assessing inhibin content. Inhibin content was measured by a FSH suppressing activity in cultured rat anterior pituitary cells using aquous extract of testes. Five days' treatment with PRL, HMG, or testosterone did not influence testicular inhibin content in male rats. The possibility that these treatments had transiently affected testicular inhibin content, or that inhibin content did not reflect inhibin production was not ruled out.     

Development of a radioimmunoassay for human seminal plasma inhibin.

Using a homogeneous inhibin preparation from human seminal plasma with a molecular weight of about 19 000, a sensitive and specific radioimmunoassay (RIA) for inhibin has been developed. None of the purified hormones tested, such as LH, FSH and prolactin from different species, showed any cross-reaction in this RIA. Steroid hormones such as testosterone, dihydrotestosterone, oestradiol-17 beta and progesterone did not interfere with the assay. The antiserum had an affinity constant (Ka) of 2.379 X 10(9). The assay sensitivity was 10-15 ng per tube and the intra- and inter-assay coefficients of variation were 5-7% (n = 6) and 15% (n = 10) respectively. The recovery for inhibin added to the serum of a castrated man was 95-110%. Using this RIA, inhibin levels in various biological fluids and tissues were measured. Normo-spermic semen contained significantly higher levels of inhibin than did oligospermic semen. Human prostate contained a substantial quantity of inhibin. Monkey semen, rat serum, and bovine, ovine and porcine follicular fluids cross-reacted in the RIA, while ram testicular inhibin and bull semen did not do so. In developing (9-28 days of age) male rats, circulating inhibin levels showed an inverse relationship with serum FSH levels. In female rats of this age endogenous inhibin concentrations changed in parallel with those of serum FSH.     

Expression and regulation of testicular inhibin alpha-subunit gene in vivo and in vitro

Inhibin, a gonadal peptide, selectively suppresses FSH release from the pituitary. The cDNAs coding for ovarian inhibin have been isolated and characterized. However, little is known about testicular inhibin. In this study we have isolated inhibin alpha-subunit cDNA from human testicular cDNA libraries and determined inhibin alpha-subunit mRNA levels in testes. The longest cDNA isolated from human testis was 1380 nucleotides long and contained a nucleotide sequence identical to that of human placental inhibin alpha-subunit and isolated human inhibin alpha-subunit gene, but different from human ovarian inhibin alpha-subunit in two amino acids in the signal peptide. A single 1.5-kilobase species of inhibin alpha-subunit mRNA was identified in the testes of several species. This mRNA was the same size as those in human ovary and placenta. The regulation of inhibin alpha-subunit mRNA in rat testis was next examined. The concentration of testicular inhibin alpha-subunit mRNA peaked between 20-25 days of age and gradually declined thereafter. Hypophysectomy decreased testicular inhibin alpha-subunit mRNA levels. Supplementation of hypophysectomized animals with FSH restored inhibin alpha-subunit mRNA levels to those in intact controls. By contrast, treatment with testosterone had no effect. Similarly, in Sertoli cell-enriched cultures, FSH, but not testosterone, increased inhibin alpha-subunit mRNA levels. We conclude that 1) human testicular inhibin alpha-subunit mRNA is similar to that of human ovary and placenta; and 2) inhibin alpha-subunit mRNA in Sertoli cells is regulated by FSH, but not testosterone, both in vivo and in vitro.     

Effect of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium crustosum broths

The conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT) has been demonstrated in Penicillium crustosum broth obtained from fermented pistachios, lemons and corn tortillas. Furthermore, the presence of 5alpha-reductase enzyme, which is responsible for this conversion, has been established by electrophoretical techniques in these cultures.5alpha-Reductase enzyme is also present in animal and human androgen-dependent tissues as well as in prostate and seminal vesicles. The increase of the conversion of T to DHT in prostate gland, has been related to some illnesses such as benign prostate hyperplasia and prostate cancer. Furthermore, treatment with 5alpha-reductase inhibitors such as finasteride reduces the prostate growth. These data have stimulated research for the synthesis of new molecules with antiandrogenic activity, whose biological effect needs to be demonstrated.The purpose of this study is to determine the inhibition pattern of 5alpha-reductase in P. crustosum by finasteride and the new steroidal compound PM-9. K(m) and V(max) values for T, were determined in the broths by Lineweaver-Burk plots using different testosterone concentrations. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk using different concentrations of T and inhibitors. Results show that finasteride and PM-9 inhibit 5alpha-reductase present in the broth in a competitive manner.     

Effects of anabolic steroid (19-nortestosterone) on the secretion of testicular hormones in the stallion.

The aim of this study was to clarify the effect of anabolic steroids on the testicular endocrine function of mature stallions. Mature thoroughbred stallions were treated with 800 mg nandrolone decanoate every 3 weeks for 3 months. After the first treatment, plasma concentrations of LH, immunoreactive inhibin and testosterone decreased rapidly to the nadir. These hormones were maintained at significantly lower concentrations compared with concentrations in intact stallions. Histology of the testicular tissue indicated the arrest of advanced spermatogenesis in the seminiferous tubules and a severe depletion of the number of Leydig cells in the interstitial compartment as a result of treatment. Most of the immunopositive cells for the inhibin alpha-subunit and steroidogenesis enzymes in the interstitial compartment decreased below detectable amounts, whereas immunopositive reactions of inhibin alpha-subunit in the seminiferous tubules were clearly observed. In conclusion, the treatment of mature stallions with nandrolone decanoate caused a decrease in the secretion of ir-inhibin and testosterone from the testis, the depletion of the number of Leydig cells and a decrease below detectable amounts of inhibin alpha-subunit and steroidogenesis enzymes. The concentration of ir-inhibin in the peripheral blood may be a useful marker for the examination of testicular activity in stallions being treated with anabolic steroids.     

L'andropause

In men, aging is associated with progressive impairment of testicular function. Decrease in total testosterone levels with aging has been reported in many studies in normal healthy men. This decrease has a primarily testicular origin, as shown by decreased number of Leydig cells in histological studies, increased basal gonadotropin levels and decreased response to hCG. A greater decrease in bioavailable testosterone rather in than total testosterone concentrations is explained by the age-dependent increase in the SHBG concentration. Although immunoreactive gonadotropin levels are higher than in young men, a relative alteration of bioactive gonadotropin secretion by the pituitatry occurs in ederly men. Althought the definitive demonstration of an alteration of GnRH secretion by the hypothalamus cannot be established in healthy ederly men, such an alteration might be responsible for a decompensation of the testicular function in case of intercurrent illness. Increased FSH and decreased inhibin plasma levels are indicating a similar alteration in seminiferous tubules as directly demonstrated by histological data showing a decrease of Sertoli cell number and daily sperm production with aging. Although the incidence of sexual dysfunction increases with aging, the relationship between sexual behaviour and testicular endocrine function remained a mater of controversy. A threshold of testosterone action on sexual behavior might be responsible for the difficulty in establishing this relationship. Although some controled studies are available, the risk-to-benefit balance of androgen substitution in older male remained a controversial issue. A positive effect on sense of well-being and/or libido has been and the lack of adverse effect on lipid and carbohydrate metabolism had been demonstrated in short term studies, however, the role of androgens in the benign hypertrophy of the prostate and in stimulating the growth of latent prostate adenocarcinoma remained to be more clearly established by longterm controlled studies.     

Androgen deprivation therapy in combination with radiotherapy for high-risk clinically localized prostate cancer

Androgen deprivation therapy (ADT) has remained the main therapeutic option for patients with advanced prostate cancer (PCa) for about 70 years. Several reports and our findings revealed that aggressive PCa can occur under a low dihydrotestosterone (DHT) level environment where the PCa of a low malignancy with high DHT dependency cannot easily occur. Low DHT levels in the prostate with aggressive PCa are probably sufficient to propagate the growth of the tumor, and the prostate with aggressive PCa can produce androgens from the adrenal precursors more autonomously than that with non-aggressive PCa does under the low testosterone environment with testicular suppression. In patients treated with ADT the pituitary-adrenal axis mediated by adrenocorticotropic hormone has a central role in the regulation of androgen synthesis. Several experimental studies have confirmed the potential benefits from the combination of ADT with radiotherapy (RT). A combination of external RT with short-term ADT is recommended based on the results of phase III randomized trials. In contrast, the combination of RT plus 6 months of ADT provides inferior survival as compared with RT plus 3 years of ADT in the treatment of locally advanced PCa. Notably, randomized trials included patients with diverse risk groups treated with older RT modalities, a variety of ADT scheduling and duration and, importantly, suboptimal RT doses. The use of ADT with higher doses of RT or newer RT modalities has to be properly assessed.     

Protein kinase C pathway is involved in regulating the secretion of prostatic acid phosphatase in human prostate cancer cells

The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4Alpha-phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5alpha-dihydrotestosterone (DHT) at the same concentration of 50 nM. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be involved in DHT action on PAcP secretion.     

Testosterone restores testicular inhibin content in cryptorchid rats

The effects of testosterone administration on testicular inhibin content and histology were studied in bilaterally cryptorchid rats, in which a marked decrease in testicular inhibin content had been observed. Mature male Wistar rats weighing approximately 300 g were made bilaterally cryptorchid by placing the testes in the abdominal cavity. Testosterone in oil, 0.1, 1.0 or 10 mg, was given i.m. each week. Testicular inhibin and testosterone content, histology and plasma LH, FSH and testosterone were studied 2 weeks later. Abnormally decreased testicular inhibin in cryptorchidism was restored toward normal by testosterone in a dose dependent manner in 2 weeks after surgery. Sertoli cell structure also recovered toward normal with increasing amount of testosterone. Decreased testicular testosterone content and Leydig cell atrophy were observed with suppressed plasma LH and FSH after testosterone. These results showed that the increased plasma concentration of testosterone had a stimulatory effect on the Sertoli cell function in cryptorchidism, in which compensated Leydig cell failure was demonstrated.     

Effects of estradiol on prostate epithelial cells in the castrated rat.

There is evidence that estrogens can modulate the activity of prostate epithelial cells. To determine whether estradiol can have a direct influence on rat prostate, this study examined the effects of estradiol-17beta (E(2)) administered alone or in combination with dihydrotestosterone (DHT) to castrated rats for 3 weeks on prostate binding protein (PBP) C1 mRNA expression and androgen receptor (AR) localization. PBP C1 mRNA levels were measured by semi-quantitative in situ hybridization using a (35)S-labeled cDNA probe. In intact animals, strong hybridization signal could be observed in prostate sections after 12 hr of exposure to Kodak X-Omat films. In castrated rats, no PBP C1 mRNA could be detected even with longer exposure times, an effect that was prevented by administration of DHT. E(2) administered alone induced a detectable hybridization signal, and the concomitant administration of E(2) and DHT induced an increase in PBP C1 mRNA that significantly exceeded that obtained in animals that received only DHT. In prostate epithelial cells of intact animals, AR immunostaining was restricted to the nucleus. In castrated animals the alveoli were decreased in size and the epithelial cells were atrophied. AR staining was weak and was detected in both cytoplasm and nucleus. DHT administration completely obviated the effect of castration on epithelial cell histology and on AR immunostaining distribution and intensity. Interestingly, E(2) administration alone induced moderate hypertrophy of epithelial cells compared to the histological appearance of cells in untreated castrated rats. Moreover, in E(2)-treated animals the nuclear staining was much stronger than that detected in untreated castrated rats, whereas the cytoplasmic staining was not modified by the treatment. In animals that received both DHT and E(2), the staining was similar to that seen in DHT-treated rats. These results suggest that E(2) can influence the activity of rat prostate epithelial cells by mechanisms that remain to be fully clarified.     

The clinical development of a 5 alpha-reductase inhibitor, finasteride.

Finasteride, a 4-aza steroid compound, is an orally active inhibitor of the 5 alpha-reductase enzyme. 5 alpha-Reductase is necessary for the metabolism of testosterone (T) to dihydrotestosterone (DHT) and is found in high levels only in certain tissues such as the prostate. Finasteride has been shown to markedly suppress serum DHT levels in man without lowering testosterone levels. In patients with benign prostate hyperplasia (BPH), finasteride was found to decrease prostate volume by a mean of 28% over a period of 6 months, without causing clinically significant adverse effects. DHT appears to be the primary androgen for prostatic growth. Selective inhibition of 5 alpha-reductase by finasteride may provide a novel approach to BPH therapy by reducing prostate size without affecting T-dependent processes such as fertility, muscle strength, and libido. The clinical development of finasteride for the treatment of benign prostate hyperplasia is reviewed.     

Evidence for a major role of inhibin in the feedback control of FSH in the male rat

Adult rats (16-18/group) received a single intratesticular injection of 25, 100 or 400 microliters glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1.5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studied in Paraplast-embedded tissue stained with PAS and haematoxylin-eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = -0.70, P less than 0.001) and testicular (r = -0.66, P less than 0.001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0.48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 microliters groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7-14%) of testicular testosterone and unaltered serum FSH concentrations.     

Beta-endorphin regulation of FSH-stimulated inhibin production is a component of a short loop system in testis

Inhibin, a hormone produced by Sertoli cells in response to FSH, regulates androgen production in nearby Leydig cells. Beta-endorphin synthesized by Leydig cells under LH control is also known to regulate Sertoli function. To delineate whether beta-endorphin might constitute part of a short loop regulatory system between these two testicular cells, the effect of this opiate on inhibin secretion was examined. Beta-endorphin alone did not alter basal inhibin accumulation in primary Sertoli cell-enriched cultures, however it did significantly reduce FSH-induced inhibin production and adenylyl cyclase activity but had no effect on forskolin-stimulated inhibin accumulation or adenylyl cyclase activity. Other opioid peptides (ACTH, dMSH, methionine-enkephalin) were without effect. These observations suggest that beta-endorphin regulates inhibin secretion by inhibiting FSH receptor coupling to adenylyl cyclase.