Effect of 5α-Reduced Androgens on Prolactin Secretion

Der Einfluß von 5α-reduzierten Androgenen auf die Prolaktinsekretion
An langzeitkastrierten männlichen und weiblichen Ratten wurden die Auswirkungen einer 6-tägigen Behandlung mit jeweils 2 mg/Tag von Testosteron, Dihydrotestosteron (DHT), 5α-androstan 3α, 17β-diol (3α-diol) und 5α-androstan-3β, 17β-diol (3β-diol) auf die Serumprolaktinspiegel untersucht.
Die Steroide wurden entweder in alkoholischer Form oder in propionierter Form verabreicht (Mono- und Dipropionat für die 3α- und 3β-diole). Keines der Steroide hatte irgendeinen Effekt auf die behandelten männlichen Tiere. Bei weiblichen Tieren hemmten DHT und 3α-diol die Prolaktinsekretion, wohingegen Testosteron und 3β-diol ohne Effekt blieben. Diese Ergebnisse lassen den Schluß zu, daß möglicherweise 5α-reduzierte Verbindungen, DHT und 3α-diol eine Rolle bei der Kontrolle der Prolaktinsekretion bei dem weiblichen Geschlecht spielen.     

Testosterone and 5α-dihydrotestosterone Concentrations in Human Seminal Plasma

Testosterone and dihydrotestosterone (DHT) were estimated by radioimmunoassay in human seminal plasma. Testosterone concentrations showed no significant differences between fertile and infertile semen samples, whereas DHT concentrations were significantly lower in azoospermic and oligozoospermic samples. It is concluded that testosterone derives essentially from the accessory sex glands, whereas DHT is mainly of testicular or epididymal origin. The low DHT concentrations found in seminal plasma of oligozoospermic and azoospermic patients is probably due to defective epididymal conversion of testosterone to DHT.     

The Extragonadal Origin of 5α-Reduced Androgens in the Peripheral Circulation of the Adult Male Rabbit

The extragonadal source of the high levels of 5α-reduced androgens in the peripheral blood of the male rabbit has been investigated. Adult 3-day castrated rabbits each received a single intravenous injection of ³H-labelled testosterone, and blood plasma was collected at various intervals thereafter. Two h post injection, animals were killed, various accessory sex organs and peripheral tissues removed, and radioactively labelled testosterone and its metabolites quantified by radio-gas-liquid chromatography (radio-g.l.c.). Five min after the injection of ³H-labelled testosterone, the major labelled metabolite in blood plasma was 5α-dihydrotestosterone with small amounts of 3α/3/β-androstanediols and androstenedione. After 2 h significant quantities of ³H-labelled 5α-dihydrotestosterone had accumulated in the accessory sex organs, whereas only small amounts were found in 'non-sexual' peripheral tissues such as skeletal muscle. However, substantial amounts of 5α-dihydrotestosterone were formed by several peripheral tissues of 3-day castrated rabbits, when they were incubated in vitro with ³H-labelled testosterone: the most active were skeletal muscle, liver and abdominal skin. Some formation of 3α/3/β-androstanediols occurred in all tissues. It is concluded that in the male rabbit skeletal muscle and skin are likely to be a significant extragonadal source of 5α-reduced androgens for the maintenance of accessory sex organs such as the prostate and epididymis, which have low 5α-reductase activity but a high capacity to retain 5α-dihydro-testosterone.     

Percutaneous absorption of 5α-dihydrotestosterone in man

The distribution of 5α-dihydrotestosterone to the blood following application of a solution of this androgen to the skin in a hydro-alcoholic gel was studied in order to evaluate the adequacy of the percutaneous route in correcting androgen deficiencies. In 14 adult men, daily percutaneous administration of 5α-dihydrotestosterone (125 mg in 5 g gel) increases, on the average, 4 to 5 times its initial concentration in plasma. On the 14th day of treatment, repeated evaluations of plasma 5α-dihydrotestosterone, between 2 and 21 h after final administration of gel, demonstrated the stability of diurnal 5α-dihydrotestosterone levels and showed the regular distribution of the steroid from a presumably cutaneous reservoir. Plasma 5α-androstane-3α, 17β-diol levels evolve parallel to those of 5α-dihydrotestosterone. Plasma testosterone and luteinizing hormone, on the contrary, decrease considerably. No variation of follicle-stimulating-hormone is observed during treatment. The percutaneous absorption represents an interesting method for administration of a natural androgen in men, particularly because one avoids the deleterious effects of supra-physiological levels in the liver achieved with oral administration.     

In Vivo Effects on Spermatogenesis and In Vitro Metabolism of 5α-Androstan-3β,17β-Diol by Testes, Prostate Glands and Seminal Vesicles

It has been shown that 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone, DHT), an irreversible metabolite of testosterone, is capable of maintaining spermatogenesis and sex accessory organs of adult rats. The same property has been displayed by 5α-androstan-3α,17β-diol (3α-diol). We have studied the effects of 5α-androstan-3β,17β-diol (3β-diol) in hypophysectomized rats. This steroid is also capable of maintaining spermatogenesis as well as the accessory glands of hypophysectomized rats. These effects of 3β-diol are quantitatively better than with 3α-diol. Incubation of teased testicular tissue, prostate glands and seminal vesicles with tritiated 3β-diol showed that 3β-diol is preferentially converted to 3α-diol in the testis and DHT in the prostate glands but is not metabolized by the seminal vesicles. This would tend to indicate that the biological activity may be attributed to any one of the 5α-reduced metabolites since the testes and prostate glands can metabolize 3β-diol and the seminal vesicles could get the appropriate form of the steroid by conversion at a peripheral site with transport to the seminal vesicles. Incubation of testicular tissue with tritiated 3β-diol and a steroidal inhibitor of 3β-hydroxysteroid dehydrogenase showed 90% inhibition of 3β-diol metabolism. In vivo studies with this inhibitor are underway to assess if 3β-diol is biologically active without being converted to other steroids.     

Localization of integrin β1, α1, α5 and α9 subunits in the rat testis

Very late activation ( VLA, β₁; α₁; α₅, α₉) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti β₁ antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, β₁ integrin was localized to the acrosome. The α₁ subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the α₁ subunit. Immunoreactivity for α₁ integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The α₅ subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III–VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII–VIII. The α9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.     

Stable maintenance of 5α-reductase activity in long-term subcultures of fibroblasts derived from the foreskin

BACKGROUND: There is up to a 50-fold variation in control subjects in current assays of 5alpha-reductase activity which makes interpretation difficult. It was therefore attempted in this study to establish an assay method which produced stable 5alpha-reductase activity in long-term subcultured foreskin fibroblasts. METHODS: Foreskin fibroblasts were obtained from three boys with phimosis (control subjects), three patients with Reifenstein syndrome and one patient with 5alpha-reductase deficiency (due to mutation L113P in exon 2 of the SRD5A2 gene). To maximize the number of cells in the DNA synthesis phase, cells were subcultured consistently to approximately 70% confluency. Thawed cells, frozen after the third subculture, were incubated for 24 h with [1beta,2beta-3H] testosterone. 5alpha-Reductase activity was expressed as the sum of formed [3H] 5alpha-reduced metabolites (separated by thin-layer chromatography). RESULTS: The full range of 5alpha-reductase activity in controls and patients with Reifenstein syndrome was 3.44-15.59 pmol/h per mg protein: a 4.53-fold variation. The activity in the patient with 5alpha- reductase deficiency was 0.52 pmol/h per mg protein. CONCLUSION: By the cell culture methods used in this study, which aimed to increase the number of cells in the DNA synthesis phase, foreskin fibroblasts maintained a considerably stable level of 5alpha-reductase activity during long-term subculture. Therefore, this assay method can be used for differential diagnosis of 5alpha-reductase deficiency from other relevant entities.     

Inhibition of Spermatogenesis in House Rat (Rattus rattus Rufescens) Following the Administration of α-Chlorohydrin

Hemmung der Spermatogenese bei der Hausratte (Rattus rattus Rufescens) nach Applikation von alpha-Chlorohydrin
Die Langzeitapplikation von alpha-Chlorohydrin (25 mg/kg Körpergewicht) verursachte Schäden der Testes der Hausratte. Es zeigten sich degenerative Veränderungen der Keimzellen. Der Durchmesser der Tubuli seminiferi und der Kerndurchmesser der Leydigschen Zellen waren verringert. Die Zellhöhe der Nebenhodenzellen war beträchtlich vermindert. Das Lumen war frei von Spermatozoen.
Gaben von alpha-Chlorohydrin hemmten die Synthese von RNS, Eiweiß, Sialinsäure in den Hoden und akzessorischen Geschlechtsorganen. Das Gesamtcholesterin in den Testes war erhöht. Auffällig war die Verarmung der Nebennieren an Ascorbinsäure. Alpha-Chlorhydrin wirkt antagonistisch auf die stimulierende Wirkung des Testosterons bei kastrierten Hausratten. Alpha-Chlorohydrin kann als eine biologisch wirksame Verbindung betrachtet werden, um die Zahl der Hausratten in menschlichen Behausungen zu kontrollieren. Seine dosisabhängige Wirksamkeit wird diskutiert.     

Effect of α-Chlorohydrin on the Male Reproductive Organs of the Indian Langur: (Presbytis entellus entellus Dufresne)

Eine subtoxische Dosis von α-chlorhydrin (3-chloro-1, 2-propanediol, U-5897) wurde erwachsenen männlichen Schlankaffen (Presbytis) sucutan injiziert (10 mg/kg Körper-gewicht über 30 Tage). Die Tiere verloren kein Gewicht; es ließ sich aber feststellen, daß das Gewicht der Hoden und der accessorischen Drüsen zurückging. Histologisch war die Spermatogenese unterdrückt, die interstitiellen Zellen schienen normal zu sein. Im Nebenhodenkopf ergab sich eine Reduzierung des Tubulusdurchmessers um die Hälfte gegenüber der unbehandelten Kontrollgruppe. Ebenfalls zeigte sich eine Reduzierung der Höhe der kubischen Zellen; auch im Nebenhodenschwanz waren Verände-rungen nachzuweisen: die Zilien fehlten entweder oder sie waren stark reduziert. Die Sialinsäurekonzentration war sowohl im Hoden als auch im Nebenhoden der behan-delten Tiere herabgesetzt; Fruktose in der Bläschendrüse und Zitronensäure in der Prostata waren ebenfalls reduziert, obwohl die Zellen des Interstitiums morphologisch normal waren, sie waren funktionell antianabolisch. Dieses scheint ein neuer Gesichts-punkt der α-chlorhydrin wirkung bei einem Primaten zu sein.     

Effect of α-chlorohydrin on metabolism and testosterone secretion by rat testicular interstitial cells

The direct effect of alpha-chlorochydrin (alpha-CH) on basic metabolism (glucose utilization and oxygen consumption) and testosterone secretion by isolated rat interstitial cells (I-cells) has been studied. In the range of concentrations between 5 and 100 microliter/ml, only the highest doses of alpha-CH decreased cell vitality and their histochemical stain for 3 beta-HSD. Oxygen consumption of I-cells was depressed at all doses higher than 10 microliter/ml and this effect was reversible only with doses lower than 50 microliter/ml. glucose utilization by I-cells was depressed significantly by alpha-CH and this effect was particularly dramatic with doses higher than 50 microliter/ml. alpha-CH decreased testosterone secretion by I-cells, with maximal effects at 100 microliter/ml. I-cells responded to hCG challenge by increasing testosterone secretion, and hCG prevented the toxic effect of alpha-CH at the lowest dose (10 microliter/ml) of alpha-CH, but failed to overcome the effects of a high dose (100 microliter/ml).     

Physico-Chemical Characterization of the NADPH Dependent Soluble 3α-Hydroxysteroid Oxidoreductase in the Rat Epididymis

The present paper describes some important physico-chemical characteristics of the NADPH dependent, 3α-hydroxysteroid oxidoreductase (3α-oxidoreductase) in the rat epididymis. This enzyme activity is stable in the presence of reducing agents (1–10 mM 2-mercaptoethanol), EDTA (1 mM), and glycerol (10% v/v). The pH optium is in the physiological range (pH 6.0–8.0).
Chromatography of epididymal cytosol fractions on calibrated G-200 columns indicated a molecular weight of 34 000, and sucrose gradient centrifugation a sedimentation coefficient of 3.3 S20,w. The enzyme has a Stokes radius of approximately 25 Å. Calculation of molecular weight using both the sedimentation rate and the Stokes radius indicated a molecular weight of 34 400 and a frictional ratio (f/f₀) of 1.17. The enzyme migrated with an R_x of 0.37 (relative to bromophenol blue) in 6.5% polyacrylamide gels and the iso-electric point (pI) determined by iso-electric focussing in 3.5 % polyacrylamide gels was 5.9. Further indication that the enzyme is negatively charged at neutral pH was obtained by DEAE-cellulose ion exchange chromatography, from which the enzyme activity was eluted between 0.05 and 0.08M KCl.
The epididymal 3α-oxidoreductase is temperature sensitive, showing a greatly reduced activity after preincubation at 50°C for 30 min. Preincubation at 60°C caused a complete loss of enzyme activity. Interestingly, preincubation at 25°C for 30 min seemed to cause a significant increase in enzyme activity. Whether this is due to "temperature activation", "cold inactivation" or to a temperature dependent disappearance of inhibitors is not known.     

Effects of α-Interferon on Sperm Production, Concentration, and Motility in the Rat

Background :
We evaluated possible effects of α-interferon (α-IFN) on testicular spermatogenesis and epididymal sperm quality in the nude rat.
Methods :
Nude male rats were administered subcutaneous injections of human α-IFN daily for 3 months. The luminal content of the cauda epididymidis was collected by micropuncture. Daily sperm production was determined by Amann's method and sperm concentrations were determined by microassay. Progressive motility was judged by evaluating the linear distance traveled by the sperm in a diluent. Serum levels of testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were also measured at the end of the experiment.
Results :
Daily sperm production and epididymal sperm concentrations were significantly increased after administration of α-IFN, while progressive motility of the spermatozoa was not altered. α-IFN significantly increased serum testosterone levels, while it decreased serum LH levels and left serum FSH levels unchanged.
Conclusion :
α-IFN may improve testicular spermatogenesis and increase the epididymal sperm concentration in the rat. These promising results with α-IFN may pave the way for a new approach to treating male infertility.     

Studies on the Effect of Corticosterone Treatment on Testicular Activity, Serum Concentration of Gonadotrophins, Testosterone and α2u-Globulin in Rats Pre-Treated with Estrogen

Adult male rats were treated with estradiol-17 beta (50 micrograms/100 g body wt./day) for 7 days. When the animals were killed 14 days later, the levels of serum gonadotrophins, testosterone and alpha 2u-Globulin as well as the weight of sex organs were reduced, testicular 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was also suppressed; spermatogenesis was inhibited. Administration of corticosterone (2 mg/day) for 14 days to estrogen-treated rats increased the concentration of gonadotrophins, testosterone and alpha 2u-globulin in the serum. The weight of accessory sex organs and spermatogenesis appeared to be normal while 17 beta-HSD activity increased in estrogen-treated rats after treatment with corticosterone. It is concluded that corticosterone has an effect on testicular function by inducing alpha 2u-globulin and gonadotrophins in estrogen treated rats.     

Effect of Androgens on Antipyrine Metabolism in the Rabbit

The inactivation of endogenous steroids and foreign compounds by the liver hydroxylative pathway, can be modified by androgenic substances. The inhibitory or inductory activity, can be estimated by measuring the half-life of antipyrine. Androgens (testosterone propionate, 5α-dihydro-testosterone and mesterolone) reduce antipyrine metabolism in male rabbits. In females, androgens are not inhibitors, but one of them, 5α-dihydrotestosterone, shows an inductive effect on liver oxidative mechanisms.     

Physico-Chemical Characterization of the Soluble 3α-Hydroxysteroid Oxidoreductase in the Rat Testis and Prostate

The present paper describes some physicochemical properties of the soluble 3α-oxidoreductases in the rat testis and prostate, and comparison with rat epididymal 3α-oxidoreductase, published previously (Hastings & Hansson 1979). The testicular enzyme shows properties very similar to that in the epididymis (size, stability, pH optium) except for minor differences in charge (iso-electric point). The prostatic enzyme revealed a slightly higher molecular weight, and was more sensitive to heating than those in the testis and epididymis, whereas the iso-electric point was the same as that in the testis (pI-5.25). The enzymes in all tissues exhibit very similar shapes (f/fo 1.14-1.17).
The similar properties of the testicular and prostate 3α-oxidoreductases to those previously reported for that in the epididymis may indicate that these enzymes represent identical peptide chains. The small differences observed in size, temperature stability and change may be due to their presence in different environments.     

α1-Adrenoceptor pharmacome: α1L-Adrenoceptor and α1A-adrenoceptor in the lower urinary tract

α₁‐Adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several α₁ antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical α₁‐adrenoceptor subtypes (α_1A, α_1B, and α_1D) have been identified, among which the α_1A and α_1D‐adrenoceptor subtypes have been regarded as the main targets of α₁ antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize α₁‐adrenoceptors pharmacologically, (i.e. all classical α₁‐adrenoceptor subtypes show high‐affinity for the drug). However, we found that α₁‐adrenoceptors in the LUT show atypical low‐affinity for prazosin. Therefore, the concept α_1L‐receptor, which indicates α₁‐adrenoceptor(s) showing low‐affinity for prazosin has been introduced. A recent study demonstrated that the α_1L‐adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the α_1L‐adrenoceptor in the LUT is now re‐defined as α_1A(L)‐adrenoceptor. The physiological and pharmacological difference between classical α_1A(H), and α_1A(L) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological α_1A‐adrenoceptor blocking pharmacotherapy. Here, we briefly review the α₁‐adrenoceptors in the LUT with special reference to phenotype‐based (pharmacome) analysis.     

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.     

Molecular diagnosis of 5α-reductase-2 gene mutation in two Indian families with male pseudohermaphroditism

Aim: To identify the genotype of two Indians with male pseudohermaphroditism. Methods: Standard radioimmu-noassay procedure was used for estimating hormonal levels. Conventional cytogenetic analysis was carried out for diagnosing the genetic sex in these subjects with genital ambiguity. Molecular analysis was carried out by standard polymerase chain reaction procedure using different sets of primers and reaction conditions specific for the 5α-reductase type 2 gene (SRD5A2) gene. Direct sequencing was carried out using the ABI Prism dye terminator sequencing kit and the ABI 310 sequencing apparatus. Results: We found an SRD5A2 gene mutation in exon 5, where arginine is substituted with glutamine (R246Q), in two males with pseudohermaphroditism and ambiguous genitalia from unrelated families. This is the first time this mutation has been reported in individuals from India. Conclusion: Identification of the R246Q mutation of the SRD5A2 gene from two unrelated Indian families possibly extends the founder gene effect.     

Zinc arginine, a 5α-reductase inhibitor, reduces rat ventral prostate weight and DNA without affecting testicular function

Summary. Zinc has been implicated in steroid endocrinology of the prostate gland; and 5α-dihydrotestosterone (DHT) is believed to express androgenic responses in the prostate. To note the effect of neutralized zinc (zinc gluconate + arginine) on the prostate, 50 sexually mature rats, weighing 325 ± 20 g, were divided into five groups as follows: (1) control, (2) sham, (3) castrated, and injected intraprostatically with (4) 10 mg neutralized zinc, and (5) 20 mg neutralized zinc. Results indicated significant reduction ( P <0.05) of prostate weight, 5α-reductase activity, and total protein and DNA concentrations in treated prostate tissue; no significant change in weight and histological structure of testes, epididymides, and seminal vesicles; and no significant effect on progeny and blood testosterone level of treated animals. These results suggest that direct application of neutralized zinc to the prostate offers a new modality for treatment of prostatitis without affecting spermatogenesis.     

Direct gene replacement of the mouse α(1,3)-galactosyltransferase gene with human α(1,2)-fucosyltransferase gene: Converting α-galactosyl epitopes into H antigens

Abstract: The chronic donor organ shortage has led to the production of transgenic animals. We assume that cells or organs derived from possible animal donors carrying a large amount of α-galactosyl epitopes should not be transplanted into humans, because a corresponding amount of immunosuppressants would be needed to prolong the survival of such xenografts in the recipients. This may not only make the recipients compromised hosts but also introduce some unknown or uncontrollable pathogens into society at large. We also assume that gene manipulation itself should not be a detriment to possible transgenic animals. To explore possibilities that not only can minimize the possible detrimental factors to humans, such as α-galactosyl epitopes, but also can minimize the possible detriment to transgenic animals, such as random integration of the extraneous genes with or without uncontrollable regulatory sequences, we have produced a DNA construct that replaces the mouse oc(1,3)-galactosyltransferase gene (GT) with the human a(1,2)-fucosyltransferase (FT) minigene (promoterless for the expression of FT) at the GT locus. The mouse fibrosarcoma cell line, L929, was transfected with the construct. Colonies were obtained after incubation with non-heat-inactivated human serum. Southern blot analysis demonstrated that one allele of the mouse GT gene was replaced with the FT minigene at the GT locus without integration of any selectable marker genes. The immunostaining analysis with lectins showed that the transfectants expressed H antigens, which suggested that H antigens were expressed by the intrinsic GT promoter. Thus gene replacement, knock-in, of the mouse GT with the human FT without integration of any selectable marker genes in the GT locus was shown to be possible. This is especially important in producing transgenic animals for the clinical application of xenografts into humans.