V. Hansson

Prolactin binding in rat testis: specific receptors in interstitial cells.

Specific receptors for [125I]hPrl (human prolactin) are present in membrane preparations of rat testis. The receptors are specific for lactogenic hormones (prolactin and human growth hormone) but do not bind gonadotropins. The prolactin receptors are localized exclusively in the interstitial cell tissue, and are not present in membrane preparations from isolated seminiferous tubules. The localization of prolactin receptors interstitial tissue suggests that the effect of prolactin on LH/hCG-stimulated testosterone production is due to a direct effect of prolactin of Leydig cells.

Sertoli cell origin of testicular androgen-binding protein (ABP)

In this report it is suggested that the specific adrogen-binding protein (ABP), previously shown to originate in the testis of rat and other species, is produced by the Sertoli cells. This suggestion is based upon the following experimental findings: 1) ABP was found in high concentrations in testicular efferent duct fluid but only in trace amounts in inter-tubular lymph. i) ABP could be recovered from crude preparations of testis tubules, but not from Leydig cells from the same testes. 3) Testes whose germinal epithelium had been severly damaged by gamma irradiation showed no decrease in ABP content. The transport of ABP to epididymis was also preserved as judged from the levels of ABP in caput epididymis. 4) Testes that were completely devoid of germ cells following prenatal gamma irradiation showed high levels of ABP, These high levels approached zero following hypophysectomy, but could be restored by FSH administration to the hypophysectomized animals. ABP has been well characterized and now provides a valuable experimental tool as an indicator of Sertoli cell function.

An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis

Abstract The cytosol fraction of rat testicular homogenates contained a specific androgen binding protein (t-ABP), indistinguishable from, the androgen binding protean in. the epididymis(e-ABP) in terms of its chromatographic behaviour by gel filtration, sedimentation rate, mobility on polyacrylamide electrophoresis and steroid specificity(5α-diaydrotestosterone(DHT) > testosterone > estradiol-17β > parogesterone and estradiol-17α). The stability of t-ABP as well was similar to that of e-ABP. The aBP-DHT complexes were stable between pH 6.5–10, exhibiting the highest degree of binding between pH 8–9. The binding activity persisted after heating at 50°C for 30 min., whereas heating at 60°C for 30 min. completely destroyed the binding. DHT did not significantly increase the stability towards pH and temperature denaturation. Binding activity was not reduced by 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS), whereas similar treatment with 1 nM N-ethyl-maleimide(NEM) or 1 mM Ellmans reagent reduced it by some 10–50 per cent. Exposure to 10 mM β-mercaptoethanol(βME) reduced the binding by 60–70 per cent. These studies strongly indicate that t-ABP and e-ABP are identical proteins. Hemicastration for 4 weeks eliminated the ABP from the epididymal cytosol fraction on the castrated side, indicating that this protein is produced by the testis.

Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic recptor (CR) in rat epididymal 105,000 g supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-3H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration of hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant for 0 degrees C for 6 h, heating at 50 degrees C for 30 min, or exposure to the sulfhydryl blocking reagent, p-chloromercuriphenylsulfonate (1mM) at 25 degrees C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5alpha-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 degrees C is greater than 2 days), while dissociation from ABP was rapid (half-time at 0 degrees C is similar to 6 min). Cyproterone acetate (250 mg/100 g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.

Maturational changes in testicular steroidogenesis: Hormonal regulation of 5α-reductase

Abstract Studies were undertaken to compare the capacity of the human and rat testis to convert progesterone-3H (3H-P) both to testosterone (T) and 5α-androstane-3α,17β-diol (5α-diol) at various stages of development, and to determine the possible mechanisms regulating the maturational changes in 5a-reductase activity in the rat testis. Testes from 1- and 13-yr-old males converted 3H-P to T (7–13%) and 17-hydroxyprogesterone (3–20%) and several unidentified metabolites. A similar metabolic pattern was obtained when 3H-P was incubated with testicular homogenate of 20-yr-old subjects, with the exception that the formation of T (27%) was higher than in 13-yr-old testis. Very little 5α-reduced androstane products, however, accumulated in these three incubations. Under the same incubation conditions, 90-day-old rat testis converted 3H-P to T, while 28-day-old testis converted 3H-P mainly to 5α-diol (58%). To determine whether 5α-reductase activity is dependent on gonadotropic stimulation, 5α-reductase was measured in seminiferous tubules and interstitial cells following various periods of hypophysectomy. In both testicular compartments, 5α-reductase activity decreased rapidly, reaching a minimal level 7 days following hypophysectomy. Treatment with either FSH or LH (3 days) 2 days following hypophysectomy increased significantly 5α-reductase activity in whole testis. Large doses of testosterone propionate, however, did not appear to increase the activity above the level of the untreated hypophy-sectomized control, indicating that this effect of gonadotropins is not mediated through androgens. These studies clearly demonstrated that neither the immature nor the mature human testis possesses a high level of 5α-reductase activity as compared with the rat testis, and that 5oc-reductase activity in the rat testis is dependent on gonadotropic stimulation, in contrast to the androgen-dependent 5α-reductase in other target tissues.

Further characterization of the 5α-dihydrotestosterone binding protein in the epididymal cytosol fraction. In vitro studies

Physicochemical properties of the DHT binding protein in the epididymal cytosol fraction (ABP) have been studied in vitro using gel filtration, sucrose gradient centrifugation, ion exchange chromatography on DEAE cellulose, isoelectric focusing, polyacrylamide gel electrophoresis and equilibrium dialysis. The DHT-ABP complex had an Einstein-Stokes radius of 47–48 A and a sedimentation coefficient of 4.6 S, indicating a molecular weight of approximately 90 000 and a frictional ratio (ffo) of 1. 61 (assuming v = 0.725 cm3/g). This complex became eluted with the bulk protein at 0.1 M NaC1 from the columns of DEAE cellulose. The DHT-ABP complex had an isoelectric point of 4.6–4.7, and moved as one sharp zone upon polyacrylamide electrophoresis, with Rf: 0.42. The epididymal ABP was organ specific, had high affinity (5.5 × 10−9M) and limited capacity for binding androgen (12 pmoles/100 mg tissue). The ligand specificity of ABP was pertinent to the biological action of the steroids upon the epididymis. (DHT > testosterone > estradiol-17β > progesterone > estradiol-17α). The physicochemical properties of the DHT-ABP complex were different from those of the cytosol receptors for DHT in the ventral prostate and seminal vesicles.

Intracellular receptors for 5α-dihydrotestosterone in the epididymis of adult rats. Comparison with the androgenic receptors in the ventral prostate and the androgen binding protein (ABP) in the testicular and epididymal fluid

Abstract Epididymal cytosol fractions of adult short-time castrated rats contained at least two different androgen protein complexes by experiments in vivo (Complex I and II). Complex I is probably located intracellularly in the epididymal cells. It was specific for 5α-dihydrotestosterone (DHT) and appeared to be very similar to the cytoplasmic DHT-receptor complexes in rat ventral prostate. By ultracentrifugation on sucrose gradients, it sedimented as heavy aggregates 8–10 S complexes and 3–4 S complexes, which dissociated into 3–4 S complexes at high ionic strength. Complex I was eluted in the void volume from columns of Sephadex G-200. Complex II was also specific for DHT and showed physical properties similar to those of the androgen binding protein (ABP) in the testicular fluid. It was eluted between immunoglobulin G (IgG) (53 A) and albumin (36 A) by gel filtration on Sephadex G-200. The sedimentation coefficient was 4.5–5 S (mean 4.6 SW, 20) at both high and low ionic strength. Complex I and the cytosol receptors for DHT in the rat ventral prostate were both destroyed by heating at 50° C for 30 min, addition of 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS) and charcoal absorption (1 mg/mg protein) overnight, whereas complex II was not influenced by similar treatment. Hemi-castration for 4 weeks caused complex II to disappear completely from the castrated side, confirming the intraluminal localization of this complex. Complex I was not influenced by such treatment, indicating that this protein is located within the epididymal cells. The similarity between complex I and the cytoplasmic DHT-receptor complexes in the ventral prostate also suggests that complex I represents the cytoplasmic receptors for DHT in the epididymis.

Androgen stimulation of prolactin receptors in rat prostate

Specific receptors for iodine-labelled human prolactin ([125I]hPrl) are present in membrane preparations of the rat ventral prostate. The binding is saturable with an apparent association constant (Ka) of 2.2 X 10(9) M-1 and a binding capacity of about 1 pmol/100mg prostatic tissue. The binding of [125I]hPrl is inhibited by hPrl, ovine Prl (otprl) and human growth hormone, but not by ovine FSH or LH. Serum from rats having Prl-producing pituitary tumors caused a displacement of the [125I]hPrl from the receptors, and the displacement curve was parallel with that of the hPrl standard. Treatment of immature rats with varying doses of dihydrotestosterone propionate (10-5000 microng) causes a dose-dependent stimulation of Prl receptors calculated both as binding sites per mg of membrane protein and as binding sites per prostate. Androgen stimulation of prostatic Prl receptors increases the tissue sensitivity for circulating Prl and may be one reason for the known increases in endogenous cAMP levels in prostatic tissue after androgen treatment in vivo.

Endocrine status of the testicular feminized male (TFM) rat

Abstract The plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), androstenedione (A), dihydrotestosterone (DHT), corticosterone and corticosterone binding globulin (CBG) were determined in 160- and 350-day testicular feminized male (tfm) and normal littermate (NL) rats. In the younger group the concentrations of T, A, DHT and 5α-androstan-3α,17β-diol (Adiol) were also determined in testis tissue. Tfm rats showed a greatly elevated plasma LH indicating lack of androgen feedback. Plasma FSH, however, was normal in both age groups, suggesting that inhibin production from tfm testes was relatively unaffected. Intratesticular concentrations of A were greatly elevated in the tfm animals at both ages whilst the testicular levels of T were highly reduced when compared to NL rats. This indicates a gonadal deficiency of the 17β-hydroxy steroid dehydrogenase (17β-HSD). Furthermore, the relatively low T: DHT ratio and high concentrations of Adiol in the tfm testes confirmed previous reports that the high 5α-reductase typical for the immature rat testis is maintained into adulthood in rats with the tfm condition. A considerable degree of peripheral conversion (A → T) probably helps to maintain normal or supranormal plasma levels of T. Gonadectomy rendered all plasma androgens undetectable, indicating that the adrenal contribution was negligible. Increasing age (350 days) was associated with a marked increase in circulating androgens in tfm rats. This is probably the reason for the observed significant reduction in circulating LH in this age group when compared to the 160-day animals. The aging tfm rat is predisposed to testicular tumors which, on the basis of histology, specific [125I]hLH binding and in vitro responsiveness to hCG, appear to be of Leydig cell origin. Microflow fluorometry (MFF) of tfm testis cell suspensions revealed the presence of haploid cells, suggesting that meiosis proceeds to a limited extent. Plasma corticosterone levels in the tfm rat were normal although plasma CBG levels were highly significantly elevated at both ages when compared to the levels in NL rats. We suggest that the adrenal hyperplasia observed in tfm rats is secondary to reduced corticosterone production and diminished free corticosterone in the circulation.

5α-Reduced androgens and testicular function in the immature rat: Effects of 5α-androstan-1 7β-ol-3one (DHT) propionate and 5α-androstan-3α,17β-diol

Abstract Increasing doses of dihydrotestosterone propionate (DHTP) and 5α-androstan-3α,17β-diol (ADIOL) were given to 21-day-old rats for 10 days. Various parameters of reproductive function were assessed including testis weight, epididymal androgen binding protein (ABP) (Sertoli cell activity), plasma LH and FSH and the testicular levels of androstenedione (A), testosterone (T), dihydrotestosterone (DHT) and 5α-androstan-3α,17β-diol (ADIOL). In the control population, of the androgens measured, only the testicular concentration of DHT was highly correlated with epididymal ABP levels. After treatment with varying doses of DHTP, testis weight and epididymal ABP exhibited a biphasic response with maximum suppression occurring at the 100–250gmg/day dose level. Recovery of both of these parameters to control levels with 5,000–10,000 μg/day DHTP was associated with an increase in testicular DHT to normal or Supranormal levels. Plasma LH and FSH (pituitary function) and testicular A and T (Leydig cell function) were suppressed in a dose-dependent manner at doses including and higher than 50 μg DHTP/day. The testicular content of ADIOL was returned to control levels with 500 μg DHTP/day, when Sertoli cell function and spermatogenesis were still markedly suppressed. At DHTP doseshigher than 500 Jug/day, ADIOL was the predominant intratesticular androgen. Treatment with ADIOL also evoked a biphasic response from testis weight and epididymal ABP comparable to that observed with DHTP and testosterone propionate (TP) (Weddington et al., 1976). Doses from 20–500 μg/day resulted in a gradual, dose-dependent suppression of all the parameters studied. Administration of 1000 μg/day ADIOL returned the testicular ADIOL content to the control level. At the same dose, epididymal ABP, testis weight and the other intratesticular androgens were still maximally suppressed. Doses of 5000 and 10,000 μg/day ADIOL elevated the endogenous ADIOL in the testis to 4 and 25 times the normal level respectively. These higher doses also caused a dose-dependent increase in intratesticular DHT which was paralleled by an increase in epididymal ABP and testis weight. Even at the highest dose of ADIOL injected, DHT, epididymal ABP and testis weight did not completely return to normal levels. It is concluded that the previously observed stimulation of Sertoli cell function and spermatogenesis by androgens, in the rat, is mediated through DHT. Furthermore, the findings indicate that the apparent direct stimulation of Sertoli cell function by ADIOL is mediated through its conversion to DHT. The sensitivity of the testis to various androgens cannot be clearly interpreted on the basis of the administered doses, but only in relation to the levels of endogenous androgens in the testis achieved by the hormone treatment. In this regard, the study emphasizes the need for careful interpretation of data involving androgen treatment, especially ADIOL, and suggests that the relative potency of androgens in a bioassay system does not necessarily provide clear information about their relative potency at the target cell level.