Copulation in castrated male rats following combined treatment with estradiol and dihydrotestosterone.

Castrated male rats injected daily with 2 micrograms of estradiol benzoate (EB) combined with 200 micrograms of dihydrotestosterone propionate (DHTP) displayed masculine mating behavior which was indistinguishable from that of other castrates treated with 200 micrograms of testosterone propionate (TP). Significantly less copulation was seen in rats treated with either 4 micrograms of TP plus 200 micrograms of DHTP or 2 micrograms of EB. Mating in male rats may depend on the action of both estrogenic and 5alpha-dihydro metabolites of testosterone.

Maturation and survival of spermatozoa in the epididymis of pregnenolone treated hypophysectomized rats.

Adult hypophysectomized male rats were injected daily with 2 mg pregnenolone for 30 days. In these rats the testosterone concentrations in peripheral blood (0.5 ng/ml) and testes (46 ng/g) were appreciably lower than in intact rats (4.1 ng/ml and 167 ng/g, respectively). In hypophysectomized animals treated with pregnenolone the testes and epididymides were much better maintained than the prostates and seminal vesicles. High concentrations of dihydrotestosterone were found to be present in the head (12 ng/g) and body (8 ng/g) of the epididymis of these rats. Although the number of spermatozoa in the distal part of the epididymis of pregnenolone-treated hypophysectomized rats was only 23% of the number found in intact control animals, the spermatozoa were fertile.

Specific, high-affinity binding of 17beta-estradiol in cytosols from several brain regions and pituitary of intact and castrated adult male rats.

Specific 17beta-estradiol binding capacities of cytosols from several brain regions and pituitary were determined in intact and castrated adult male rats. The binding capacity of the pituitary was approximately 10 times higher than that of any of the 5 brain region studied. Of these brain regions, the highest 17beta-estradiol binding capacities were present in the anterior hypothalamus followed by progressively lower capacities in the posterior hypothalamus, amygdala, midbrain, and cerebral cortex. The specific 17beta-estradiol binding capacity of cytosol from the anterior hypothalamus was significantly higher in castrated males than in intact rats. No such difference was found in any of the other tissues studied. Using sucrose gradient ultracentrifugation, an 8S sedimentation coefficient was found for the specific estradiol binding macromolecules present in cytosols from pituitary as well as anterior and posterior hypothalamus of castrated rats. The affinity for estradiol of cytosols from anterior and posterior hypothalamus was very high, with the mean association constants being 2.9 and 2.4 X 10(10) M-1, respectively. In competition experiments the 17beta-estradiol binding molecules present in cytosols from pituitary and anterior hypothalamus showed a higher affinity for 17beta-estradiol than for either estrone or estriol. In both tissues these 17beta-estradiol binding molecules showed a moderate affinity for the anti-estrogens MER-25 and cis-clomiphene citrate as well as for the androgen 3beta-androstranediol, but almost no affinity for 3alpha-androstanediol, 5alpha-dihydrotestosterone, testosterone, or corticosterone. These findings suggest that a true cytoplasmic receptor for estradiol exists in the male rat brain and pituitary which may play an important role in regulating reproductive function.

Effects of a prolactin- and adrenocorticotropin-secreting tumor on gonadotropin levels and accessory sex organ weights in adult male rats: a possible role of the adrenals.

The presence of the transplantable PRL- and ACTH-secreting tumor 7315a in intact male rats resulted in very high serum PRL levels, decreased levels of LH and FSH, and reduced weights of testes and accessory sex organs. In gonadectomized rats bearing a small testosterone-filled capsule, the tumor inhibited the postcastration rise in gonadotropins and reduced the weights of the prostate and seminal vesicles. However, after adrenalectomy of intact or gonadectomized male rats bearing a small testosterone-filled capsule, inhibitory effects of the tumor on serum gonadotropin levels or on reproductive organ weights were totally absent. These results show that in adrenalectomized male rats hyperprolactinemia in itself doses not affect gonadotropin secretion and the androgenic action of testosterone. Rather, the tumor might exert a gonadotropin inhibitory action through elevated levels of PRL combined with progesterone, which is secreted by the ACTH-stimulated adrenal.

Effects of adrenocorticotropin and corticosterone on the negative feedback action of testosterone in the adult male rat.

Injections of ACTH (Synacthen depot) to intact male rats resulted in high serum levels of corticosterone and progesterone, and decreased levels of LH, FSH, and testosterone. In gonadectomized rats with low serum testosterone levels (approximately 1 ng/ml) induced by a small testosterone-filled silicone elastomer capsule, ACTH inhibited the postcastration rise in LH and FSH and reduced the wts of the prostate and seminal vesicles. After adrenalectomy the inhibitory effects of ACTH on serum gonadotropins and organ wts were almost totally absent. Administration of corticosterone acetate (10 mg/day) to gonadectomized and adrenalectomized male rats resulted in high serum levels of corticosterone (approximately 400 ng/ml) which were about 3 times higher than those measured in intact control animals. Nevertheless, in these rats the serum levels of LH and FSH were as high as those measured in gonadectomized and adrenalectomized oil-treated rats. However, when in addition to the injections of corticosterone acetate, a small testosterone-filled capsule was implanted, the postcastration rise in FSH was fully inhibited, whereas the serum levels of LH were below the level of detection. Significant inhibition of the postcastration rise in LH and FSH also occurred when smaller quantities of corticosterone acetate were given. Since in gonadectomized and adrenalectomized male rats similar testosterone-filled capsules did not prevent the postcastration rise in LH and FSH, it is concluded that a high serum level of corticosterone increases the sensitivity to the negative feedback effects of testosterone.

Serum inhibin B as a marker of spermatogenesis.

Inhibin B is produced by Sertoli cells, provides negative feedback on FSH secretion, and may prove to be an important marker for the functioning of seminiferous tubules. The purpose of the present study was to examine the relationship between the spermatogenic function of the testis of subfertile men and the plasma concentrations of inhibin B and FSH. These parameters were estimated in a group of 218 subfertile men. Serum inhibin B levels were closely correlated with the serum FSH levels (r = -0.78, P < 0.001), confirming the role of inhibin B as feedback signal for FSH production. The spermatogenic function of the testis was evaluated by determining testicular volume and total sperm count. Inhibin B levels were significantly correlated with the total sperm count and testicular volume (r = 0.54 and r = 0.63, respectively; P < 0.001). Testicular biopsies were obtained in 22 of these men. Inhibin B was significantly correlated with the biopsy score (r = 0.76, P < 0.001). Receiver operating characteristic analysis revealed a diagnostic accuracy of 95% for differentiating competent from impaired spermatogenesis for inhibin B, whereas for FSH, a value of 80% was found. We conclude that inhibin B is the best available endocrine marker of spermatogenesis in subfertile men.

Distribution of testosterone and 5alpha-dihydrotestosterone in rat epididymis and their concentrations in efferent duct fluid.

5alpha-Dihydrotestosterone concentrations were found to be appreciably higher in the proximal portion of the rat epididymis than in the distal portion. Following ligation of the efferent ducts of a testis, the 5alpha-dihydrotestosterone concentration diminished in the proximal half but not in the distal half of the epididymis. Epididymal fluid and spermatozoa were washed out from minces of epididymal tissue. The numbers of spermatozoa present in the wash fluid and in the homogenate of washed minces of epididymal tissue were used as measures for the amounts of epididymal fluid present in these fractions. Of the total amount of 5alpha-dihydrotestosterone (30-2 ng/g tissue) in the proximal portion, 20-5 ng were localized in the epididymal fluid, whereas of the total amount of 5alpha-dihydrotestosterone (8-6 ng/g tissue) in the distal portion, 2-8 ng were found in the epididymal fluid. The amounts of testosterone present in the fluids of both proximal and distal portions were 1-2 ng/g tissue. In contrast to the epididymal fluid, efferent duct fluid had a high concentration of testosterone (28-8 ng/ml) and a low concentration of 5alpha-dihydrotestosterone (1-9 ng/ml). These data suggest that the fluid surrounding the spermatozoa in the testis and the epididymis contains a high concentration of androgen and that as the fluid moves from the testis to the epididymis there is a clear change in the ratio of testosterone to 5alpha-dihydrotestosterone.

Effects of prolactin on testicular functions using an intratesticular pituitary graft.

Pituitary glands were grafted under the capsule of the left testis of rats to induce high levels of prolactin in this organ. One hundred days after implantation, significantly increased levels of prolactin were found in the tissue and the venous plasma of the left testis. Although the levels of testosterone in testicular venous plasma were not raised, the testicular content of testosterone was increased when compared to the right testis. The ratio of testosterone and dihydrotestosterone was not affected in the pituitary-grafted testis. Since the seminiferous tubules adjacent to the pituitary graft appeared to be completely normal, it is concluded that in the rat high levels of prolactin have no direct inhibitory effect on testicular functions.

The contribution of corticosterone and testosterone to the suppression of serum LH in hyperprolactinaemic adult male rats with pituitary transplants.

The effects of hyperprolactinaemia on serum levels of LH were investigated in adult male rats of the R X U strain. Hyperprolactinaemia was induced by three pituitary grafts under the kidney capsule, transplanted on day 0 of each experiment. Special attention was paid to the contribution of prolactin-stimulated testes, adrenals and corticosterone. In experiment 1, hyperprolactinaemia significantly reduced the serum concentrations of LH in intact rats. In spite of a significant increase in the serum levels of corticosterone, serum testosterone was not significantly affected by hyperprolactinaemia. The weights of both the adrenals and accessory sex glands were significantly increased at autopsy. In experiment 2, treatment with 10 mg corticosterone s.c. daily from day 14 to day 28 after pituitary grafting significantly reduced serum levels of both LH and testosterone. The suppression of testosterone in the hyperprolactinaemic corticosterone-treated animals was significantly less than in the corticosterone-treated control animals. The weights of the accessory sex glands were significantly increased in the hyperprolactinaemic animals. In experiment 3, rats were adrenalectomized and half of them were substituted with corticosterone. Serum testosterone levels significantly increased in both hyperprolactinaemic adrenalectomized rats and in adrenalectomized corticosterone-treated animals without any significant effect on serum LH. Again the weights of the accessory sex glands were significantly increased in the hyperprolactinaemic animals. In experiment 4, rats were adrenalectomized, gonadectomized and corticosterone treated on day 0 and then implanted with a 2, 1.5 or 1 cm silicone elastomer capsule containing testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the new prolactin-producing tumour 7315b on gonadotrophin secretion in adult male and female rats.

The effects of the transplantable purely prolactin-secreting tumour 7315b on serum gonadotrophins were studied in adult rats. Possible contributions of the adrenals to the tumour-induced inhibition of serum LH and FSH were evaluated. The suppressive actions of tumour 7315b on serum gonadotrophins in gonadectomized plus adrenalectomized male and female rats were compared. Within 4 weeks after inoculation of tumour 7315b in intact male rats very high levels of prolactin and decreased serum levels of gonadotrophins and testosterone were recorded. At autopsy reduced weights of testes and accessory sex organs and slightly increased adrenal weights were found. In addition, in animals treated with a small testosterone-filled capsule after castration, tumour 7315b reduced serum concentrations of LH and FSH. Adrenalectomy did not prevent this suppressive action of the tumour on the post-castration rise of serum gonadotrophins. Suppression of serum gonadotrophins during hyperprolactinaemia was greater in gonadectomized plus adrenalectomized female rats than in male rats, indicating that the degree of the tumour-induced suppression of LH and FSH after castration is determined to a large extent by the sex of the animal. The purely prolactin-secreting tumour 7315b has therefore been shown to be a suitable model for studying the effects of severe hyperprolactinaemia on the pituitary-gonadal axis in rats.

Effects of prolactin-secreting tumour on copulatory behaviour in male rats.

The effect of the transplantable prolactin- and ACTH-secreting tumour 7315a on male copulatory behaviour was investigated. Castrated tumour-bearing rats implanted with testosterone-filled capsules exhibited significantly longer latencies to first mount or intromission and to ejaculation than castrated control animals. In contrast to the number of intromissions, the number of mount before ejaculation of the tumour-bearing rats was considerably increased. However, when castration was carried out in addition to adrenalectomy, the differences in copulatory behaviour between tumour-bearing rats and control rats were no longer present. During the last tests for copulatory behaviour the tumour-bearing rats had serum prolactin concentrations or more than 4000 ng/Ml while control rats had less than 100 ng/ml. Plasma testosterone levels produced by silicone elastomer capsules were neither affected by the presence of the tumour nor by adrenalectomy. It was concluded that hyperprolactinaemia does not suppress the copulatory behaviour of adrenalectomized rats.

Prenatal and early postnatal sex differences in plasma and gonadal testosterone and plasma luteinizing hormone in female and male rats.

Plasma levels of testosterone and LH were estimated in female and male rats at gestational ages of 19, 20 and 21 days and at 1, 3, 6, 12, 18 and 24 h after birth. Concentrations of testosterone in the gonads were also estimated in 20-day-old fetuses and at various times after birth. Before birth female fetuses had significantly lower levels of testosterone and higher levels of testosterone and higher levels of LH than had male fetuses. During the first 24 h after birth female rats also had lower levels of testosterone and higher levels of LH than male rats. The pattern of levels of testosterone in the hours after birth was significantly different between female and male rats in that high levels were observed 1 and 3 h after birth in male rats (3.0 and 2.2 ng/ml respectively). This finding, as well as the relatively high levels of testosterone in female fetuses (about 50% of the levels found in male womb-mates) is discussed.

Androgens in the fetal guinea-pig after maternal infusion of radioactive testosterone.

Testosterone was measured in plasma pools from male and female fetal guinea-pigs between the ages of 30 and 55 days of pregnancy. Between days 33 and 36 the testosterone concentration in the plasma of males (1.4 ng/ml) was several times higher than that found at other ages or that measured in female fetuses. After infusion of tritiated testosterone for 2 h into pregnant guinea-pigs at day 36 of pregnancy, high levels of testosterone and androstenedione were found in maternal plasma. Nevertheless, tritiated testosterone and androstenedione could hardly be shown in the fetuses. Similar large differences in plasma progesterone levels appeared to exist between the maternal and the fetal circulation. Therefore, only a very small fraction of these steroids can penetrate from the maternal circulation into that of the fetus. This finding might be explained by the large difference in androgen-binding capacity between maternal and fetal plasma, as was shown by equilibrium dialysis.

Evidence that a placental factor other than androsterone or dihydrotestosterone inhibits oestrogen-induced lordosis behaviour in pregnant rats.

Daily administration of oestradiol benzoate, beginning 10 days after mating, stimulates lordosis behaviour in deciduomata-bearing pseudopregnant rats, but not in pregnant rats. The inhibition of this behaviour during pregnancy was not prevented by reducing the number of conceptuses to two, by removing the fetuses while leaving the placentas in utero, or by removing the ovaries and administering progesterone to prevent abortion. Removal of the uterus or fetuses and placentas on day 12, however, led to high levels of lordosis behaviour. Thus, it is likely that the placenta produces a factor which inhibits the behavioural responsiveness to oestrogen. Plasma levels of progesterone, androsterone and dihydrotestosterone were higher during the second half of pregnancy than in the second half of pseudopregnancy prolonged by uterine decidualization. The possible involvement of these steroids in the inhibition of lordosis behaviour was investigated by increasing their levels in deciduomata-bearing pseudopregnant rats and determining the effect on oestrogen-induced lordosis behavior. Little suppression of this behaviour was seen when the pseudopregnant rats were treated with progesterone or androsterone whereas treatment with dihydrotestosterone resulted in a significant inhibition of lordosis behavior. However, the dose of dihydrotestosterone required to do so resulted in high, non-physiological plasma levels of this steroid. No inhibition of lordosis behaviour was observed when dihydrotestosterone levels were approximately threefold those normally present in pregnant rats. It is concluded that none of these three steroids is primarily responsible for the suppression of lordosis behaviour during pregnancy.

Effects of androgens and estrogens on the vasopressin and oxytocin innervation of the adult rat brain.

Recently we reported that castration of rats eliminates vasopressin immunoreactivity in the lateral septum and other areas that appear to receive vasopressin innervation from the bed nucleus of the stria terminalis. Testosterone treatment counteracts this effect of castration. In the present study, we investigated whether this action of testosterone depends on its androgenic or estrogenic metabolites by treating long-term castrated rats with estradiol (E) and/or 5 alpha-dihydrotestosterone (DHT) or testosterone. The brains were then processed for immunocytochemistry or radioimmunoassay. DHT did not increase vasopressin staining in the lateral septum, although it fully restored the size of the seminal vesicles. E did restore the original fiber density, but individual fibers stained more weakly than in sham-operated males. Only treatment with both E and DHT fully restored the vasopressin innervation. This pattern was also reflected in the radioimmunoassay data. The vasopressin content of the lateral septum decreased about 90% after castration but was fully restored by either testosterone or E + DHT treatment. E alone, however, was only half as effective as E + DHT. The treatments had no effect on the oxytocin content of the septum, or on the vasopressin or oxytocin content of the dorsal vagal complex. The results suggest that E mediates most of the effects of testosterone on the vasopressin innervation of the lateral septum. DHT enhances the response to E but has little effect on its own.

Testicular regulation of epididymal protein secretion.

The effects of different androgen and testicular fluid levels on the protein synthesis and secretion of the epididymis of mice and rats were examined. In mice, the in vitro protein synthesis and secretion of five epididymal segments were measured from 3 days up to 8 weeks after efferent duct ligation. During the entire period, alterations in the rate of protein secretion per milligram tissue were small. At 8 weeks, the mean rate of protein synthesis per milligram ligated epididymal tissue was 80% of that of the control side. As a consequence of the weight loss of the ligated epididymis, however, the protein secretion per organ can be estimated to be reduced by 50%. Changes in the protein profile were only found in the proximal segment, where a 40-kd protein appeared and a 29-kd protein disappeared. In rats, the effects of efferent duct ligation were studied in vivo for up to 8 months. Structural changes were present both in the proximal and in the distal epididymis. The most conspicuous change in the protein profile of secretory proteins was the disappearance of a 27-kd protein from the proximal segment. In the distal epididymis, a 32-kd protein was no longer secreted. In mice, the effects of castration on the profile of secreted proteins demonstrated that, without androgen stimulation, some proteins are still secreted 6 weeks after castration. Administering low or high doses of testosterone propionate to castrated mice resulted in almost similar profiles of secretory proteins. However one protein secreted in the proximal epididymis was preferentially stimulated by the high dose of testosterone propionate.

Androgen receptor immunoexpression in the testes of subfertile men.

The localization and intensity of androgen receptor immunostaining was studied in the testes of 37 subfertile men with oligozoospermia and normal serum gonadotropin levels using a polyclonal antibody raised against a synthetic peptide corresponding to the first 20 N-terminal amino acid residues of the androgen receptor (AR). Furthermore, we investigated whether or not the immunoexpression of the AR in human Sertoli cells, in histologically normal testis tissue, is dependent on the stage of the spermatogenic cycle, as has been found in the rat. In the human testis, AR immunoexpression was observed in Sertoli cells, peritubular myoid cells, Leydig cells, and periarteriolar cells, but not in germinal cells. We found no evidence for a stage-dependent immunoexpression of AR in Sertoli cells. The intensity of AR immunoexpression varied substantially between biopsy specimens of different patients. There was, however, no correlation of the intensity of AR immunoexpression in either Sertoli cells or peritubular myoid cells with spermatogenic adequacy as measured by the method of Johnsen. When, in this study, the intensity of peritubular myoid cell staining was used as a standard to evaluate the intensity of Sertoli cell staining, no correlation was detected as well. Furthermore, serum gonadotropin levels were not correlated with AR immunoexpression levels in Sertoli cells and peritubular myoid cells. These results indicate that immunodetectability of the AR is not related to the condition of the spermatogenic epithelium in patients with oligozoospermia. Inappropriate expression of the AR is neither a cause nor a consequence of idiopathic infertility in the present group of patients.

Comparison of the response of rat testis and accessory sex organs to treatment with testosterone and the synthetic androgen methyltrienolone (R1881).

We have studied the ability of the synthetic androgen methyltrienolone (R1881) to maintain testis and accessory organ weights, as compared to the effect of testosterone propionate (TP). In contrast to TP, R1881 is not metabolized and does not significantly bind to androgen-binding protein (ABP). Thirty-six rats were treated with ethane dimethane sulphonate (EDS) and GnRH antagonist (Org30267) to abolish all testicular androgen production, and recombinant human FSH (rec-hFSH, Org32489) was administered to ensure adequate FSH levels. Of these rats, five groups of four rats were treated daily with 0-, 50-, 100-, 200-, and 400-microgram TP, s.c., and four groups of four rats were treated daily with 150-, 300-, 600-, and 1200-microgram R1881, s.c. One control group of four rats received vehicle injections only. EDS treatment, followed by GnRH antagonist and rec-hFSH treatment for 17 days, significantly reduced testis, prostate, and seminal vesicle weights (P < 0.001, P < 0.01, P < 0.001, respectively). Simultaneous treatment with androgens prevented this organ weight decrease, in a dose-dependent manner. In all TP-treated animals, relative weights (% of control) of the acces, sory sex organs were significantly higher than the relative testis weights (P < 0.001). However, there was no difference in relative weights between testis and accessory sex organs in the R1881-treated animals. In another series of experiments, we investigated the effect of treatment with Finasteride, a 5 alpha-reductase inhibitor, on testis and accessory sex organ weights in rats treated with EDS and TP. Treatment with EDS, TP (300 micrograms/day) and Finasteride (40 mg/kg/day) did not alter testis weight as compared to the effect of treatment with EDS and TP alone. Prostate and seminal vesicle weights were, however, markedly reduced (significantly different from rats treated with EDS and TP alone; P < 0.01 and P < 0.05, respectively). Immunohistochemical analysis of androgen-receptor (AR) expression in the testis revealed that testicular AR immunoexpression is androgen dependent and that FSH alone is not able to maintain AR immunoexpression. Furthermore, the stage-dependent pattern of AR immunoexpression in Sertoli-cell nuclei, during the spermatogenic cycle, is identical in all TP- and R1881-treated rats. It is concluded that testes, prostate, and seminal vesicles are equally stimulated when the androgen receptor in these tissues is exposed to the same intracellular concentration of free androgen and that the low 5 alpha-reductase activity in the testis plays a critical role in the differential response of the testis and the accessory sex organs to T. Furthermore, stage-dependent AR immunoexpression in Sertoli cells does occur in the absence of testicular androgen production and is not due to androgen metabolism or local differences in androgen concentration.