Androgen-dependent accumulation of carnitine by rat epididymis after injection of [3H]butyrobetaine in vivo.

After i.m. injection of [3H]butyrobetaine into rats, the accumulation of carnitine into the epididymis, prostate gland, seminal vesicles, testis and heart was studied. The concentration of radiolabeled carnitine into the cauda epididymis increased linearly with time up to 72 h after the injection of the precursor, while its level in the prostate and seminal vesicles decreased rapidly. Very low levels of carnitine were found in the testis. Castration reduced the carnitine accumulation by cauda epididymis to 6% of the control levels while treatment of castrated animals with testosterone propionate (500 mug/day) partly restored the carnitine uptake. Similar treatment with 17beta-oestradiol valerate or 17alpha-hydroxyprogesterone had no effect. Surprisingly, cyproterone acetate (5 mg/day) also significantly stimulated carnitine accumulation by the epididymis to a level above that of the castrated controls. Simultaneous injection of both cyproterone acetate and testosterone propionate to castrated animals caused an additive effect of these steroids. This indicated that cyproterone acetate in this system is working as a weak androgen. Treatment of rats with 17beta-oestradiol valerate also reduced carnitine accumulation by the cauda epididymis. This is due to suppression of pituiatry gonadotrophin secretion, since concommitant treatment with testosterone propionate (500 mug/day) caused a normalization of the carnitine uptake. Treatment of intact rats with cyproterone acetate significantly reduced the epididymal weight, but not the carnitine accumulation. 17alpha-Hydroxyprogesterone treatment had no effect either on the epididymal weight or the accumulation of the carnitine. Unilateral orchiectomy reduced the carnitine accumulation by the cauda epididymis to about 40% of that occurring in the non-operated control side. This indicates that the luminal contact between the testis and epididymis or the luminal content of the epididymis itself is of importance for the androgen-dependent metabolic process occurring in the cauda epididymis. Castration or hormone treatment did not change the conversion of butyrobetaine to carnitine, or the carnitine uptake by heart. Carnitine uptake by the testis after [3H]butyrobetaine injection was rather low and this would exclude the possibility of synthesis of carnitine in the testis as a source of epididymal carnitine. Carnitine only accumulated in the cauda epididymis in vivo 4 to 96 h after injection of [3H]butyrobetaine. The presence of radioactively labeled butyrobetaine or methylcholine was not detected.     

The effect of testosterone propionate on the concentration of testicular and epididymal androgen binding activity in the hypophysectomised rat.

The effect of testosterone propionate on the level of androgen binding activity in both testis and epididymis was investigated by both a dextran coated charcoal (DCC) method and by equilibrium polyacrylamide gel electrophoresis (PAGE) which measures specifically androgen binding protein.There was a decrease in androgen binding activity in both testis and epididymis after hypophysectomy. Testosterone propionate treatment (1 mg/day), begun at one day posthypophysectomy, maintained androgen binding activity above the level in testes from hypophysectomised rats for 30 days (PAGE) or 60 days (DCC) and 60 days in epididymides (PAGE). The ability of testosterone propionate treatment (250, 500 and 750 μg/day) to restore androgen binding activity in testis and epididymis of chronically hypophysectomised rats (30 days) was tested using a 3-day treatment period. These doses restored testicular androgen binding activity to 60, 36 and 75% of the levels in control animals, respectively. The degree of restoration of androgen binding activity in epididymis in this time interval was inferior to that in the testis at all doses. Prolonged treatment with testosterone propionate (1 mg/day for 10, 30 and 60 days) resulted in complete restoration of androgen binding activity in testis, expressed per mg protein (PAGE and DCC), and over 50% restoration in the epididymis (PAGE).It was concluded that testosterone propionate is able to maintain and restore testicular and epididymal androgen binding activity and, in particular, androgen binding protein, after hypophysectomy and that this hormone may, in the short term, influence testicular rather than epididymal levels of binding activity.     

Effect of testosterone propionate on levels of carnitine and testicular androgen binding protein (ABP) in rat epididymis.

Twenty-one day old rats were treated for 10 days with various doses of testosterone propionate (TP) (10 micrograms to 10 mg/day) and the levels of L-carnitine and testicular androgen binding protein (ABP) were measured in the 105,000 x g supernatant fractions of epididymis. Treatment with TP in increasing doses had a biphasic effect on the level of ABP in the epididymis; thus, with doses of TP of 10-100 micrograms/day, the ABP level was reduced in a dose-dependent way, whereas with higher doses of TP (0.2 to 1 mg/day) the extent of reduction of ABP levels was less as the dose of TP increased. Treatment with high doses (5 mg or 10 mg/day) did not change the ABP level compared with non-treated control rats. The concentration of carnitine increased linearly (log dose-response) with increasing doses of TP (10-200 micrograms/day) and there was no further increase after treatment with higher doses of TP. In adult rats TP (175 micrograms and 17.5 mg/day) reduced the level of ABP but not the level of carnitine in the epididymis. These studies suggest, therefore, that ABP is of minor importance for the supply of androgens to the carnitine-concentrating cells in the corpus and cauda epididymis.     

The effect of castration and testosterone replacement on specific proteins and androgen levels of the rat epididymis.

The normal weight increase of the epididymis during sexual maturation and its maintenance through adulthood were found to be dependent on the provision of androgens. Binding of [3H]dihydrotestosterone (DHT) to the epididymal 8S cytoplasmic receptor gradually decreased after castration to become undetectable after 25 days. Binding to the androgen binding protein (ABP) was absent 4 days after castration and was not reinduced by 3 weeks of testosterone (T) administration. Unilateral castration for periods of up to 27 days showed the disappearance of ABP with preservation of the 8S receptor on the castrated side, indicating a testicular source for ABP and the epididymal origin of the 8S receptor. The tissue concentrations of T and DHT in the epididymis became undetectable 30 days after castration and were restored to normal values by administration of testosterone in large doses (1.5 mg/100 g BW). Similar results were obtained in rats castrated at 10 days of age and injected with testosterone until 60 days old. The ratio DHT/T was depressed in the castrate and increased with testosterone treatment. The protein content of the epididymis (mg of protein/g wet weight) was also found to be influenced by androgens. Our results show evidence of some mechanisms involved in the trophic effect of androgens upon the epididymis and suggest the possible androgenic control of epididymal 5alpha-reductase activity. They also indicate that a testicular factor is required for the maintenance of the 8S cytoplasmic androgen receptor. It is not known whether this factor is testosterone or some other testicular secretion.     

Sexual differentiation of positive feedback: effect of hour of castration at birth on estradiol-induced luteinizing hormone secretion in immature male rats

In the male rat, a dramatic increase in hypothalamic testosterone and estradiol concentrations occurs during the first few hours of postnatal life. These experiments sought to determine whether such increases participate in the defeminization of positive estrogen feedback effects on LH secretion. Newborn male rats were castrated either in utero (0 h males), or 10 or 24 h after birth. Some males were castrated at 0 h in utero and injected at the time of surgery with 1,2.5, or 5 micrograms testosterone propionate. A group of females was ovariectomized at 0 h in utero (0 h females). The control group consisted of male and female rats sham gonadectomized at 0 h in utero which were either gonadectomized at 21 days of age or left intact. The experimental groups were challenged before puberty to determine if estrogen induced a release of LH using two different types of estrogen treatment. The first treatment consisted of an injection of 0.2 microgram estradiol benzoate (EB) on day 28 followed by a second 10 micrograms injection of EB on day 29. This treatment resulted on the afternoon of day 30 in a surge of LH in intact females. Normal males, 0 h males, or females castrated at 21 days did not have a significant LH surge. The second test consisted of the daily injection of 0.05 microgram EB on days 23-27; on day 28 the rats were injected with 2.5 micrograms EB. Zero hour male and female rats showed a large LH surge on the afternoon of day 29; sham castrated males never responded to this treatment. No sex difference was observed in the mean size of the LH surge providing the males were castrated at 0 h in utero. The effect of the hour of castration on the day of birth also was studied. Males castrated at 10 or 24 h after birth showed either no LH surge or the magnitude of the surge was greatly reduced compared to that obtained in the 0 h males (P less than 0.001). The fact that 0 h males injected with 1 microgram testosterone propionate never showed an LH surge after prepuberal treatment with estrogen suggests that 0 h is a time during which the newborn is sensitive to the defeminizing effect of androgens. These results are consistent with the idea that the testicular hyperactivity which occurs at the time of birth could influence the defeminization of the LH surge mechanisms.     

Nuclear accumulation of androgens in perfused rat accessory sex organs and testes.

The uptake of androgens into the nuclei of caput epididymis, ventral prostate, seminal vesicle and testis was studied by recirculating physiological and pharmacological concentrations of [3H]testosterone in an artificial medium through the lower half (hemicorpus) of castrated or hypophysectomized rats. The accumulation of dihydrotestosterone in accessory sex organ nuclei was saturable, inhibited by perfusion of excess testosterone or cyproterone acetate, and associated with binding to 3S salt-extractable molecules. In castrated preparations the mean saturation levels (pmol/mg DNA) were different in the three organs: seminal vesicle, 2.8; ventral prostate, 1.8; caput epididymis, 0.9. The saturation level was significantly lower in ventral prostate of hypophysectomized rats (1.2) treated with testosterone to regenerate the accessory sex organs. Testosterone was the major nuclear androgen in the testes of mature hypophysectomized preparations perfused with testosterone. Although there was a large amount of nonspecific accumulation, testosterone binding to 3S molecules was shown by sucrose gradient centrifugation. Binding of dihydrotestosterone to 3S molecules in testicular nuclei was also demonstrated. The ratio of dihydrotestosterone to testosterone was different in immature and mature testicular nuclei and was altered by treatments known to affect testicular 5 alpha-reductase activity. The results suggest that in rat accessory sex organs and immature testis the major active androgen is dihydrotestosterone, whereas in mature testis it is testosterone. The shift in the predominant nuclear androgen in the testis from dihydrotestosterone to testosterone is most simply explained by the maturational change in 5 alpha-reductase activity.     

Social behaviour and testicular activity of juvenile rats

The juvenile stage of ontogeny is often characterized as a time of inactivity and quiescence for the immature reproductive system. The principle social behaviour by juveniles of many mammalian species is a rough-and-tumble activity known as play-fighting. An experiment is reported in which play-fighting by male rats was observed after various manipulations of gonadal steroids. Rats were housed in groups and castrated either on day 1 or day 10, times which are respectively during and after the sensitive period for androgen-induced organization of neural tissues in rats. Animals were injected with either 40 micrograms testosterone propionate or vehicle between 21 and 45 days of age, and play-fighting with unoperated, unfamiliar rats was examined. Castration on days 1 and 10 suppressed play-fighting. The behaviours remained suppressed in rats castrated on day 1 and injected with testosterone propionate, but testosterone propionate restored play-fighting to near-normal levels until 35 days of age in rats castrated on day 10. Moreover, gonadally intact juvenile males exposed to the androgen antagonist flutamide play-fought less than intact control males, although their social activity increased appreciably after day 35. It is concluded that the juvenile is not experiencing endocrine quiescence. Behavioural and physiological data suggest a reproductive system which is active, although differently from that in the adult. Hypersensitivity to testosterone and surges of gonadotrophin-releasing factors and LH at 35 days of age may be the events responsible for changes in play-fighting resulting from manipulations of gonadal steroids in juvenile rats.     

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

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.     

Peripheral and central androgenic stimulation of sexual behaviour of castrated male rats

The effects of androgens on the maintenance and restoration of sexual behaviour (mounts, intromissions and ejaculations) of castrated male rats were studied. In the maintenance study the rats were treated during 5 weeks, starting one day following castration. Testosterone propionate maintained sexual behaviour at an almost normal level. The androgenoestrogen intermediate 19-hydroxytestosterone propionate was unable to prevent the decline in the number of ejaculations over the weeks although this hormone maintained the post-ejaculatory refractory period in those rats that ejaculated and also maintained normal sexual latencies. In the restoration study administration of testosterone propionate during 7 weeks to long-term castrated rats restored sexual behaviour to normal. 19-Hydroxytestosterone propionate treated rats displayed mounts but no other signs of sexual behaviour. The 5alpha-reduced androgen dihydrotestosterone propionate did not restore sexual behaviour. Testosterone propionate and dihydrotestosterone propionate stimulated peripheral target organs; 19-hydroxytestosterone propionate was ineffective in this respect. It has been suggested that testosterone might stimulate sexual behaviour in rats in two ways, i.e., via its aromatization to oestradiol in the brain, andy by stimulating growth of peripheral tissues via its 5alpha-reduction to dihydrotestosterone. In support for this view we have found that the combination of 19-hydroxytestosterone propionate and dihydrotestosterone propionate was effective in restoring the full pattern of sexual behaviour in castrated male rats.     

L-prostaglandin D synthase expression and regulation in mouse testis and epididymis during sexual maturation and testosterone treatment after castration

Lipocalin-type prostaglandin D synthase (L-PGDS) is highly expressed in the adult testis and epididymis of many mammals. The present study was to investigate L-PGDS expression in mouse testis and epididymis during sexual maturation, and the effects of testoster-one replacement on L-PGDS expression in epididymis by in situ hybridization and immunohistochemistry. Both L-PGDS mRNA and protein were highly expressed in the interstitial tissue of adult testis. L-PGDS mRNA was first detected on d 30 after birth and exhibited an abundant signal in adult caput and cauda epididymis. L-PGDS immunostaining was first observed on d 30 after birth. There was a strong level of L-PGDS immunostaining in adult epididymis. Castrated male mice were treated with either vehicle or testosterone propionate following 3 d postcastration. L-PGDS expression steadily declined in a time-dependent fashion in control groups. No L-PGDS mRNA expression or immunostaining was detected in the controls for 12 d. When the castrated mice were treated with testosterone propionate for 5 or 12 d, L-PGDS expression was significantly increased in the whole epididymis. These data suggest that L-PGDS expression in mouse epididymis gradually declined in parallel to the declining concentration of endogenous androgen after castration and increased with the treatment of exogenous testosterone, indicating that L-PGDS expression in mouse epididymis was modulated by androgen levels. However, differential expression in different areas of the epididymis may also be influenced by factors derived from the testis.     

Effect of androgens on hypothalamic pro-opiomelanocortin

Testosterone and estradiol have been shown to affect the hypothalamic content of several pro-opiomelanocortin (POMC)-derived peptides in castrated male and female rats, respectively. It was unclear, however, whether the effects of testosterone on hypothalamic POMC were due to conversion by aromatization to estradiol or whether there were independent androgen actions on hypothalamic POMC. In order to answer this question, the effect of treatment with the nonaromitizable androgen 5-alpha-dihydrotestosterone (DHT) on the concentration of beta-endorphin (beta-EP) in the medial basal hypothalamus (MBH) was studied in castrated male rats and compared to the effect of treatment with testosterone or estradiol. The concentrations of two other POMC-derived peptides, corticotropin-like intermediate lobe peptide (CLIP) and alpha-MSH were measured as well. Adult male rats were castrated and received either no treatment or treatment with subcutaneously implanted silastic capsules, containing either DHT, testosterone or estradiol, designed to produce steroid levels in a physiological range. After 4 weeks the mean concentration of beta-EP in the MBH of the untreated castrated rats was 1,640 +/- 56 fmol/mg protein. This was reduced significantly to 1,184 +/- 74 fmol/mg protein after DHT treatment (p less than 0.001). Similar reductions to 1,340 +/- 95 and 1,130 +/- 85 fmol/mg protein were noted after testosterone and estradiol treatment, respectively. The mean CLIP concentration of 1,870 +/- 73 fmol/mg protein in the untreated animals fell to 1,390 +/- 95 after DHT (p less than 0.001) compared to 1,520 +/- 105 and 1,260 +/- 101 after testosterone and estradiol treatment, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Castration induces apoptosis in the mouse epididymis during postnatal development

The effect of castration on apoptosis in the mouse epididymis during postnatal development was examined. The weight of the epididymis slowly increased from day 0 (day of birth) to day 20 after birth, followed by a rapid increase thereafter. Castration on days 0, 5, 10, 20, 30, 40 and 60 increased apoptotic indices (percentages of apoptotic cells) of epithelia of the caput (head), corpus (body), and cauda (tail) epididymis, their apoptotic indices reaching maximal levels on day 2 after castration with the exception of a maximal apoptotic index on day 4 in the tail after castration on day 60. The maximal levels of apoptotic indices of the head, body and tail after castration on days 0, 5, 10 and 20 were significantly lower than those after castration on days 40 and 60. DNAs extracted from the epididymides 2 days after castration on days 0, 5, 10 and 60 showed a ladder pattern on agarose gel electrophoresis, which is a characteristic of apoptosis. When testosterone propionate (10 microg/g body weight) was injected twice a day into mice which had been castrated on day 10, 30 or 60, the increases in apoptotic indices of the head, body and tail of the epididymis were completely inhibited. The weights of the paired epididymides 6 days after castration on days 0, 5, 10, 20, 30, 40 and 60 were significantly lower than those of sham-operated mice, indicating the secretion of androgen by the testes from birth to adulthood. The present results indicated that androgen deprivation caused by castration induces apoptosis in the epithelium of the epididymis of mice from birth to adulthood, and suggested that a proportion of epithelial cells, the survival of which is dependent on the testes, is smaller in the epididymides during a slow growth stage than in the epididymides after this stage.     

Pituitary responsiveness to LH-RH in intact and ovariectomized androgen-sterilized rats.

Serum LH changes in response to LH-RH injection were measured in intact and ovariectomized, steroid-treated female rats which were androgenized neonatally with 1,250 microgram testosterone propionate (TP) on day 5. At a dose of 20 ng LH-RH/100 g b.w., serum LH levels in intact rats increased over pre-injection levels, and at a dose of 100 ng LH-RH/100 g b.w., LH concentrations 15 min after injection were higher in nembutal-blocked proestrous rats than in androgen-sterilized rats. However, the ovulation response was not different between the groups. In ovariectomized estradiol benzoate (EB)-treated, androgen-sterilized rats, serum LH concentrations 15 and 60 min after LH-RH injection were lower than in similarly treated control rats. This effect was not secondary to the anovulatory state of the animal, since it also occurred in ovariectomized EB-treated prepuberal rats and in rats ovariectomized prepuberally and treated with EB in adulthood. Also, after treatment with 5alpha-dihydrotestosterone propionate (5alpha-DHTP), pituitary responsiveness to LH-RH in androgen-sterilized rats was lower than in control rats, which suggests that the subnormal response in the estrogen-treated rats was not due to a relative insensitivity to estrogen in the androgen-sterilized rats. The relatively high pituitary responsiveness to LH-RH in intact androgen-sterilized rats is probably due to the high circulating estrogen levels. The subnormal pituitary responsiveness to LH-RH after ovariectomy and estradiol treatment suggests, in addition to an effect on the hypothalamus, also a direct effect of neonatal androgen administration on the pituitary.     

Androgen-estrogen synergy in rat levator ani muscle: glucose-6-phosphate dehydrogenase

The effects of castration and hormone administration on the activity of glucose-6-phosphate dehydrogenase in the rat levator ani muscle were studied. Castration caused a decrease in enzyme activity and in wet weight of the levator ani muscle. Chronic administration of testosterone propionate increased glucose-6-phosphate dehydrogenase activity in the levator ani muscle of castrated rats; the magnitude of the recovery of enzyme activity was related to the length of time of exposure to testosterone propionate after castration as well as to the length of time the animals were castrated. The longer the period of castration before exposure to testosterone propionate, the greater the effect. This result may be related to previously reported castration-mediated increases in androgen receptor binding in muscle. Dihydrotestosterone was less effective than testosterone propionate in enhancing glucose-6-phosphate dehydrogenase activity in the levator ani muscle from castrated rats; estradiol-17 beta alone was ineffective. Combined treatment with estradiol-17 beta and dihydrotestosterone, however, was as effective as testosterone alone. Thus, androgens and estrogens may exert synergistic effects on levator ani muscle.     

Sex steroids deficiency impairs glucose transporter 4 expression and its translocation through defective Akt phosphorylation in target tissues of adult male rat

There is a substantial body of evidence suggesting that altered level of sex steroids in male is associated with insulin resistance and type 2 diabetes mellitus. However, the mechanism of this effect is not apparent. Our recent study indicated that testosterone deprivation decreases insulin receptor expression and glucose oxidation in insulin target tissues. The present study was designed to assess the impact of deficiency of testosterone and estradiol on Akt phosphorylation, glucose transporter expression, and glucose uptake in skeletal muscle, adipose tissue, and liver of adult male rat. Adult male albino rats of Wistar strain were orchidectomized and supplemented with testosterone (100 μg/100 g body weight per day), estradiol (5 μg/100 g body weight per day), and their combination (100 μg testosterone plus 5 μg estradiol per 100 g body weight per day) for 15 days from the 11th day postorchidectomy. On the day after the last treatment, animals were perfused; and blood was collected for the assay of plasma glucose, serum insulin, testosterone, and estradiol. Gastrocnemius muscle, adipose tissue, and liver were dissected out and used for the assay of various parameters such as Akt phosphorylation, glucose transporter (GLUT) 2 and 4 expression, glucose uptake, and glycogenic and glycogenolytic enzymes activity. Castration elevated the blood glucose level, which was accompanied by inhibitory effect on serum insulin, Akt phosphorylation, GLUT4 expression and its plasma membrane population, glucose uptake, glycogen and glycogen synthase activity, and stimulatory effect on GLUT2 expression and glycogen phosphorylase activity in tissues studied. After testosterone and its combination with estradiol supplementation to castrated rats, a normal pattern of all these parameters was restored. Estradiol administration to castrated rats increased the Akt phosphorylation without altering other parameters studied. It is concluded from the present study that sex steroids deficiency–induced defective glucose uptake in skeletal muscle and adipose tissue is mediated through defective Akt phosphorylation and GLUT4 expression in plasma membrane.     

The in-vitro transformation of [3H]dehydroepiandrosterone into its principal metabolites in the adrenal cortex of adult castrated male rats and following steroid treatment

The adrenal gland of castrated adult male rats metabolized [3H]dehydroepiandrosterone in vitro to delta 4-androsten-3,17-dione (4AD), testosterone, dihydrotestosterone (DHT) and 5 alpha-androstane-3,17-dione (5 alpha AD). Despite the low testosterone values, DHT and 5 alpha AD were higher 30 and especially 60 days after castration, with raised 4AD:testosterone and decreased testosterone:DHT ratios. The 5 alpha-reductase activity thus appears to increase with time after castration. Fourteen days after castration, 4AD was the only metabolite that was raised compared with intact animals, and testosterone was comparable in sham-operated and castrated rats. The administration of testosterone propionate to castrated rats restored testosterone values to those of intact rat adrenals, whereas 4AD values were greater. The administration of dihydrotestosterone propionate also yielded higher levels of 4AD, in the presence of a lower testosterone value. After administration of oestradiol benzoate, 4AD values were lower especially compared with the other hormone-treated groups, and there was an unexpectedly high testosterone value. These data indicate that the adrenal gland contributes to the production of androgens, as previously noted by Andò, Canonaco, Beraldi et al. (1988) who showed increased plasma 4AD and testosterone levels in adult male rats 30 days after castration. Furthermore, adrenal androgen production in castrated animals is differentially regulated by sex steroids.     

Region-specific expression of androgen and growth factor pathway genes in the rat epididymis and the effects of dual 5alpha-reductase inhibition

Dihydrotestosterone (DHT) is the primary androgen acting in the epididymis, the site of sperm maturation. Previously, we showed that the treatment of male rats with PNU157706, an inhibitor that acts on both isoforms of 5alpha-reductase to prevent DHT formation, has effects on the expression of genes implicated in processes that create the optimal luminal microenvironment required for sperm maturation, and on sperm maturation itself. However, signaling pathways involved in regulating or mediating DHT actions in the epididymis remain largely unknown. The goals of this study were to determine the expression profiles of potential signaling systems in the epididymis and assess their DHT-dependence using two different dual 5alpha-reductase inhibitors. Rats were untreated or gavaged with vehicle, 10 mg/kg per day PNU157706 or 32 mg/kg per day FK143 for 28 days and epididymal gene expression was analyzed. Gene array analysis revealed analogous effects of FK143 on overall epididymal gene expression when compared with previous PNU157706 studies. Quantitative RT-PCR analysis of the expression of the 5alpha-reductase isozymes, androgen receptor, and members of the IGF, FGF, TGF, and VEGF families revealed novel region-specific expression profiles in the epididymis that were differentially affected by 5alpha-reductase inhibition; the two inhibitors had parallel effects. Specifically, in proximal regions, 5alpha-reductase 1, androgen receptor, and TGF-beta1 expression increased after treatment, while in distal regions expression of IGF-I, IGFBP-5, IGFBP-6, and FGF-10 decreased. These results provide insight into epididymal signaling mechanisms and indicate potential candidates acting either upstream or downstream of DHT to regulate and/or mediate its actions in the epididymis.     

Androgen and estrogen effects on protein synthesis by the adult rabbit epididymis

The effects of castration and hormone replacement on [35S]methionine incorporation into newly synthesized proteins by the adult rabbit epididymis were studied in vitro. The proteins were analyzed using two-dimensional polyacrylamide gel electrophoresis. Short term (4-day) castration resulted in a few changes in the pattern of radiolabeled proteins observed in the caput, but no effect was seen in the corpus or cauda. The changes in the caput could be reversed if the samples were incubated with testosterone. The epididymis of short term castrates failed to respond to exogenous estradiol. Long term castration (4-6 weeks) resulted in changes in protein synthesis among all three epididymal segments. Short term (4-h) incubation with testosterone restored the pattern of proteins secreted by the caput and cauda to that in intact rabbits. Short term incubation with estradiol did not restore the pattern of radiolabeled secreted proteins, but it did slightly intensify a 28K protein (pI 5.2) that was present in the caput and cauda of castrated animals. No clear-cut effect of the hormones on proteins secreted by the corpus was observed. Short term incubation with testosterone or estradiol restored the patterns of tissue proteins synthesized by the caput and corpus of castrated rabbits to that in intact animals. In the cauda, estradiol also enhanced the presence of a small group of high mol wt proteins present in the control castrate sample, while testosterone inhibited these proteins. This group of proteins was absent in cauda tissue samples from intact rabbits.     

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 receptor (CR) in rat epididymal 105,000 $\text{g}$ 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 01 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 at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °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 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g 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.