Effects of testosterone and estrogen on hepatic levels of cytochromes P450 2C7 and P450 2C11 in the rat.

The effects of exogenous hormone treatment on the expression of cytochromes P450 2C7 and P450 2C11 were studied in neonatally gonadectomized and sham-operated male and female rats. Hepatic levels of cytochrome P450 2C7 were found to be two- to threefold higher in intact adult female versus male rats. Neonatal gonadectomy resulted in a reversal of the relative cytochrome P450 2C7 levels in male and female animals at maturity. Expression of this isozyme was restored in ovariectomized females by estradiol treatment. Furthermore, neonatal and/or pubertal administration of estradiol to intact male rats induced cytochrome P450 2C7 to adult female levels. On the other hand, administration of testosterone at all times examined had no effect in intact female rats, but decreased cytochrome P450 2C7 to normal levels in neonatally castrated males treated during adulthood. Neonatal testosterone treatment also increased hepatic cytochrome P450 2C7 content in both ovariectomized females and intact males. These results indicate that estrogen is required for full expression of cytochrome P450 2C7 while the effect of testosterone is ambiguous. In comparison, neonatal gonadectomy of male rats abolished the adult expression of cytochrome P450 2C11. Normal levels were restored only by treatment with testosterone during adulthood. Neonatal testosterone treatment did not induce cytochrome P450 2C11 levels in gonadectomized rats of either sex. In contrast, neonatal estrogen treatment suppressed cytochrome P450 2C11 expression in intact adult male rats to the same extent as neonatal castration. These results indicate that androgen exposure during the adult, and not the neonatal, phase is essential for full expression of cytochrome P450 2C11.     

Regulation of urine marking in male and female mice: effects of sex steroids

Effects of sex steroids on urine-marking activity were studied in male, female, and neonatally androgenized female mice. Urine marking was estimated by suspending ceramic tubes that were connected in a horizontal row with a steel rod into the home cage of an isolated mouse. Intact males showed high marking activity, which was diminished after castration. Both testosterone propionate (TP) and estradiol benzoate (EB) were effective in restoring the marking activity of castrated males, while 5-alpha-dihydrotesterone (DHT) did not have any stimulative effects. Intact normal females showed quite low marking activity and ovariectomy further depressed it. TP and DHT enhanced the marking of ovariectomized females, but EB restored the activity only to the preovariectomy level. In intact females which were neonatally androgenized, the marking activity was much higher than that of normal females. The pattern of the change induced by gonadectomy and hormone treatment in these females resembled that in males. Thus, ovariectomy reduced the activity and both TP and EB restored the level. These results indicate that the sexual dimorphism in the urine marking in mice is primarily determined by hormonal environment during early postnatal age. Hormonal control of scent marking is discussed in relation to the studies in other rodents.     

Stem cell activation in adults can reverse detrimental changes in body composition to reduce fat and increase lean mass in both sexes

Detrimental changes in body composition are often associated with declining levels of testosterone. Here, we evaluated the notion that multipotent mesenchymal stem cells, that give rise to both fat and muscle tissue, can play a significant role to alter existing body composition in the adult. Transgenic mice with targeted androgen receptor (AR) overexpression in stem cells were employed. Wild-type littermate and AR-transgenic male and female mice were gonadectomized and left untreated for 2 months. After the hypogonadal period, mice were then treated with 5α-dihydrotestosterone (DHT) for 6 weeks. After orchidectomy (ORX), wild-type males have reduced lean mass and increased fat mass compared to shams. DHT treatment was beneficial to partially restore body composition. In wild-type females, ovariectomy (OVX) produced a similar change but there was no improvement with DHT. In targeted AR transgenic mice, DHT treatment increased lean and reduced fat mass to sham levels. In contrast to wild-type females, DHT treatment in female transgenic mice significantly ameliorated the increased fat and decreased lean mass changes that result after OVX. Our results show that DHT administration reduces fat mass and increases lean mass in wild-type males but not females, indicating that wild-type females are not as sensitive to androgen treatment. Because both male and female transgenic mice are more responsive than wild-type, results suggest that body composition remains linked to stem cell fate in the adult and that targeted androgen signaling in stem cells can play a significant role to reverse detrimental changes in body composition in both sexes.     

Androgens are not major down-regulators of androgen receptor levels during growth of the immature rat penis

This study was undertaken to investigate the prevalent hypothesis that androgens are responsible for the organ-specific down-regulation of penile androgen receptors (ARs) and decline of penile growth in the rat during sexual maturation. Sexually immature male rats (21 days old) were castrated and treated for 3 days (“short-term”), with high doses of: (a) testosterone and the -reductase inhibitor finasteride (T/F); (b) dihydrotestosterone (DHT); or (c) finasteride alone (F). Intact and castrate controls received vehicle only. PolyA + RNA was analysed by Northern blot hybridization and ARs were estimated in the penis and ventral prostates by (3-H)R-1881 binding in the cytosol. Short-term castration, with or without F, increased penile AR mRNA, whereas high doses of T/F and DHT reduced it considerably. Although penile cytosol AR concentration in the control castrates, with or without F, paralleled the AR mRNA rise, treatment with androgens left cytosol AR content per organ and AR concentration above those of the intact rat penis despite the drop in AR mRNA. A “long-term” treatment (10 days) on 19-day-old rats with either medium or high doses of T/F and DHT also failed to down-regulate penile cytosol ARs below the intact controls. Western blot analysis of penile cytosol AR levels confirmed these results. Block of pituitary FSH and LH release by a GnRH antagonist in castrates receiving T/F or DHT at high doses did not modify the response. In the case of intact rats, high doses of T/F or DHT actually increased penile cytosol AR content. No difference was observed between T/F and DHT effects. In contrast to what occurs during sexual maturation, the prostate ARs and growth rate responded to all treatments in a similar way to what was observed in the penis. Our results suggest that increases in serum T or DHT are not major factors in the physiological down-regulation of ARs and androgen-dependent growth in the rat corpora cavernosa.     

Ontogeny of an 8S androgen binding protein in rat liver cytosol

Studies were performed to elucidate the ontogeny of a single class of androgen binding protein in male rat liver cytosol which exhibits characteristics of a ligand specific, high affinity (Kd = 2.3 nM), 8S-receptor capable of nuclear translocation. Detectable levels of receptor first appear at 45 days of age in the male and reach maximum concentration at 65 days. Barely detectable levels are seen in females throughout the duration of study (80 days). Gonadectomy in both sexes (65 days) and androgen treatment of oophorectomized females do not alter the normal development of sexual differentiation of the high affinity androgen receptor. After neonatal castration (2 days) and DES replacement however, receptor sites do not undergo differentiation and adult males exhibit female levels. Conversely, neonatal androgen replacement in 2-day castrates partially restores the level of binding sites to control males values (TP, 71%; DHT, 51%). Neonatal castration without replacement retards but does not fully eliminate sexual differentiation of levels of receptor sites in adult males. Likewise, neonatal androgen treatment in females results in a partial masculinization of binding sites. Following hypophysectomy, levels of receptor sites in females are similar to intact or hypophysectomized males; sexual differences in the adult are abolished. These studies suggest that sexual differentiation of specific liver cytosol androgen binding sites in the adult may be partially programmed at birth by testicular androgen and furthermore, adult sexual dimorphism is maintained through an inhibitory influence of the pituitary in the female.     

MicroRNAs are mediators of androgen action in prostate and muscle

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3' UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues.     

Regulation of sexual odor preference by sex steroids in the posterodorsal medial amygdala in female rats

Our previous study in male rats demonstrated that bilateral administration of flutamide, an androgen receptor (AR) antagonist, into the posterodorsal medial amygdala (MePD) increased the time sniffing male odors to as high as that sniffing estrous odors, eliminating the preference for estrous odors over male odors. This made us speculate that under blockade of AR in the MePD, testosterone-derived estrogen acting on the same brain region arouses interest in male odors which is otherwise suppressed by concomitant action of androgen. In cyclic female rats, endogenous androgen has been thought to be involved in inhibitory regulation of estrogen-activated sexual behavior. Thus, in the present study, we investigated the possibility that in female rats the arousal of interest in male odors is also normally regulated by both estrogen and androgen acting on the MePD, as predicted by our previous study in male rats. Implantation of either the estrogen receptor blocker tamoxifen (TX) or a non-aromatizable androgen 5α-dihydrotestosterone (DHT) into the MePD of ovariectomized, estrogen-primed female rats eliminated preference for male odors over estrous odors by significantly decreasing the time sniffing male odors to as low as that sniffing estrous odors. The subsequent odor discrimination tests confirmed that the DHT and TX administration did not impair the ability to discriminate between male and estrous odors. These results suggest that in estrous female rats estrogen action in the MePD plays critical roles in the expression of the preference for male odors while androgen action in the same brain region interferes with the estrogen action.     

Androgen Excess Produces Systemic Oxidative Stress and Predisposes to β-Cell Failure in Female Mice

In women, excess production of the male hormone, testosterone (T), is accompanied by insulin resistance. However, hyperandrogenemia is also associated with β-cell dysfunction and type 2 diabetes raising the possibility that androgen receptor (AR) activation predisposes to β-cell failure. Here, we tested the hypothesis that excess AR activation produces systemic oxidative stress thereby contributing to β-cell failure. We used normal female mice (CF) and mice with androgen resistance by testicular feminization (Tfm). These mice were exposed to androgen excess and a β-cell stress induced by streptozotocin (STZ). We find that following exposure to T, or the selective AR-agonist dehydrotestosterone (DHT), CF mice challenged with STZ, which are normally protected, are prone to β-cell failure and insulin-deficient diabetes. Conversely, T-induced predisposition to β-cell failure is abolished in Tfm mice. We do not observe any proapoptotic effect of DHT alone or in the presence of H₂O₂ in cultured mouse and human islets. However, we observe that exposure of CF mice to T or DHT provokes systemic oxidative stress, which is eliminated in Tfm mice. This work has significance for hyperandrogenic women; excess activation of AR by testosterone may provoke systemic oxidative stress. In the presence of a prior β-cell stress, this may predispose to β-cell failure.     

Differential maintenance of penile responses and copulatory behavior by gonadal hormones in castrated male rats

Sexually experienced male rats were castrated and immediately received implants of Silastic tubing containing either testosterone (T), dihydrotestosterone (DHT), estradiol (E), or nothing (blank). The ability of these hormone treatments to maintain precastration levels of copulatory behavior and ex copula penile responses was assessed for 40 days after castration. Throughout the study T- and E-treated males, but not males with DHT or blank implants, maintained normal copulatory behavior. In contrast males treated with T and DHT, but not E or blanks, maintained penile responses ex copula. In blank-treated males, penile-response latencies increased more rapidly than did intromission latencies. These results, together with those of previous studies, appear to rule out a role for estradiol and reinforce the role of androgens in the activation of rats' penile-response potential ex copula. Similarly, the results support the conclusion that in castrated male rats estradiol treatment is sufficient for the activation of masculine copulatory behavior, and that the penile actions necessary for intromission are not dependent on androgen. Thus, the evocability of penile actions and their relative androgen dependence are context sensitive.     

Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats

The fine modulation of gonadotropin gene expression and secretion is well recognized to be regulated by sex steroids through their direct action both at the anterior pituitary level and on the pulsatile pattern of GnRH secretion at the hypothalamic level. Since the influence of sex steroids on hypothalamic GnRH mRNA levels remains to be elucidated, quantitative in situ hybridization was used to study the effect of sex steroids on cellular levels of pro-GnRH mRNA in adult rats of both sexes. The effects of 14-day gonadectomy as well as administration of 17 beta-estradiol (E2, 0.25 micrograms) or dihydrotestosterone (DHT, 100 micrograms) twice a day during 14 days to gonadectomized animals were evaluated. In addition, the effect of progesterone (P, 2 mg, twice daily) alone or in the presence of E2 was also studied in ovariectomized animals. Hybridization was performed using a 35S-labeled cDNA probe encoding rat pro-GnRH and the corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In male rats, castration induced a highly significant 65% increase (compared to intact rats) in the mean number of grains per neuron. Administration of E2 or DHT to castrated animals completely prevented the post castration rise in pro-GnRH mRNA levels. In female animals, the effect of ovariectomy was less striking than in the male, a 25% increase (P less than 0.001) being observed. Treatment with E2 or DHT also completely prevented the increase in pro-GnRH mRNA levels induced by ovariectomy. Moreover, treatment with P in ovariectomized animals markedly potentiated the inhibitory effect of E2 on pro-GnRH mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of castration and androgen treatment on androgen-receptor levels in rat skeletal muscles.

The effects of castration and dihydrotestosterone (DHT) treatment on levels of skeletal muscle androgen receptor (AR) were examined in three groups of adult male rats: 1) intact normal rats, 2) rats castrated at 16 wk of age, and 3) rats castrated at 16 wk of age and given DHT for 1 wk starting at week 17. All animals were killed at 18 wk of age. Castration caused a decrease (P < 0.05) in the weights of the levator ani and bulbocavernosus muscles. The administration of DHT to the castrated rats increased (P < 0.05) the weights of the levator ani and bulbocavernosus muscles. Castration caused a significant downregulation of AR levels in the bulbocavernosus (P < 0.05) but had no significant effect on AR levels in the levator ani muscle. DHT administration to the castrated group upregulated AR levels in the bulbocavernosus and levator ani muscles. The plantaris muscle did not significantly (P > 0.05) change for any of the treatments. These findings suggest that the effects of castration and androgen replacement differentially affect skeletal muscle mass and AR levels.     

Synthesis and phosphorylation of androgen receptor of the mouse brain cortex and their regulation by sex steroids during aging

To examine the synthesis and phosphorylation of androgen receptor (AR) and their regulation by sex steroids, adult (24 weeks) and old (65 weeks) male and female mice were gonadectomized and administered with testosterone and estradiol. AR amount, synthesis and phosphorylation were measured in the brain cortex by immunoblotting and immunoprecipitation using antibody raised against rat AR transactivation domain (TAD) which was expressed in E. coli as a fusion protein. We found that the amount of AR was high in adult and declined in old mice of both sexes. Administration of testosterone and estradiol significantly down-regulated the level of AR in old male and adult female. Similarly, the rate of AR synthesis also declined with age. Exogenous treatment of gonadectomized mice with testosterone and estradiol reduced the extent of synthesis significantly in all groups except in old female. No sex-dependent variation was noticed either in the level or synthesis of AR. In contrast, the extent of phosphorylation was higher in old mice of both sexes as compared to their adult counterparts. Testosterone and estradiol supplementation resulted in remarkable increase in AR phosphorylation in all groups. Thus it is evident from our findings that the amount and synthesis of AR decrease but phosphorylation of AR increases in the brain cortex with advancing age of mice and they are regulated by testosterone and estradiol in age and sex-specific manner.     

Induction of male-typical aggression by androgens but not by estrogens in adult female mice

Ovariectomized adult CF-1 female mice were implanted with silastic capsules containing either testosterone (T), dihydrotestosterone (DHT), methyltrienolone (R1881), estradiol (E2), diethylstilbestrol (DES), or oil vehicle and were tested for aggressive behavior. The androgenic treatments (T, DHT, R1881) were highly effective in promoting male-like aggression while the estrogens (DES, E2) were completely ineffective. Subsequent receptor-binding studies confirmed assumptions about the specificity of DES, DHT, and R1881 binding to estrogen and androgen receptors in mouse hypothalamus.     

Influence of neonatal diethylstilbestrol treatment on androgen and estrogen receptor levels in the mouse anterior prostate, ventral prostate and seminal vesicle

Perinatal exposure to the synthetic estrogen, diethylstilbestrol (DES), affects the structure of both male and female reproductive systems. Changes may also occur in the levels of steroid hormone receptors. Cytosolic and nuclear androgen and estrogen receptor levels (expressed per mg DNA) from the sex accessory glands of male BALB/c mice exposed neonatally to DES were analyzed by exchange assays. Neonatal DES exposure caused significant decreases in: (1) cytosolic androgen and cytosolic and nuclear estrogen receptor levels in the anterior prostate and (2) cytosolic estrogen receptor levels in the ventral prostate. A significant increase was seen in the cytosolic estrogen receptor levels in the seminal vesicle. Significant decreases in cytosolic protein levels occurred in all DES-exposed glands.     

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.     

Sexual orientation, proceptivity, and receptivity in the male rat as a function of neonatal hormonal manipulation

The influence of neonatal androgen on the potential to exhibit feminine sexual behavior was investigated. Male rats castrated on Day 0 but not those castrated on Day 4 or later showed hop/darting, ear wiggling, and lordotic behavior in response to treatment with estrogen and progesterone in adulthood at a frequency equal to that of females. Neonatal treatment with testosterone propionate (1 mg/rat for 4 days) abolished the capacity to show these behaviors. In subsequent experiments, involving castration of male rats at 0 or 4 hr after cesarean delivery, the effect of the postnatal surge of testicular secretions on the expression of female sexual behavior was investigated. No differences were seen in the frequency of hop/darting, ear wiggling, and receptivity between males castrated immediately or 4 hr after delivery. In a preference test where the experimental male could choose between an estrous female and a sexually active male, the neonatally castrated males preferred the company of a male when treated with estrogen and progesterone. The implantation of testosterone resulted in a preference for an estrous female. It was concluded that testicular secretions in the newborn male influence adult sexual orientation and suppress the ability to show proceptive and receptive behaviors.     

Decidualization in rats given 5alpha-dihydrotestosterone or its propionate neonatally.

In Experiment 1, female rats were given a single subcutaneous injection of 1.25 mg 5alpha-dihydrotestosterone (DHT) or its propionate (DHTP) on day 5 of postnatal life. All of them showed regular estrous cycles as adults like untreated control animals. At about 60 days of age, the rats were ovariectomized and given 7 daily injections of 2 mg progesterone (P) plus 0.2 mug estradiol-17beta (ED). Uterine trauma applied on the 4th day of P-ED injections resulted in well developed deciduomata in all animals by the day after the last injection. This made a sharp contrast to the failure of female rats receiving testosterone propionate (TP) neonatally to give a positive response under similar experimental conditions (Takewaki and Ohta, 1974). The mean weight of traumatized horns was significantly larger in DHTP-treated rats (but not in DHT-treated rats) than in controls. In Experiment 2, rats were ovariectomized on day 4 and given a dose of 1.25 mg DHT or DHTP on day 5. Controls were ovariectomized on day 4 but not given any steroid on the next day. A series of 7 daily injections of 2 mg P plus 0.2 mug ED was started at about 60 days of age, after the animals had received 3 daily injections of 0.2 mug ED or 30 daily injections of 0.1 mug ED. Incidence of deciduomata following uterine traumatization was markedly lowered only in animals treated with DHTP neonatally and given 0.1 mug ED for 30 days as adults, no significant differences being found in both incidence and size of deciduomata among the other groups. It was suggested that the effects of neonatal steroid administration on uterine responsiveness in adulthood are specific to the steroid. The previous conclusion that persistent estrus in androgen-sterilized rats plays a part in the reduction of uterine responsiveness was confirmed. An exposure of rats to estrogen for a prolonged postpuberal period was without effect, unless the animals had received enough androgen neonatally.     

Influence of androgen on the development of sexual behavior in rats : I. Time of administration and masculine copulatory responses, penile reflexes, and androgen receptors in females

The objective of the present study was to investigate the effect of the time of administration of androgen, during the neonatal period, on the development of masculine copulatory behavior in female rats. In addition, the influence of androgen, administered neonatally, on the development of penile reflexes and cytoplasmic androgen receptor levels in the hypothalamic-preoptic area (HPOA) was examined. Female rats were injected with 0.5 mg testosterone propionate (TP) at either 1, 8, or 24 hr after birth and again 24 hr after the first injection. Fifty percent of the females treated with TP at 1 and 8 hr after birth displayed the ejaculatory response when tested in adulthood. In contrast, 93 and 87.5% of oil-treated males and females, respectively, which were androgenized at 24 hr after birth exhibited this response. The results indicate that a considerable amount of masculinization occurs postnatally in the rat. However, none of the androgenized females displayed any penile reflexes even when tested following the display of an ejaculatory response. HPOA androgen receptor levels were somewhat higher in the oil-treated females than in males but were not correlated with the ability to exhibit ejaculation patterns.     

Neonatal hormonal influences on the development of proceptive and receptive feminine sexual behavior in rats

The objective of this study was to examine the influence of androgen and of the inhibiting of aromatization of androgen to estrogen during the early neonatal period on the development of receptive (lordosis and acceptance of stimulus male mounting attempts) and proceptive (affiliation with and solicitation of stimulus males) feminine sexual behavior. Within 8 hr of birth, male rats were castrated or received subcutaneous implants of the aromatase inhibitor androst-1,4,6-triene-3, 17-dione (ATD) while females received injections of testosterone propionate (TP). At 90 days of age all treated animals and controls were tested for receptive and proceptive feminine sexual behavior. It was found that androgen present neonatally blocked proceptive as well as receptive behavior patterns in adult rats. The proceptive and receptive feminine sexual behavior patterns displayed by adult males deprived of the effects of androgen neonatally either by castration or by treatment with ATD were comparable to those of normal females.