Dihydrotestosterone Propionate Research Articles

Efecto de la orquidectomización y la administración exógena de la testosterona en la cuantificación de las plaquetas en ratas wistar macho

Introduction: Anabolic-androgenic steroids (AAS) modify the physiological functioning of the cardiovascular system and have possible effects on the origin of cardiovascular thrombosis. Objective: To determine the impact of the testosterone metabolite on platelet quantification in ORX rats with or without DHT replacement. Material and methods: 24 male 45-day-old Wistar rats underwent orchidectomy with a simple scrotal incision. At 2.5 months of age, the 24 rats were divided into 4 study groups. Half of the 12 male ORX rats received hormone replacement with dihydrotestosterone propionate (DHT) at a 2 mg/kg dose via subcutaneous injection for 7 days and the other half received a physiological solution (0.9% NaCl). The same occurred in the 12 non-ORX males (SHAM). After 7 days of the administration, blood was collected by orbital puncture, and platelet quantification was performed. Results: A significant difference (ANOVA, p < 0.005) was found between the 4 groups. When performing the Dunn Method, a significant difference (p < 0.05) was found in the endogenous administration of DHT between Sham rats and rats ORX + 0.9% NaCl but not with ORX + DHT. Conclusions: DHT induces an increase in platelet quantification inrats Sham and not in ORX rats with DHT, which may affect the increase in platelet quantification.

Urine from domesticated male goats (Capra hircus) provides attractive olfactory cues to estrous females

During the breeding season, the male goat urinates onto his face, beard, and front legs in a behavior known as self-enurination (SE). Whereas it has been suggested that the behavior may be used by the buck to present chemical signals in the urine to attract estrous does for mating, this hypothesis lacks empirical evidence. Two experiments were conducted to determine if estrous female goats (does) are attracted to male goat (buck) urine and if there is a difference in attractivity of buck urine emitted during SE versus ordinarily emitted urine. In addition, the ability of androgens to activate the attractive chemical signals in buck urine was investigated in experiment 2. In the first experiment, a 5-minute preference test allowed estrous does (n = 16) to choose between ovariectomized (OVX) does that had either buck SE urine or wether (prepubertally castrated male goat) urine applied to their coats. The does preferred buck SE urine-treated OVX does over wether urine-treated OVX does (P < 0.05). In the second experiment, recycling fountains were used to present urine in a continuous stream. The urine choices were as follows: buck SE vs. wether; dihydrotestosterone propionate (DHTP)-treated wether vs. estradiol benzoate (EB)-treated wether; and buck SE vs. buck. Estrous does (n = 10) were subjected to a 5-minute preference test. The does chose buck SE urine over wether urine, DHTP-treated wether urine over EB-treated wether urine, and buck urine over buck SE urine (P < 0.05). In general, estrous females preferred urine from bucks over urine from wethers.

Differential effects of androgens, estrogens and socio-sexual context on sexual behaviors in the castrated male goat

The behavioral and endocrine activation of sexual behaviors exhibited by male goats, especially self-enurination (SE), is poorly understood. In the first experiment, to assess the influence of socio-sexual context on SE in bucks, the effects of distance from does, the presence of estrous versus non-estrous does and the presence of another buck on SE and courtship frequencies of intact male goats (bucks; n = 12) were tested using a unique behavior test apparatus. For experiments 2 and 3, to test the relative contributions of sex steroid hormones and socio-sexual context on SE, castrated male goats (wethers; n = 20) were randomly divided into five groups and injected for seven weeks with one of the following: 25 mg testosterone propionate (T), 25 mg dihydrotestosterone propionate (DHT), 100 μg estradiol benzoate (E), 100 μg E and 25 mg DHT (E + DHT), or oil (CON). The effects of these treatments on frequency of SE and courtship were assessed using the behavior test apparatus (social scenarios) adapted from the findings in experiment 1. In one scenario, a wether could observe (from 4.6 m) a buck and estrous female (doe) together in a wire mesh holding pen. In a different scenario, the wether could observe (from the same distance) a buck that could only court the estrous doe through a wire mesh barrier. Finally, to observe the effects of steroid treatment on mounting and ejaculation frequencies, in addition to SE and courtship, each wether was placed in a pen with an estrous doe for 10 min. After a five-week, treatment-washout period, wethers were randomly assigned to different treatment groups and retested. In experiment 1, bucks that were distanced from females displayed more SEs than those with fence-line contact, while those with fence-line contact displayed more bouts of courtship (P < 0.05). In experiments 2 and 3, courtship frequencies displayed in all three scenarios were greater than CON only for groups exposed to estrogen directly or via aromatization (T, E + DHT, E; P < 0.05). Frequencies of SE exhibited during behavior tests in which the wether was watching were greater than CON only for androgen-treated groups (T, E + DHT, DHT; P < 0.05). In contrast, when the wether was free to interact with the female, only the DHT group displayed SE at a higher frequency than CON (P < 0.05). Treatment had no effect on mount frequencies in this test scenario, however ejaculation frequencies were highest for T and E + DHT (P < 0.05). These studies suggest that the courtship behaviors of the male goat are estrogen-dependent. However, SE appears to be activated by androgens. It was also demonstrated that social context contributes as much to behavior expression as steroid treatment, as in social scenario 2 some sexual behaviors were displayed in similar frequencies across groups, despite differing sex steroid treatments.

Prenatal influence of an androgen agonist and antagonist on the differentiation of the ovine sexually dimorphic nucleus in male and female lamb fetuses.

The ovine sexually dimorphic nucleus (oSDN) is 2 times larger in rams than in ewes. Sexual differentiation of the oSDN is produced by testosterone exposure during the critical period occurring between gestational day (GD)60 and GD90 (term, 147 d). We tested the hypothesis that testosterone acts through the androgen receptor to control development of the male-typical oSDN. In experiment 1, pregnant ewes received injections of vehicle, androgen receptor antagonist flutamide, or nonaromatizable androgen dihydrotestosterone (DHT) propionate during the critical period. Fetuses were delivered at GD135. Both antagonist and agonist treatments significantly reduced mean oSDN volume in males but had no effects in females. Experiment 2, we analyzed the effect of treatments on the fetal hypothalamic-pituitary-gonadal axis to determine whether compensatory changes in hormone secretion occurred that could explain the effect of DHT. Pregnant ewes were injected with vehicle, flutamide, or DHT propionate from GD60 to GD84, and fetuses were delivered on GD85. Flutamide significantly increased LH and testosterone in males, whereas DHT significantly decreased both hormones. In females, LH was unaffected by flutamide but significantly reduced by DHT exposure. DHT significantly decreased pituitary gonadotropin and hypothalamic kisspeptin mRNA expression in males and females. These results suggest that androgen receptor mediates the effect of testosterone on oSDN masculinization, because this process was blocked by the androgen receptor antagonist flutamide in eugonadal males. In contrast, the reduction of oSDN volume observed after DHT exposure appears to be mediated by a negative feedback mechanism exerted on the hypothalamus to reduce LH and testosterone secretion. The reduced androgen exposure most likely accounted for the decreased oSDN volume. We conclude that, during the critical period, the male reproductive axis in long gestation species, such as sheep, is sufficiently developed to react to perturbations in serum androgens and mitigate disruptions in brain masculinization.

Developmental Programming: Impact of Prenatal Testosterone Excess on Ovarian Cell Proliferation and Apoptotic Factors in Sheep1

Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.

Androgen inhibition of sexual receptivity is modulated by estrogen

Sexual receptivity induced in ovariectomized rats by the long-term administration of estradiol benzoate (EB) can be inhibited by concurrent administration of androgens. Experiment 1 examined the role of time course and dose of androgens in the inhibition of estrogen-induced sexual receptivity. Ovariectomized rats were treated with EB (2.0 microg per rat per day) for 6 days and tested for sexual receptivity (Test Day I). EB treatment continued for 15 days concomitant with daily administration of one of three doses of dihydrotestosterone propionate (DHTP; 7.5, 0.75, 0.075 mg/kg) or 3α-androstanediol (3α-Adiol; 3.75, 1.0, 0.375 mg/kg). Four tests for sexual receptivity were conducted on days 3, 6, 14, and 15 of the androgen/vehicle treatment period (Test Days II-V). On Day 15 (Test Day V), the rats received progesterone (1.0 mg per rat) 4 h before testing. Using the same experimental design, Experiment 2 examined the effect of increasing the dose of estrogen on the androgenic inhibition of sexual receptivity. Ovariectomized rats were treated with one of two doses of EB (2.0 or 10.0 microg per rat per day) concomitant with daily administration of DHTP (7.5 mg/kg) or 3α-Adiol (3.75 mg/kg). In Experiment 1, the highest doses of both DHTP and 3α-Adiol significantly inhibited estrogen-induced sexual receptivity. Data from Experiment 2 indicate that the inhibitory effects of DHTP but not 3α-Adiol can be moderated by an increased dose of EB.

Control of Cell Number in the Bed Nucleus of the Stria Terminalis of Mice: Role of Testosterone Metabolites and Estrogen Receptor Subtypes

The bed nucleus of the stria terminalis (BNST) exhibits several sex differences that may be related to male sexual behavior and gender identity. In mice and rats, sex differences in the principal nucleus of the BNST (BNSTp) are due to sexually dimorphic cell death during perinatal life. Although testosterone treatment of newborn female rats increases BNSTp cell number, the relevant hormone metabolite(s) are not known, and the effect of testosterone on the development of BNSTp cell number in mice has not been examined. To identify the sex hormone metabolites and receptors controlling cell number, volume, and cell size in the BNSTp of mice. In the first experiment, C57BL/6J male mice were injected on the day of birth with peanut oil; females were injected with testosterone propionate (TP), estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), or oil alone, and the BNSTp of all animals was examined in adulthood. In the second experiment, to compare effects of EB to the effects of estrogen receptor subtype specific agonists, newborn female mice were injected with EB, propyl-pyrazole-triol (PPT, a selective estrogen receptor alpha [ERalpha] agonist), or diarylpropionitrile (DPN, a selective estrogen receptor beta [ERbeta] agonist). Nuclear volume measurements and stereological cell counts in the BNSTp in adulthood. TP treatment of newborn females completely masculinized both BNSTp volume and cell number. EB masculinized neuron number, whereas DHTP had no effect on volume or cell number. In the second experiment, EB again fully masculinized neuron number in the BNSTp and in this study also masculinized BNSTp volume. PPT and DPN each significantly increased cell number, but neither completely mimicked the effects of EB. We conclude that estrogenic metabolites of testosterone control sexually dimorphic cell survival in the BNSTp and that activation of both ERalpha and ERbeta may be required for complete masculinization of this brain region.

Neonatal DHT but not E2 speeds induction of sexual receptivity in the musk shrew

Neural aromatization of testosterone (T) to estrogen during development is thought to be important for sexual differentiation of many altricial mammals. We evaluated the effects of neonatal injections of the non-aromatizable androgen dihydrotestosterone propionate (DHTP) and estradiol (E2) on the copulatory behavior of the female musk shrew, an altricial insectivore. Following adult ovariectomy and replacement T, animals were paired with a stimulus female for two 60-minute copulatory behavior tests. The latency to induce sexual receptivity (in the form of tail-wagging by the female), mount latency and total number of mounts were recorded in experimental females and in a group of untreated control males. While neither hormone treatment significantly affected mounting behavior, DHTP-treated animals induced receptivity faster and with latencies not significantly different from intact males, suggesting that early non-aromatizable androgens can have masculinizing actions by either increasing sexual motivation or making the treated animal more attractive to the stimulus female. Reliance on androgenic rather than estrogenic metabolites for the differentiation of courtship behaviors conforms to the pattern seen more typically in primates than rodents.

The in vitro conversion of 3H androstenedione to testosterone and dihydrotestosterone in the adrenal gland of castrated male rat: influence of gonadal steroid administration.

In the present study we considered the effects of bilateral orchiectomy on the metabolism of androstenedione in the adrenal gland of the adult male rat. Adrenal glands of intact animals incubated in the presence of 3H androstenedione resulted in its conversion to testosterone and dihydrotestosterone (the two principal metabolites investigated). The value of the latter metabolite increased with the elapsing of time from castration despite the decrease of testosterone with respect to that observed in the sham operated rats of the same age. This effect is reversed in the presence of testosterone treatment with androstenedione and testosterone levels similar to those obtained in intact animals. Androstenedione adrenal metabolism is greatly lowered following the administration of dihydrotestosterone propionate, resulting in high substrate residue values with respect to untreated castrated rats. Contemporarily, testosterone metabolite value remains unchanged while the production of dihydrotestosterone is significantly lower. Finally a decrease of DHT was also encountered in the presence of estradiol benzoate. The above results clearly point to a progressive activation of the 5 alpha-reductase of the adrenal gland after castration utilizing androstenedione as substrate. The metabolic pathway leading to the production of dihydrotestosterone from androstenedione appears to be modulated differently following the administration of gonadal steroids.

Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor balance

Steroid hormones play an important role in reproduction and the receptors through which they signal change in a developmental time, follicle stage, and cell-specific manner. Disruption in steroid receptor expression affects follicle formation and differentiation. In this study, using prenatal testosterone (T) and dihydrotestosterone (DHT)-treated female sheep as model systems, we tested the hypothesis that prenatal androgen excess disrupts the developmental ontogeny of ovarian steroid receptor protein expression. Pregnant Suffolk ewes were injected twice weekly with T propionate or DHT propionate (a non-aromatizable androgen) in cottonseed oil from days 30 to 90 of gestation. Changes in ovarian estrogen receptors (ER; ESR1, ESR2), androgen receptor (AR) and progesterone receptor (PGR) proteins were determined at fetal (days 90 and 140), postpubertal (10 months), and adult (21 months; only prenatal T-treated sheep studied) ages by immunohistochemistry. Prenatal T and DHT treatment induced selective increase in AR but not ER or PGR expression in the stroma and granulosa cells of fetal days 90 and 140 ovaries. An increase in ESR1 and decrease in ESR2 immunostaining coupled with increased AR expression were evident in granulosa cells of antral follicles of 10- and 21-month-old prenatal T but not DHT-treated females (analyzed only at 10 months). These findings provide evidence that an early increase in ovarian AR is the first step in the altered ovarian developmental trajectory of prenatal T-treated females, and manifestations of postnatal ovarian dysfunction are likely facilitated via altered equilibrium of antral follicular granulosa cell ER/AR protein expression.

Androgen regulation of corticotropin-releasing hormone receptor 2 (CRHR2) mRNA expression and receptor binding in the rat brain

Stress-induced affective disorders, such as depression and anxiety, are more prevalent in females than in males. The reduced vulnerability to these disorders in males may be due to the presence of androgens, which are known to dampen the stress response and reduce anxiety-like behaviors. However, a neurobiological mechanism for this sex difference has yet to be elucidated. Corticotropin-releasing hormone receptor 2 (CRHR2) has been implicated in regulating anxiety-type behaviors and is expressed in stress-responsive brain regions that also contain androgen receptors (AR). We hypothesized that androgen may exert its effects through actions on CRHR2 and we therefore examined the regulation of CRHR2 mRNA and receptor binding in the male rat forebrain following androgen administration. Young adult male Sprague/Dawley rats were gonadectomized (GDX) and treated with the non-aromatizable androgen, dihydrotestosterone propionate (DHTP) using hormone filled Silastic capsules. Control animals received empty capsules. Using quantitative real-time RT-PCR, CRHR2 mRNA levels were determined in block-dissected brain regions. DHTP treatment significantly increased CRHR2 mRNA expression in the hippocampus, hypothalamus, and lateral septum ( p < 0.01) when compared to vehicle-treated controls. A similar trend was observed in amygdala ( p = 0.05). Furthermore, in vitro autoradiography revealed significantly higher CRHR2 binding in the lateral septum in androgen-treated males, with the highest difference observed in the ventral lateral region. Regulation of CRHR2 mRNA by AR was also examined using an in vitro approach. Hippocampal neurons, which contain high levels of AR, were harvested from E17–18 rat fetuses, and maintained in primary culture for 14 days. Neurons were then treated with dihydrotestosterone (DHT; 1 nM), DHT plus flutamide (an androgen receptor antagonist), or vehicle for 48 h. CRHR2 mRNA levels were measured using quantitative real-time RT-PCR. Consistent with in vivo studies, DHT significantly increased CRHR2 mRNA expression in hippocampal neurons ( p < .02) compared to vehicle-treated controls. Flutamide treatment prevented the effect of DHT on CRHR2 mRNA indicating that DHT's effect on CRHR2 expression is AR-mediated. Thus, the CRHR2 gene appears to be a target for regulation by AR and these data suggest a potential mechanism by which androgen may alter mood and anxiety-related behaviors.

Developmental Programming: Postnatal Estradiol Amplifies Reproductive Defects Induced by Fetal Exposure to Excess Testosterone and Dihydrotestosterone in Sheep.

Prenatal testosterone (T) excess causes defects in gonadotropin surge and follicular dynamics culminating in infertility. Prenatal dihydrotestosterone (DHT) treatment, while not affecting gonadotropin surge dynamics, leads to luteal defects. Considering that humans are exposed to estrogenic environmental steroids from variety of sources throughout their lifespan, we tested the hypothesis that constant exposure to estradiol-17β (E) postnatally would exaggerate the defects in periovulatory hormonal and follicular dynamics induced by prenatal T- and DHT excess. Pregnant sheep were treated with either T propionate or DHT propionate (100 mg im twice weekly) in cottonseed oil from 30 to 90 days of gestation. At nine weeks of age, a subset of control C (n=7), T (n=7), and DHT (n=6) females received a 10 mm Silastic implant containing E (produces circulating levels of <1 pg/mL E) to generate CE, TE, and DHTE groups. Six C, 7 T and 6 DHT females served as corresponding prenatal controls (no postnatal E). All animals were treated with 2 prostaglandin injections 11 days apart during their first breeding season to synchronize estrus. Blood samples were taken at 2h-intervals for 8 days to assess changes in periovulatory LH/FSH dynamics and daily for an additional 28 days to assess luteal progesterone dynamics during two cycles. Transrectal ultrasonography was performed daily to monitor ovarian follicular dynamics. Temporal changes in hormone dynamics and follicular size classes were analyzed by ANOVA, Wilcoxon test and linear mixed model. As expected, prenatal T but not DHT treatment severely dampened primary LH surges. Postnatal E treatment did not affect primary LH/FSH surge dynamics of C, T or DHT females, but obliterated secondary FSH surges in TE females. Postnatal E treatment increased (P<0.05) the incidence of subluteal cycles (≤2 ng/ml) in TE and tended to do so in DHTE females relative to their corresponding prenatal only counterparts (T: 0% vs TE: 80%, P=0.01; DHT: 16.7% vs DHTE: 50%). Postnatal E tended to increase incidence of short cycles (≤10 days) in the DHTE group (DHT: 16.7%, DHTE: 50%). Transrectal ultrasonography revealed fewer TE females developed corpora lutea relative to T (T: 85.7, TE: 14.3%, P=0.03). Postnatal E treatment tended to decrease the number of medium sized follicles (3 to ≤4mm) in all groups. Postnatal E treatment also reduced number of follicles >4 to ≤8 mm in the DHTE relative to DHT group (6.7±0.8 vs 3.7±0.9, P<0.05). All the T and 60% of the TE females had at least one luteinized follicle. Only few CE (16.6%), DHT (16.6%) and DHTE (25%) and none of C females had luteinized follicles. The luteinized follicle also tended to persist longer in the TE relative to T group (T: 31.5±3.5 vs TE: 22.1±2.4 days, P=0.07). These findings suggest that postnatal E treatment further deteriorates periovulatory hormonal/ovarian follicular dynamics and subsequent luteal outcome in prenatal steroid-treated females with the quality of prenatal steroid treatment determining the type and nature of disruption. Supported by NIH P01 HD44232.

Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic with males having more AVP-immunoreactive cells/fibers than females. This neuropeptide neuronal system is highly sensitive to steroids and has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory. We demonstrate here that gonadally intact male aromatase knockout (ArKO) mice, which cannot aromatize androgens into estrogens due to a targeted mutation in the aromatase gene, showed severe deficits in social recognition as well as a reduced AVP-immunoreactivity in several brain regions. To determine whether this reduction is due to a lack of organizational or activational effects of estrogens, we assessed social recognition abilities and AVP-immunoreactivity in male ArKO and wild-type (WT) mice when treated with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP) in adulthood. Adult treatment with EB and DHTP restored social recognition abilities in castrated ArKO males since they showed normal female-oriented ultrasonic vocalizations and were able to recognize an unfamiliar female using a habituation–dishabituation paradigm. Furthermore, adult treatment also restored AVP-immunoreactivity in the lateral septum of ArKO males to levels observed in intact WT males. These results suggest that social recognition in adulthood and stimulation of AVP expression in the adult mouse forebrain depend predominantly on the estrogenic metabolite of testosterone. Furthermore, our results are in line with the idea that the organization of the AVP system may depend on androgen or sex chromosomes rather than estrogens.

Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice

Little is known about the hormonal regulation of sexual behavior and about the pattern of expression in the brain of sex-steroid receptors in the BALB/c AnN strain of mice (Mus musculus). In this study, 8-week old male BALB/c AnN mice were castrated and the temporal course of decline of sexual behavior was studied, as well as the effects of daily treatment with either testosterone propionate (TP), estradiol benzoate (EB), or dihydrotestosterone propionate (PDHT). Castration resulted in rapid decline of sexual behavior, in both control or vehicle-treated mice. TP maintained full sexual behavior of castrated mice, while PDHT or EB did not have this effect. The expression of ER-alpha dropped nearly 50% after castration, and this pattern remained in TP or PDHT-treated mice, while EB increased the ER-alpha mRNA levels to almost the same values as in intact control mice. The same pattern was found when ER-beta mRNA levels were analyzed. The expression of the PR-A/B gene in the different brain regions in intact mice and after castration, or among the differently treated mice, showed significant differences between normal and castrated mice at all times in all brain regions studied, with the exception of the frontal cortex. Castration reduced the expression of AR by 10-fold, as compared to intact control mice, while TP or PDHT treatment returned its expression to the same levels as in intact control mice, in all brain areas studied. The changes are more prominent in POA-HIP than in HYP and CF. These results demonstrated a rapid decline of sexual behavior in this strain of mice after castration, and show that only TP was able to maintain male sexual behavior, with no correlation with the pattern of expression of sex hormone receptors in specific areas of the mouse brain.

Intraprostatic Lymphocyte Profiles in Aged Wistar Rats With Estradiol Induced Prostate Inflammation

We report LS phenotypes in aged Wistar rats (Charles Rivers, Wilmington, Massachusetts) with EPI. EPI was induced in 36 to 40-week-old male rats by castration with subcutaneous injections of 17beta-estradiol (0.25 mg/kg daily) plus dihydrotestosterone propionate (2.5 mg/kg daily) in sesame oil for 30 days. Controls were sham castrated, aged rats that were injected with sesame oil. Prostate, spleen and blood LSs in aged and young (10 to 12-week-old) rats were identified by flow cytometry in a cluster of differentiation system. All prostates in 6, 17beta-estradiol plus dihydrotestosterone propionate treated rats and in 3 of 7 controls (43%) showed inflammation foci. All studied LSs in Aged-SPIs and Aged-EPIs were similar. Blood LSs in Aged-SPIs, Aged-EPIs, Aged-NPIs and Youngs showed no differences. Levels of lymphocytes bearing the natural killer marker were decreased, and levels of total T and CD4(+) T cells were increased in prostates with age. Intraprostatic and splenic levels of CD4(+) natural killer T cells were down-regulated significantly in Aged-SPIs and Aged-EPIs compared to those in Aged-NPIs and Youngs. Levels of CD45RC(+)CD4(+)alphabetaTCR(+) T cells were decreased 2-fold in the spleen and up-regulated 2-fold in the prostate of Aged-SPIs and Aged-EPIs compared to those in Aged-NPIs and Youngs. Similar LS features in Aged-SPIs and Aged-EPIs may indicate that the EPI model is appropriate for studies of the immune aspects of prostate inflammation. Imbalance between suppressive CD4(+) natural killer T cells and autoreactive CD45RC(+)CD4(+)alphabetaTCR(+) T cells in Aged-SPIs and Aged-EPIs may suggest their role in prostate inflammation in this model.

Restoration of male sexual behavior by adult exogenous estrogens in male aromatase knockout mice

We previously found that male aromatase knockout (ArKO) mice that carry a targeted mutation in exons 1 and 2 of the CYP19 gene and as a result cannot aromatize androgen to estrogen show impaired sexual behavior in adulthood. To determine whether this impairment was due to a lack of activation of sexual behavior by estradiol, we studied here male coital behavior as well as olfactory investigation of sexually relevant odors in male ArKO mice following adult treatment with estradiol benzoate (EB) or dihydrotestosterone propionate (DHTP). Again, we found that gonadally intact ArKO males show pronounced behavioral deficits affecting their male coital behavior as well as their olfactory investigation of volatile body odors but not that of soiled bedding. Deficits in male coital behavior were largely corrected following adult treatment with EB and the androgen DHTP, suggesting that estradiol has prominent activational effects on this behavior. By contrast, adult treatment with EB to either castrated or gonadally intact ArKO males did not stimulate olfactory investigation of volatile body odors, suggesting that this impairment may result from a lack of proper organization of this behavior during ontogeny due to the chronic lack of estrogens. In conclusion, the present studies suggest that the behavioral deficits in sexual behavior in male ArKO mice result predominantly from a lack of activation of the behavior by estrogens. This is in contrast with earlier pharmacological studies performed on rats and ferrets that have suggested strong organizational effects of estradiol on male sexual behavior.

Androgen inhibits, while oestrogen enhances, restraint-induced activation of neuropeptide neurones in the paraventricular nucleus of the hypothalamus.

The hormonal response to stress is enhanced by oestrogen but inhibited by androgens. To determine underlying changes in activity of neuropeptide neurones in the paraventricular nucleus of the hypothalamus (PVN), we examined the effect of oestrogen and androgen treatment on restraint-induced c-fos mRNA, corticotropin-releasing hormone (CRH) heteronuclear RNA, and arginine vasopressin hnRNA expression in the PVN. Male rats were gonadectomized and injected with oestradiol benzoate (EB) or dihydrotestosterone propionate (DHTP; s.c., daily for 4 days). Rats were stressed by restraint for 10 min or 30 min before killing. Other rats were stressed for 30 min and then returned to their home cage for 20 min before killing. Corticosterone and adrenocorticotropic hormone responses to restraint stress were significantly greater in EB-treated rats and lower in DHTP-treated rats at the 30-min timepoint compared to controls. c-fos mRNA increases following stress were augmented by EB but inhibited by DHTP. CRH hnRNA expression increased significantly in the PVN in response to restraint stress, and this increase was augmented by EB treatment, but decreased by DHTP treatment. Vasopressin hnRNA expression was also increased in response to stress, and this increase was attenuated by DHTP. These findings indicate that gonadal hormones influence the reactivity of the hypothalamic-pituitary adrenal axis to stress.

Effect of Dihydrotestosterone on Pituitary Follicle-Stimulating Hormone Isoforms in Adult Male Rats Treated with a Gonadotropin-Releasing Hormone Antagonist

The effect of androgens on the oligosaccharide structure of follicle-stimulating hormone (FSH) isoforms was studied in adult male rats treated with a potent gonadotropin-releasing hormone (GnRH) antagonist. Animals were castrated (Cx), and 24 h later were treated with physiological doses of either testosterone propionate (T) or dihydrotestosterone propionate (DHT) (0.25 mg/rat in corn oil, daily, s.c.) for 7 days. The antiandrogen flutamide (F; 5 mg/day/rat, twice a day, s.c.) was administered to either Cx-T or Cx-DHT rats to block androgen action. The GnRH antagonist Org 30276 (Ant; 1 mg/kg/day, s.c.) was injected to both Cx- and Cx-DHT treated rats. FSH serum levels reached normal values in Cx rats treated with either T or DHT, whereas those treated with F retained Cx conditions. Both Cx-Ant and Cx-Ant-DHT treated animals presented normal serum FSH levels. Concanavalin A affinity chromatography was used to isolate pituitary FSH isoforms according to their carbohydrate inner structure. An increased proportion of FSH isoforms bearing complex-type oligosaccharides was observed in Cx rats treated with T or DHT, whereas the proportion of these isoforms was reduced in Cx and Cx-T-F or Cx-DHT-F rats. These results demonstrate that functional androgens are needed to complete the oligosaccharide processing of FSH. In addition, the proportion of the different pituitary FSH isoforms after DHT replacement was equivalent to that found when GnRH action was blocked, indicating that androgens are involved in the regulation of carbohydrate incorporation to the FSH molecule by acting mainly at the pituitary level, independently of GnRH action.

Sex differences in progesterone receptor expression: a potential mechanism for estradiol-mediated sexual differentiation.

The differential exposure of males and females to testosterone (T) and its metabolite estradiol (E) contributes to the development of sex differences in the brain. However, the mechanisms by which T and E permanently alter neural development remain virtually unknown. Two regions of the rat preoptic area, the anteroventral periventricular nucleus (AVPv) and the medial preoptic nucleus (MPN), are sexually dimorphic and serve as models for studying the hormonal mechanisms of sexual differentiation. Around birth, these regions express dramatically higher levels of progesterone receptor immunoreactivity (PRir) in males than they do in females. The present study examined the possibility that sexually dimorphic induction of PR expression in these two regions constitutes a potential mechanism of E-mediated sexual differentiation. Prenatal exposure to either T propionate or the synthetic estrogen, diethylstilbestrol, but not dihydrotestosterone propionate, significantly increased PRir levels in the MPN and AVPv of fetal females compared with controls. Prenatal exposure to the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione, significantly reduced PRir in the MPN and AVPv of fetal males, whereas the androgen receptor antagonist flutamide had no effect. This suggests that aromatization of T into E is crucial for the sex difference in PR expression in the MPN and AVPv during development.

Ultrastructural and Quantitative Immunohistochemical Changes Induced by Nonsteroid Antiandrogens on Pituitary Gonadotroph Population of Prepubertal Male Rats

Specific blockade of the androgen receptor by the nonsteroid antiandrogens flutamide and Casodex has proven to be a useful tool for studying androgens in vivo. The aim of the present study was to investigate the effect of antiandrogen administration at the pituitary level by evaluating the ultrastructural changes in gonadotrophs, in correlation with the quantitative immunohistochemical findings, and by comparing these alterations with the effect of androgen deprivation by castration either with or without subsequent androgen replacement. Male Sprague-Dawley rats (23 days old) were grouped as follows: (1) controls, (2) flutamide-injected (10 mg/rat/day), (3) Casodex-injected (10 mg/rat/day), (4) castrated, and (5) castrated plus androgen-replaced (dihydrotestosterone propionate; 40 µg/rat/day). Groups were sacrificed after 10 days of maintenance under each condition. Pituitaries were processed for both light and electron microscopy. Serial sections (4 µm) were obtained at different levels and immunostained by means of the primary murine monoclonal antibodies anti-FSH and anti-LH and a peroxidase-mediated EnVision System (Dako). Volume density, cell density and mean cell area were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). The mean cell area (p < 0.001) and the volume density (p < 0.05) increased significantly in the flutamide- and Casodex-treated groups as well as the castrated group of FSH and LH cells. On the other hand, androgen replacement in the castrated rats, however, reduced in both parameters related to control animals. The cell density of FSH-secreting cells was increased (p < 0.05) in the Casodex and flutamide treatment as well as castrated group. The cell density of LH-secreting cells was augmented (p < 0.05) in the Casodex-treated group, while there was no increase in such parameter with flutamide and castration. The ultrastructure of all groups showed two types of gonadotrophs. Type I cells contained large (300–500 nm) and small (150–200 nm) secretory granules, while type II cells were smaller, and exhibited only small granules (100–200 nm). Flutamide-treated, Casodex-treated and castrated groups presented a decreased number of secretory granules with some exocytotic profiles, well-developed rough endoplasmic reticulum and an expanded Golgi complex of both types of cells. The gonadotrophs from the castrated group exhibited numerous mitochondria with electron-dense ring-shaped laminar figures, while in the castrated plus androgen-replaced rats only a few mitochondria had similar changes to those observed in castrated animals, as a possible residual alteration. Finally, the gonadotrophs from flutamide-treated rats showed mitochondrial alterations with clear areas and isolated electron-dense laminar figures. In summary, we conclude that lack of androgen reaction through the effects of nonsteroid antiandrogens and castration on prepubertal rats produced a hypertrophia-hyperplasia of the FSH cells, and hypertrophia of LH-secreting cells, with marked alterations at the ultrastructural level suggestive of a hyperstimulation stage.