Raloxifen prevents bone loss in castrated male mice

Raloxifen is a selective estrogen receptor modulator which prevents bone loss in ovariectomized female mice in a fashion similar to estrogens. Since testosterone-deficient male mice also lose bone mass, we were interested in testing the effects of raloxifen on bones in intact and castrated male mice. Bone density was significantly reduced in castrated animals (1.36+/-0.04 g/ml) as compared to intact animals (1.42+/-0.03 g/ml) (p<0.01). When castrated mice with extraordinarily low concentrations of testosterone and with reduced weight of seminal vesicles were treated with raloxifen, the changes in bone density and bone minerals resulting from castration (1.36+/-0.04 g/ml) were entirely prevented (1.40+/-0.01 g/ml). Cortical bone was lost in orchidectomized mice, and this decrease in cortical thickness of the femur was prevented by raloxifen administration. Raloxifen in a dose used in humans for treatment of osteoporosis decreased the weight of seminal vesicles, an organ which is highly sensitive to the androgenic effect, decreased the concentration of testosterone (12.5+/-2.8 micromol/l) (p<0.01) but not to the same level as in the case of castrated animals (0.6+/-0.3 micromol/l), and did not have any effect on bone density or mineral content in intact mice. The results of the present study may thus be interpreted as supporting the hypothesis that raloxifen is an effective agent against the deleterious effects of castration-induced osteopenia in male mice and also support the hypothesis that estrogens may have physiological skeletal effects in male mice.     

Effects of triiodothyronine and estrogen administration on bone mass, mineral content and bone strength in male rats.

Experimental hyperthyroidism had a negative effect on bone mineral density, but did not significantly alter mechanical properties of femur and femoral bone thickness. Estradiol at a dose used in humans for the treatment of osteoporosis decreased seminal vesicle weight and concentration of testosterone but increased bone density in male rats compared to intact animals. In these rats, the mechanical analysis revealed an increased mechanical femur strength higher than the increase in bone density and femoral cortical thickness. When hyperthyroid male rats with low bone density were treated with estradiol in spite of a low plasma testosterone, the changes in bone density resulting from hyperthyroidism were entirely prevented. Estrogens protect the male skeleton against resorbing action of T (3). Treatment with estradiol in male rats with hyperthyroidism did not increase mechanical bone strength or femoral cortical thickness as it did with estradiol administration alone. Our results suggest that exogenously administered estrogens may have therapeutic value in preventing bone loss accompanying triiodothyronine administration, even in male rats with a low testosterone levels. At the concentration studied, estradiol increased in spite of low plasma testosterone, bone mineral density, mechanical strength of femur, and femoral cortical thickness.     

Antiandrogenic and antirenotropic effect of spironolactone.

Spironolactone given to male mice in dose 1.5 mg for 21 days produced significant changes not only in the highly androgen dependent seminal vesicles but also in the kidneys. Spironolactone blocked the action of endogenous testosterone in intact mice and led to further significant decrease in seminal vesicles and kidney weight in castrated mice. Such an effect could be due to a blocking of androgenic action of adrenal steroids.     

The effect of chronic nicotine administration on bone mineral content and bone strength in normal and castrated male rats.

Tobacco, containing nicotine as the principal pharmacologically active chemical, has been identified as being a risk factor for the development of osteoporosis. Thirty-two male Wistar rats of two months of age were castrated or sham operated to evaluate the effects of long-term administration (four months) of nicotine in drinking water (9.0 mg/kg/day). The presence of cotinine in urine confirmed successful delivery of nicotine. The bones were tested mechanically by a three point bending test in a Mini Bionix (MTA) testing system. The bones from castrated rats were characterized by a reduction in bone density as well as ash, calcium and phosphate content. Castration significantly altered mechanical properties of bone (9%) and femoral cortical thickness. When intact rats were treated with a high dose of nicotine, nicotine had negative effect on tibial bone density as well as ash, calcium, phosphate content and significantly altered the mechanical properties of bone (12%) and femoral cortical thickness compared to intact animals. Nicotine itself does not exert any anti-androgenic effect and does not produce changes in the weight of seminal vesicles. Nicotine-induced bone loss is associated with high bone turnover in the male rats as expressed by increased TrACP and B-ALP. When castrated rats were treated with the high dose of nicotine the changes in bone density resulting from castration were not further potentiated. These results document the efficacy of nicotine at high doses to cause bone loss and loss of bone mechanical strength in intact rats. The results of the present study may be interpreted as supporting the hypothesis of nicotine as a risk factor for osteoporosis.     

Sensitivity and specificity of the bioassay of estrogenicity in mammary gland and seminal vesicles of male mice

Young intact (18 days old) and adult castrated males of CBA and C3H/Di mice were used for measuring the estrogenicity on the basis of growth response of mammary epithelial structures and the weight of seminal vesicles. It was demonstrated that heavier young males had disproportionally heavier seminal vesicles (sex steroid-responsive organs) than small animals at day 33 of age (that is on the day when experimental animals were killed and organs dissected). However, the weight of the spleen (sex steroid-nonresponsive organ) was proportionally related to body weight. To minimize variability in hormone responsiveness, all animals were weighed at the age of 18 days and only males weighing 8+/-1 g were used for hormone treatment. The percentage area of mammary fat pad occupiedby mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.01 microg x d(-1). The maximum effective dose of estradiol was 0.1 microg x d(-1) and dose 10 microg x d(-1) of estradiol decreased mammary size to control level (inverted-U-shaped dose-response curve). Progesterone alone stimulated mammary growth only in high doses (500 microg x d(-1) and higher) in young intact males, but had no effect on mammary growth in adult castrated animals. In young intact males, estradiol alone, or progesterone alone decreased the weight of seminal vesicles. No such inhibitory effect of these hormones was noted in adult castrated males. Progesterone acted synergistically with estradiol to produce higher mammary growth compared to that in males treated with estradiol alone. In the presence of progesterone seminal vesicles weight was decreased by estradiol given in such low doses as 0.001 microg x d(-1) of estradiol, which is 10 times lower than that effective in animals treated with estradiol alone. On the other hand, in the adult castrated males a combination of estradiol plus progesterone stimulated seminal vesicles weight. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate (a synthetic steroid exhibiting progestantial and estrogenic activities) and inhibited by both testosterone and cortisol. Estradiol, progesterone, norethindrone acetate, or testosterone did not affect spleen weight and size of mammary lymph nodes.However, cortisol significantly decreased not only spleen weights but also size of mammary lymph nodes. These results showthat simultaneous evaluation of mammary gland growth, seminal vesicles, and the spleen weight in the same animal is suitable for bioassay of estrogenicity as well as for detection of androgenic and antiandrogenic activities.     

Perinatal testosterone surge is required for normal adult bone size but not for normal bone remodeling

Although testosterone (T) has striking effects on mature skeletal size and structure, it is not clear whether this depends exclusively on adult circulating levels of T or whether additional early-life factors also play a role. We have compared the androgen-deficient hypogonadal (hpg) mutant mouse with intact, orchidectomized, and T-treated non-hpg mice to determine relative contributions of adult and perinatal T to bone growth and development. At 3 wk of age, although trabecular and cortical bone structure was normal, bone turnover was significantly altered in hpg male mice; osteoid volume (OV/BV) and osteoblast surface (ObS/BS) were significantly lower and osteoclast surface (OcS/BS) significantly higher in hpg mice compared with age-matched non-hpg mice, pointing to a role for the perinatal T surge in determining bone turnover levels before sexual maturity. At 9 wk of age, the hpg bone phenotype mimicked closely that of age-matched non-hpg mice that had been orchidectomized at 3 wk of age, including low trabecular bone mass and high bone turnover. These bone phenotypes of hpg and orchidectomized non-hpg mice were all prevented by replacement doses of T or dihydrotestosterone (DHT), suggesting that these are determined by adult sex steroid hormones. In contrast, a short bone phenotype that could not be prevented by T or DHT treatment was observed in 9-wk-old hpg mice yet not in intact or castrated non-hpg mice. These data suggest a role for the perinatal T surge in determining adult bone length and confirms that adult circulating T determines adult bone density.     

Changes in enzyme activities of thymidine kinase and 5'-nucleotidase for dTMP during hormonal regeneration of seminal vesicles of mice.

An increase of thymidine kinase [EC 2.7.1.21] activity and decrease of 5'-nucleotidase [EC 3.1.3.5] activity for dTMP were found during hormonal regeneration of the seminal vesicles by daily or single administration of testosterone propionate into mice castrated 2 weeks previously. Actinomycin D injected on day 0 of testosterone treatment completely inhibited both the increase of thymidine kinase and the decrease of 5'-nucleotidase. When injected on day 2, actinomycin D decreased thymidine kinase activity below the control level and 5'nucleotidase activity was not restored to the normal level. The activity of 5'-nucelotidase in a mixed sample, in which seminal vesicles of castrated mice and those of testosterone-treated mice were homogenized together, was intermediate between the activities determined separately. This indicates the absence of any inhibitor of 5'nucleotidase in the regenerating vesicles. Changes in total activity of 5'nucleotidase and total protein content in extracts during various treatments showed that the decrease in specific activity of 5'-nucleotidase in the first 2 days of testosterone treatment was not due to inhibition of enzyme activity but to dilution of the enzyme with other proteins which increased in content more rapidly than 5'-nucleotidase.     

Effect of antiandrogen cyproterone acetate on the action of testosterone on mouse kidney.

The loss of endogenous testosterone in castrated male mice leads to a marked decrease in seminal vesicle and kidney tissue weight. 21 days' administration of exogenous testosterone abolished the effect of castration on the seminal vesicles and kidney tissue. The antiandrogen cyproterone acetate produced significant changes in the target tissue for androgens, i.e. in the seminal vesicles. In every case it blocked the action of both exogenous and endogenous testosterone on the seminal vesicles, but failed to block the "renotropic" action of testosterone, expressed as relative kidney weight. Contrary to its effect on the seminal vesicles, it did not influence relative kidney weight in normal animals. It likewise did not block the effect of exogenous testosterone on kidney tissue. The mechanism of the action of cyproterone acetate in androgen-dependent tissues is known to consist in inhibition of androgen binding to specific cell receptors in the target tissues. Some of the specific androgen receptors in mouse kidney are evidently different in character from those in the accessary sex glands, that being the reason why cyproterone acetate has an antiandrogenic, but not an antirenotropic effect. In agreement with experiments on rats, adrenal weight also decreases in mice after the administration of cyproterone acetate.     

The biological activity of dimeric testosterone, a new long-acting androgen, and of testosterone enanthate in the castrated male rat.

The long-term effect of single intramuscular injections of various doses of dimeric testosterone and of testosterone enanthate into castrated male rats upon serum testosterone, luteinizing hormone (LH), pituitary LH, and on the weight of the seminal vesicles, the ventral prostate and the levator ani muscle, was investigated. The effect of the enanthate was characterized by a rapid onset and a protracted androgenic action and a suppression of serum LH, while the dimeric testosterone brought about only a moderate but very even depot effect. The injection of 5 mg of the dimeric testosterone caused a positive feedback effectu upon LH release for 16 weeks. The results indicate that the dimeric testosterone may exert is hormonal effects as intact ester.     

Roles of prepubertal androgen, estrogen or androgen plus prolactin on androgen-induced proliferative response of seminal vesicles in adult mice

Male mice castrated on day 0 after birth were pretreated daily with testosterone propionate (TP, 4 micrograms/g body weight), 17 beta-estradiol (E2, 0.2 micrograms/g body weight) or vehicle for 21 days starting from day 20. In another experiment, male mice were castrated on day 25; two pituitaries from 60-day-old females were immediately grafted under the capsule of the left kidney in one group. The castrated mice with or without grafts were pretreated daily with TP (4 or 20 micrograms/g body weight) for 36 days starting from day 25, and the left kidney was removed on day 60. Daily TP injections (4 micrograms/g body weight) were started again at 30 days after the end of pretreatments to examine androgen-induced proliferation, and incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was used as an index of proliferation. In the neonatally castrated mice, both TP and E2 pretreatments given during the prepubertal period significantly increased seminal vesicle weight even long after the end of the pretreatments. However, androgen-induced proliferative response found in the neonatally castrated adult mice (poor response; long duration with a low peak) was changed to that found in mice castrated at adulthood (good response; short duration with a high peak) by the TP pretreatment only but not at all by the E2 pretreatment. In the mice castrated on day 25, a pharmacological dose of TP or TP plus hyperprolactin could not enhance or change the adult castration type of androgen-induced proliferation induced by physiological prepubertal androgens, although both treatments significantly enhanced the prepubertal growth of the seminal vesicles.     

Effect of androgen pretreatments at adulthood on androgen-induced proliferative response of seminal vesicles in neonatally castrated mice

Male mice were castrated on days 0 and 60 after birth. The majority of the neonatally castrated mice were pretreated with androgen; the mice were given daily injections of testosterone propionate (TP; 4 or 8 micrograms/g body wt) for 20 or 30 days starting from day 60. Daily injections of TP (4 micrograms/g body wt) to examine androgen-induced proliferation were started from day 30 or 60 after the end of TP pretreatments or from day 60 after castration; on various days after starting TP injections, the weight and the incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles were determined as indices for proliferation. The seminal vesicles of neonatally castrated adult mice were characterized by long duration of androgen-induced proliferation (greater than 20 days) with a low peak (neonatal castration type), whereas the seminal vesicles of adult castrated mice were characterized by short duration of proliferation (10 days) with a high peak (adult castration type). In neonatally castrated adult mice, the neonatal castration type of androgen-induced proliferation was changed largely to the adult castration type when pretreatment with 8 micrograms/g body wt of TP had been given for 30 days. However, this effect gradually disappeared when the mice had been pretreated with decreasing amounts of TP for a shorter period. The present findings suggest that the defect in the androgen-induced proliferative response of mouse seminal vesicles induced by the absence of neonatal and prepubertal testicular androgens can be compensated by androgens given in adulthood, if enough androgen is given for a sufficiently long time.     

Pneumadin in the ventral prostate and seminal vesicles of estradiol-treated rats: RIA and immunocytochemical studies

Pneumadin (PNM) is a decapeptide (the rat peptide: Tyr-Gly-Glu-Pro-Lys-Leu-Asp-Ala-Gly-Val-NH2) isolated from mammalian lungs. Human and rat PNM differ only by substitution of one amino acid--Tyr/Ala. PNM evokes an antidiuretic effect via a potent stimulation of arginine-vasopressin (AVP) release. By means of recently established, highly specific RIA method, high concentration of PNM had been found in the rat ventral prostate. Castration resulted in a profound drop in PNM concentration, an effect prevented by testosterone replacement. The present studies were aimed at investigating the effect of prolonged estradiol administration on PNM concentration, content and localization in the prostate and seminal vesicles of the rat. Depo estradiol (estradiolum valerianicum) administration to adult male rats resulted in a notable atrophy of ventral prostate and seminal vesicles. During the entire experiment (till day 30 after administration), PNM concentration in ventral prostate was similar to that seen in intact animals, while peptide content per gland was markedly lowered. PNM immunostaining was observed in prostate epithelium of estradiol-treated rats and its localization resembled that observed in intact animals. Nearly 40 times lower PNM concentration than in ventral prostate was found in seminal vesicles. In contrast to prostate, on days 20 and 30 of estradiol treatment PNM concentration in seminal vesicles was higher than in intact rats. However, due to profound seminal vesicle atrophy, PNM content per entire gland was notably lowered in estradiol-injected rats. By immunocytochemistry, PNM-immunoreactive substances were not found in seminal vesicles of either intact or estradiol-administered rats. High PNM concentration in the rat prostate suggests its important role in the function of the gland.     

Proliferative response of seminal vesicle cells to androgen and estrogen in neonatally castrated mice

Proliferation and death of androgen- and estrogen-responsive cells in seminal vesicles were compared between neonatally and adult (on Day 60 after birth) castrated mice. Daily injections of either testosterone propionate (TP) or estradiol-17 beta (E2) were started on Day 90 after birth; the incorporation of 5-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) into the whole seminal vesicles was used as an index for proliferation. Although the peak of [125I]IdUrd uptake was observed 3 days after starting TP injections in both neonatally and adult castrated mice, the peak was lower and the period of proliferation was much longer in the former than in the latter. When TP injections were stopped, the fraction of surviving cells that synthesized DNA on Day 3 of TP injections was much larger in neonatally than adult castrated mice. The difference was attributed to the presence of TP-induced proliferation of fibromuscular cells in the neonatally castrated mice but not in the adult castrated mice; only the fibromuscular cells but not epithelial cells survived after stopping TP injections. Although injections of E2 increased the proliferation of epithelial cells but did not the weight of seminal vesicles in adult castrated mice, the same procedure increased the proliferation of both epithelial and fibromuscular cells and the weight in neonatally castrated mice. The E2-induced fibromuscular cells seemed to survive in the presence or absence of E2. The present results seem to indicate that androgen- and estrogen-induced proliferation of fibromuscular cells is irreversible in seminal vesicles of neonatally castrated mice and that the depletion of androgen in the seminal vesicle during neonatal and prepubertal periods is at least in part compensated by the administration of androgen, even after 90 days of age.     

Hormonal regulation of chemosignal-stimulated precopulatory behaviors in male housemice (Mus musculus)

Five experiments examined the hormonal regulation of the precopulatory reproductive behavior of male housemice of two genotypes (DBA/2J inbreds and C57BL/6J X AKR/J hybrids). The two precopulatory behaviors examined were preferences for female urinary odors and ultrasonic courtship vocalizations to anesthetized females. The preferences were then used to make inferences about odor attractiveness. Gonadally intact hybrid males were highly attracted to the airborne urinary odors of female mice but were either indifferent to, or exhibited less attraction to, male urinary odors. Castration decreased male attraction to female odor such that castrated males were equally attracted to male and female odors. Normal levels of attraction could be maintained in castrated hybrid males by Silastic implants of either testosterone or estradiol. While Silastic implants of dihydrotestosterone (DHT) were also effective in maintaining attraction in hybrids, this hormone was ineffective in inbreds. The effectiveness of estradiol, DHT, and testosterone in maintaining attraction following castration was paralleled in castrated hybrids by the effects of these hormones in maintaining courtship vocalizations to females. In contrast to the genotype-specific effects of DHT upon behavior, DHT was effective in both genotypes in maintaining seminal vesicle weight. Estradiol, on the other hand, which was quite effective in maintaining both precopulatory behaviors in hybrids, had little effect upon seminal vesicle weight. Thus these experiments dissociate the behavioral effects of steroids from their effects upon peripheral morphology. We suggest that testosterone can activate precopulatory behaviors following either aromatization or 5-alpha reduction but that genetic variability somehow gives rise to strain differences in DHT responsiveness.     

Effects of testosterone on the metabolism of folate coenzymes in the rat

1. The effects of castration and testosterone treatment on enzymic activities involved in folate coenzyme metabolism in the liver and in accessory sex organs of male adult rats were studied. 2. In the liver of castrated rats the concentration of 10-formyltetrahydrofolate (10-HCO-H(4)folate) synthetase and tetrahydrofolate (H(4)folate) dehydrogenase were significantly decreased whereas that of 5,10-methylenetetrahydrofolate dehydrogenase increased; the treatment with five doses of testosterone caused a return to normal values of these activities. 3. In the prostate of castrated rats a pronounced decrease in H(4)folate dehydrogenase, serine hydroxymethyltransferase and 10-HCO-H(4)folate synthetase activities was observed. The administration of testosterone restored the enzymic activities to normal values. 4. In the seminal vesicles of castrated rats only 10-HCO-H(4)folate synthetase was markedly depressed; testosterone treatment not only restored activity to normal values but raised it to higher than normal values. The slight changes observed in other enzymic activities also returned to normal values with the hormone treatment. 5. These results are discussed in relation to a possible control mechanism of folate metabolism by testosterone.     

Proliferative response of seminal vesicle cells to androgen in mice castrated neonatally and pretreated with estrogen or androgen at adulthood

Seminal vesicle cells of neonatally castrated adult mice show poor response to androgen, compared to those of mice castrated at adulthood; effects of pretreatment with androgen or estrogen at adulthood on androgen-induced proliferation of the seminal vesicle cells were examined in neonatally castrated mice. Male mice castrated at day 0 after birth were pretreated with daily injections of testosterone propionate (TP, 100 micrograms/mouse), 17 beta-estradiol (E2, 5 micrograms/mouse) or vehicle for 20 days starting from day 60; daily TP injections (100 micrograms/mouse) for 30 days were started again from day 110 in all the pretreated mice to examine androgen-induced proliferation by incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles. Both TP and E2 pretreatments significantly increased the seminal vesicle weight found before TP treatment. However, androgen-induced proliferation of the seminal vesicle found in neonatally castrated mice (poor response; long duration with a low peak on day 3) was changed at least in part to that found in mice castrated at adulthood (good response; short duration with a high peak on day 3) only following the TP pretreatment but not at all following the E2 pretreatment. The E2 pretreatment induced poor androgen-induced proliferation with a low peak on day 7.     

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment.

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM)electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

Effect of testosterone on the amount of serous-like granules in convoluted tubular cells of mouse submandibular glands.

The effect of testosterone on the amount of granules present in convoluted tubular cells of the submadibular glands of mice was studied by the following two methods; 1) an immunochemical method using antiserum specific to the granular components, and 2) a histographic method. The results obtained by these two methods were in agreement. The amounts of the granules in normal female and castrated male mice were one-tenth to one-twentieth of that in normal male mice. When male mice were castrated, the amount of granules decreased, reaching a minimum 20 days after the aperation the injection of the male hormone, testosterone, into castrated male mice caused an increase in the amount of granules; this increase reached a maximum 15 days after the injection. The increase of granules caused by testosterone injection was almost completely prevented by inhibitors of protein synthesis, actinomycin D and puromycin. This suggests that protein synthesis was indispensable to the increase in the amount of granules. In male mice, the injection of female hormones scarcely affected the amount of granules. Kinetic analysis of the decrease and increase of granules on castration and testosterone injection suggested that the male hormone stimulated granule synthesis, but it hardly influenced the loss of granules.     

Gender dependent effects of testosterone and 17β-estradiol on bone growth and modelling in young mice

This study examined the effects of estrogen (17β-estradiol) and testosterone on the growth of long bones in male and female mice, with and without gonadectomy. Weight and nose-to-tail length were determined at 3 weeks of age at time of gonadectomy, 7 days later at the onset of hormone therapy, and throughout the treatment period. Gonadectomized mice exhibited an initial weight gain during the pretreatment period but length was unaffected. Hormone treatment altered weight gain in surgical and intact animals in a gender- and hormone-dependent manner. Estradiol enhanced weight gain in intact mice, but inhibited weight gain in ovariectomized mice. Lower doses of estradiol increased weight gain in orchiectomized mice at early time points. Testosterone increased weight in intact females and males, but not in gonadectomized mice. Estradiol increased nose-to-tail length in intact females at early time points, but inhibited length in ovariectomized females at later times, and it decreased length in intact males. Testosterone increased length in normal females and normal males. Serum Ca was unaffected by ovariectomy, but orchiectomy resulted in decreased levels. Estradiol reduced serum Ca in gonadectomized animals; serum Ca was increased by estradiol treatment in intact females. Changes in tibial bone weight, ash weight and mineral composition, and relative sizes of epiphyseal and metaphyseal bone were gender-, gonadectomy- and hormone-specific. Bone weight was greater in ovariectomized mice. Ash weight per bone was comparable, but there was an increase in Ca and P content with ovariectomy. Estradiol increased bone weight, ash content, and bone Ca and P in ovariectomized and intact females. Orchiectomy alone did not alter bone weight, ash content, or Ca and P, but orchiectomized mice were sensitive to estradiol; all parameters were increased in the orchiectomized animals treated with estradiol. Analysis of the ash content and Ca and P per mg bone, rather than per bone, demonstrated estradiol and testosterone alter net bone formation, but not the amount of mineral per unit bone. Ovariectomy increased hypertrophic cartilage. While estradiol did not alter tibial area in ovariectomized mice, it caused an increase in intact females. The total amount of growth plate cartilage in ovariectomized animals was decreased by estradiol to levels typical of intact animals due to a greater decrease in the hypertrophic cartilage in the ovariectomized mice, as well as a greater increase in metaphyseal bone area. Testosterone had no effect on these parameters in the females. Orchiectomy decreased the amount of growth plate cartilage, but increased the hypertrophic zone. Estradiol increased growth plate cartilage in intact male mice, but decreased it in orchiectomized mice. This difference was also seen in the hypertrophic zone. Total growth plate cartilage and hypertrophic cartilage were increased by testosterone in intact males, whereas metaphyseal and epiphyseal bone area were decreased. The results show for the first time that there is a gender-specific response in both male and female mice to both estradiol and testosterone, whether or not the animals have been gonadectomized. For many parameters, orchiectomized mice behave like females in response to both sex steroids, indicating that the male gonad is needed for mouse bone to exhibit the male phenotypic response to estradiol and testosterone.     

Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.