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.     

Testosterone prevents orchidectomy-induced bone loss in estrogen receptor-alpha knockout mice.

To examine the role of the estrogen receptor-alpha (ERalpha) during male skeletal development, bone density and structure of aged ERalphaKO mice and wild-type (WT) littermates were analyzed and skeletal changes in response to sex steroid deficiency and replacement were also studied. In comparison to WT, ERalphaKO mice had smaller and thinner bones, arguing for a direct role of ERalpha to obtain full skeletal size in male mice. However, male ERalphaKO mice had significantly more trabecular bone as assessed both by pQCT and histomorphometry, indicating that ERalpha is not essential to maintain cancellous bone mass. Six weeks following orchidectomy (ORX), both WT and ERalphaKO mice showed high-turnover osteoporosis as revealed by increases in serum osteocalcin and decreases in trabecular (-38% and -58% in WT and ERalphaKO, respectively) and cortical bone density (-5% and -4% in WT and ERalphaKO, respectively). Administration of testosterone propionate (T, 5 mg/kg/day) completely prevented bone loss both in ERalphaKO and in WT mice. As expected, estradiol (E2, 60 microg/kg/day) replacement did not prevent cancellous bone loss in ORX ERalphaKO mice. However, E2 stimulated bone formation at the endocortical surface in ORX ERalphaKO, suggesting that osteoblasts may respond to nonERalpha-mediated estrogen action. In conclusion, although functional ERalpha may play a significant role during male skeletal development, this receptor does not seem essential for androgen-mediated skeletal maintenance in older male mice.     

Pathophysiology of bone loss in castrated animals

The pathophysiology of bone loss in castrated animals is reviewed. Both male and female rats rapidly lose metaphyseal trabecular bone from the tibia and the femur due to an imbalance between bone resorption and bone formation. The aetiology of sex hormone deficiency-induced bone loss is not fully understood. It seems unlikely that the bone loss is due to changes in the circulating levels of the calciotropic hormones or to an increase in the spontaneous release from peripheral blood monocytes of the bone resorption stimulating cytokine IL-1. Changes in the sensitivity of bone of castrated rats to calciotropic hormones may play a role as well as the lack of direct stimulatory effects of gonadal oestrogens and androgens on bone cells. In addition several data indicate that prostaglandins may be involved.     

Apigenin inhibits osteoblastogenesis and osteoclastogenesis and prevents bone loss in ovariectomized mice

Polyphenol have been reported to have physiological effects with respect to alleviating diseases such as osteoporosis and osteopetrosis. We recently reported that the olive polyphenol hydroxytyrosol accelerates bone formation both in vivo and in vitro. The present study was designed to evaluate the in vivo and in vitro effects of apigenin (4′,5,7-trihydroxyflavone), one of the major polyphenols in olives and parsley, on bone formation by using cultured osteoblasts and osteoclasts and ovariectomized (OVX) mice, respectively. Apigenin markedly inhibited cell proliferation and indices of osteoblast differentiation, such as collagen production, alkaline phosphatase activity, and calcium deposition in osteoblastic MC3T3-E1 cells at concentrations of 1–10 μM. At 10 μM, apigenin completely inhibited the formation of multinucleated osteoclasts from mouse splenic cells. Moreover, injection of apigenin at 10 mg kg⁻¹ body weight significantly suppressed trabecular bone loss in the femurs of OVX mice. Our findings indicate that apigenin may have critical effects on bone maintenance in vivo.     

Supraphysiological testosterone enanthate administration prevents bone loss and augments bone strength in gonadectomized male and female rats

High-dose testosterone enanthate (TE) may prevent hypogonadism-induced osteopenia. For this study, 3-mo-old male and female Fisher SAS rats underwent sham surgery, gonadectomy (GX), or GX plus 28 days TE administration (7.0 mg/wk). GX reduced serum sex hormones (i.e., testosterone, dihydrotestosterone, and estradiol) (P < 0.05) in both sexes and bone concentrations of testosterone (males only), and estradiol (females only). GX also elevated urine deoxypyridinoline/creatinine in both sexes and serum osteocalcin (females only), findings that are consistent with high-turnover osteopenia. GX reduced cancellous bone volume (CBV) and increased osteoid surfaces in tibia of both sexes. GX males also experienced reduced trabecular number and width and increased trabecular separation, whereas GX females experienced increased osteoblast and osteoid surfaces. Bone biomechanical characteristics remained unaffected by GX, except that femoral stiffness was reduced in females. In contrast, TE administration to GX rats elevated serum and bone androgens to supraphysiological concentrations in both sexes but altered neither serum nor bone estradiol in males. Additionally, TE did not prevent GX-induced reductions in serum or bone estradiol in females. TE also reduced markers of high-turnover osteopenia in both sexes. In males, TE prevented GX-induced changes in trabecular number and separation, CBV, and osteoid surfaces while diminishing osteoblast and osteoclast surfaces; however, these changes were not fully prevented in females. In both sexes, TE increased femoral length and femoral maximal strength to above that of Sham and GX animals while preventing the loss of femoral stiffness in females. In conclusion, TE administration appears protective of cancellous bone in male rats and augments cortical bone strength in both sexes.     

Genotype and retention of the ejaculatory reflex in castrated male mice

Genotype proves to be an important factor affecting the retention of sexual responsiveness following castration. Male F₁ hybrids between C57BL/6 females and DBA/2 males (B6D2F₁) show very prolonged retention. Experiment 1 used a diallelic design using C57BL/6, DBA/2 and BALB/c strains and showed that heterosis per se is not an adequate explanation of the superior retention of the B6D2F₁ genotype. Experiment 2 showed that neonatal injections of 100 μg TP to B6D2F₁ male mice did not result in better retention of the ejaculatory reflex after castration in adulthood. B6D2F₁ males from both experiments who exhibited the ejaculatory reflex for many months after castration went through a ‘difficult period’ in the first 10 to 12 postcastration weeks. During this time, ejaculation latency increased fourfold, and then returned to the original level. On the other hand, the proportion of males reaching the ejaculatory threshold during this period first declined then increased.     

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.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis

The objective of the present study was to explore the bone protective role of blueberry in an ovariectomized rat model. Thirty 6-month-old female Sprague-Dawley rats were either sham-operated (Sham) or ovariectomized (Ovx) and divided into three groups: Sham, Ovx (control), Ovx+blueberry (5% blueberry w/w). After 100 days of treatment, rats were euthanized, and blood and tissues were collected. Bone mineral density (BMD) and content of whole body, right tibia, right femur and fourth lumbar vertebra were assessed via dual-energy X-ray absorptiometry. As expected, Ovx resulted in loss of whole-body, tibial, femoral, and 4th lumbar BMD by approximately 6%. Blueberry treatment was able to prevent the loss of whole-body BMD and had an intermediary effect on prevention of tibial and femoral BMD when compared to either Sham or Ovx controls. The bone-protective effects of blueberry may be due to suppression of Ovx-induced increase in bone turnover, as evident by lowered femoral mRNA levels of alkaline phosphatase, collagen type I and tartrate-resistant acid phosphatase to the Sham levels. Similarly, serum osteocalcein levels were also lower in the blueberry group when compared to the Ovx control group, albeit not significantly. In summary, our findings indicate that blueberry can prevent bone loss as seen by the increases in BMD and favorable changes in biomarkers of bone metabolism.     

Steroids and sexual behavior in castrated male rheusus monkeys.

Sexual behavior of long-term castrated rhesus males was not increased by administration of the hydroxylated metabolite of testosterone (T), 17 beta, 19-dihydroxy-5 alpha-androstan-3-one diacetate (19-OH-DHTA) at a dose of 1 mg/kg/day. Simultaneous administration of 19-OH-DHTA and estradiol benzoate (EB) also failed to increase the level of sexual performance, but daily injection (1 mg/kg/day) of testosterone propionate (TP) was very effective in effective in activating sexual behavior.     

Genetic loss of Faah compromises male fertility in mice

Marijuana is the most commonly used illicit drug. Although there is some indication that reproductive functions in males are impaired in chronic marijuana users, the genetic evidence and underlying causes remain largely unknown. Herein we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah(-/-) sperm on the egg zona pellucida provides evidence that the capacity of sperm to penetrate the zona barrier is hampered by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.     

Oral administration of phytocomponent p-hydroxycinnamic acid prevents bone loss in ovariectomized rats

The preventive effect of phytocomponent p-hydroxycinnamic acid (HCA) on ovariectomy (OVX)-induced bone loss was investigated. HCA (250 or 500 μg/100 g body weight) was orally administered once daily for 30 days to OVX rats. The analysis using a peripheral quantitative computed tomography (pQCT) showed that OVX caused bone loss in the femoral-metaphyseal tissues. This change was significantly restored after the administration of HCA (250 or 500 μg/100 g body weight) to OVX rats. Mineral content, mineral density, and polar strength strain index in the femoral-metaphyseal tissues were significantly decreased in OVX rats. These decreases were significantly restored after the administration of HCA (500 μg/100 g) to OVX rats. Moreover, OVX caused a significant decrease in calcium content or alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. These decreases were significantly restored after the administration of HCA (250 or 500 μg/100 g) to OVX rats. Deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in OVX rats. These increases were significantly restored after oral administration of HCA (500 μg/100 g). This study demonstrates that HCA has preventive effects on OVX-induced bone loss of rats in vivo.     

Dietary bisphenol A prevents ovarian degeneration and bone loss in female mice lacking the aromatase gene (Cyp19 ).

We previously generated mice lacking aromatase activity by targeted disruption of Cyp19 (ArKO mice), and reported phenotypes of the female mice, showing hemorrhage formation and follicular depletion in the ovary, diminution in uterine size, and bone loss. In the present study, we examined the influence of dietary bisphenol A (BPA), a monomer used for the production of polycarbonate and known to have estrogenic activity, on these phenotypes of the ArKO mice. When ArKO mice were fed chow diets supplemented with 0.1% or 1% (w/w) BPA for 5 months, they were protected from ovarian degeneration, uterine diminution and bone loss in a dose-dependent manner. Northern blot analyses of ovarian RNA of ArKO mice showed differences in the expression levels of insulin-like growth factor (IGF)-I, IGF-I receptor, growth differentiation factor 9 and bone morphogenetic protein 15 as compared with those in the ovaries of wild-type mice. The differences in the expression levels were restored by dietary BPA. In the ArKO uteri, expression of progesterone receptor and vascular endothelial growth factor mRNAs was diminished, and was restored by BPA to the levels in wild-type mice. In contrast, BPA had little effect on the ovarian, uterine and skeletal structures of wild-type mice. In conclusion, estrogenic effects of BPA on the reproductive tract as well as skeletal tissue were evident in adult female ArKO mice. These results suggest that the ArKO mouse is an animal model suitable for studying effects of estrogenic chemicals as well as estrogen in vivo.     

Ultrastructure of STH cells of the pars distalis of castrated male mice after hepatectomy

Summary An electron microscopic study aimed at differentiation between castration gonadotrophs and posthepatectomy STH cells was performed on the pars distalis of the pituitary of castrated male mice, after partial hepatectomy. The ultrastructural features observed permit the distinction of both cell types. In the present experiments some remarkable ultrastructural changes, other than those described in a previous report, have been found in STH cells of hepatectomized mice with or without previous castration. Most of them contained masses of heterogeneous electron density, suggesting fusion of granules. These masses and some secretion granules were observed close to the plasma membrane, apparently in the process of discharging material into the pericapillary space. Granular extrusion was more frequent than normally. An increased number of lysosomes, probably related to the digestion of overproduced secretion material, was evident. The appearance of concentric lamellar formations might be related to an increase in cell movements. STH cells with severe cytoplasmic damage were also found, indicating an increased rate of cell loss.Work carried out with the financial assistance by a grant from the Comisión de Investigación Científica de la Universidad Nacional de La Plata. Thanks are due to the members of the Technical Staff of the Institute for their technical assistance.