Castrated Male Rats Research Articles

Preclinical Characterization of a (S)-N-(4-Cyano-3-Trifluoromethyl-Phenyl)-3-(3-Fluoro, 4-Chlorophenoxy)-2-Hydroxy-2-Methyl-Propanamide: A Selective Androgen Receptor Modulator for Hormonal Male Contraception

The pharmacologic effects of (S)-N-(4-cyano-3-trifluoromethyl-phenyl)-3-(3-fluoro, 4-chlorophenoxy)-2-hydroxy-2-methyl-propanamide (S-23) were characterized in male rats as an animal model of hormonal male contraception. S-23 showed high binding affinity (inhibitory constant = 1.7 +/- 0.2 nm) and was identified as a full agonist in vitro. In castrated male rats, the ED50 of S-23 in the prostate and levator ani muscle was 0.43 and 0.079 mg/d, respectively. In intact male rats treated for 14 d, S-23 alone suppressed LH levels by greater than 50% at doses greater than 0.1 mg/d, with corresponding decreases in the size of the prostate but increases in the size of levator ani muscle. In intact male rats treated for up to 10 wk with S-23 and estradiol benzoate (EB; necessary to maintain sexual behavior in rats), S-23 showed biphasic effects on androgenic tissues and spermatogenesis by suppressing serum concentrations of LH and FSH. EB alone showed no effect on spermatogenesis. In the EB + S-23 (0.1 mg/d) group, four of six animals showed no sperm in the testis and zero pregnancies (none of six) in mating trials. After termination of treatment, infertility was fully reversible, with a 100% pregnancy rate observed after 100 d of recovery. S-23 increased bone mineral density and lean mass but reduced fat mass in a dose-dependent manner. This is the first study to show that a selective androgen receptor modulator combined with EB is an effective and reversible regimen for hormonal male contraception in rats. The beneficial effects of S-23 on the muscle, tissue selectivity, and favorable pharmacokinetic properties make it a strong candidate for use in oral male contraception.

The hepatic aquaporin 9 (AQP9) expression is regulated in response to castration and PPARα‐ agonist treatment

It has previously been shown that male rats have higher hepatic expression of AQP9 than female rats (Nicchia et al, J Histochem Cytochem, 2001). The regulation of hepatic AQP9 has also been related to PPARα (Patsouris et al, J Clin Invest, 2004). This study aims to investigate the role of castration and PPARα–agonist (Wy 14643) administration in regulation of hepatic AQP9 protein expression in male rats. In castrated rats, immunoblotting showed a 1.7 fold increase in hepatic AQP9 (p < 0.01). This finding was confirmed by immunohistochemical analysis. Serum testosterone was decreased in the castrated group (p < 0.01), while serum insulin was 1.0 ± 0.3 and 1.9 ± 0.6 in castrated and controls, respectively, however this was not statistically significant and plasma glucose levels remained unchanged. In rats treated for 10 days with Wy 14643 (3 mg/kg/day), immunoblotting showed a 2.6‐fold decrease in the expression of hepatic AQP9 (p < 0.001). Immunohistochemical analysis revealed a marked decrease of AQP9 in the periportal zone, whereas AQP9 expressed in the perivenous zone appeared unchanged. There were no changes in serum insulin or glucose levels. We conclude that hepatic AQP9 protein expression increases in response to castration in male rats, whereas treatment with PPARα–agonist results in a marked decrease and redistribution of hepatic AQP9 protein.Supported by the Danish National Research Foundation

Effects of dihydrotestosterone and estradiol on rat facial nerve regeneration following a crush axotomy

Previously our laboratory has demonstrated that testosterone treatment increases the regeneration rate of the rat facial nerve following a crush axotomy, and electrical stimulation (ES) reduces the initial delay in axonal sprout formation while the combination of these two treatments has an additive effect. In these studies, testosterone propionate (TP) was given to adult rat at the time of facial nerve crush axotomy at its exit from the stylomastoid foramen. Since testosterone is enzymatically metabolized to either estradiol or the nonaromatizable androgen, dihydrotestosterone (DHT), the present studies explore whether the regenerative properties of TP on the facial motoneurons are due to the effects of androgens, estrogens, or both. We also explored the effects of these metabolites in combination with the ES. Following the facial nerve axotomy and steroid implantation, adult castrated male rats were observed daily for signs of functional recovery from the facial paralysis. The average day of return of each of the four behavioral components (semi‐blink, blink reflex, full vibrissae movement, and complete recovery) was calculated for all groups. We found that although both estradiol and DHT, alone or in combination with ES treatment, reduced the variability in facial nerve recovery, their effects on facial nerve regeneration were not as potent as TP itself.

Intraperitoneal administration of gonadotropin-releasing hormone-PE40 induces castration in male rats

To evaluate the long-term effects of gonadotropinreleasing hormone (GnRH)-based vaccine on levels of GnRH antibody and testosterone, and vaccine-induced immunocastration on sexual behavior of male rats. The rats were treated with GnRH-PE40 intraperitoneally every other day for 12 wk. GnRH antibody and testosterone level in rat blood were determined by ELISA and radioimmunoassay, respectively. Morphological changes in testes and sexual behavior of rats were evaluated. GnRH-PE40 induced a high production in GnRH antibody, decreased the serum testosterone level, testis atrophy and sexual function in rats. Intraperitoneal administration of GnRHPE40 produces structural and functional castration of male rat reproductive system by inducing anti-GnRH antibody.

Study of Furostenol Glycoside Fraction of Tribulus terresteris. on Male Sexual Function in Rats

Tribulus terrestris. Linn. (Zygophyllaceae) has long been used in the traditional Chinese and Indian systems of medicine for the treatment of various ailments and is popularly claimed to improve sexual function in men. In this study, we have examined the aphrodisiac properties of the furostenol glycoside fraction of Tribulus terrestris. extract (TT-FG) in male castrated rats. Adult Wister rats were castrated and divided into five groups of six animals each and treated with either vehicle, sildenafil (5 mg/kg, p.o.) or TT-FG (5, 10, and 25 mg/kg, p.o.) once daily for 14 days. Sexual behavioral and orientational activity was observed after acute (1 day) and subacute (7 and 14 days) treatment. Serum testosterone levels were also measured on day 14 by withdrawing blood from retroorbital plexus. The improvement in sexual behavior as noted by increase in mounting frequency (MF), intromission frequency (IF), and ejaculation latency (EL) and decrease in mounting latency (ML), intromission latency (IL), and postejaculation interval (PEI) after TT-FG treatment in castrated rats in our study implies increase in the desired component of sexuality. TT-FG was found to increase orientational activity parameters such as licking and ano-and genital grooming and decreased climbing and nongential grooming by male rats indicating increased sexual stimulation and lack of interest toward external environment after treatment with TT-FG. These results are supported by increased serum testosterone levels in male rats after 14 days of treatment by TT-FG. In conclusion, the furostenol glycoside fraction of Tribulus terrestris. exhibited good aphrodisiac properties.

Gender-Related Difference in the Toxicity of Ultraviolet Absorber 2-(3′,5′-Di-tert-butyl-2′-hydroxyphenyl)-5-chlorobenzotriazole in Rats

2-(3′,5′-Di-tert-butyl-2′-hydroxyphenyl)-5-chlorobenzotriazole (DBHCB) is widely used as an ultraviolet absorber. Previously, we showed that male rats had more than a 100 times higher susceptibility to the toxic effects of DBHCB than females. In order to investigate the role of sex steroids in the mediation of this gender-related difference, DBHCB (0 or 250 mg/kg/day) was given to male and female young intact and castrated rats by gavage for 28 days in the current study. In intact rats, relative liver weight increased to more than two times that of the control in males, while the rate of change was less than 10% in females. On histopathology, hypertrophy of hepatocytes was observed in males but not in females. In castrated rats, an approximately 40% increase in the relative liver weight was found only in males, and no histopathological changes in the liver were detected in either sex. The gender-related difference was also determined in preweaning rats administered DBHCB at 0, 250, or 500 mg/kg/day by gavage from postnatal days 4 to 21. Blood biochemical changes, including increases in the levels of AST, ALT, and ALP, 80–95% increase in the relative liver weight and histopathological changes in the liver, such as hypertrophy and single cell necrosis of hepatocytes, were observed at both doses in both sexes. In conclusion, the gender-related difference in the toxicity of DBHCB, which was observed in young rats, was markedly reduced by castration and abolished in preweaning rats.

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4′-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols

We performed a uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study [enhanced Organization for Economic Co-operation and Development (OECD) test guideline No. 407] of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols. In the uterotrophic assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), the test chemical was orally administered to castrated male SD rats at doses of 0, 50, 200, and 1,000 mg/kg/day for ten consecutive days beginning on postnatal day 56, and no changes were observed. When this chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, an increase in thyroid weight was observed in the female rats in the 125 mg/kg group, an increase in serum thyroid-stimulating hormone (TSH) values in the male and female rats in the 125 mg/kg group, and a decrease in serum T3 and T4 values in the male rats in the 125 mg/kg group, and thyroid follicular epithelial cell hypertrophy was observed in some of the female rats in the 125 mg/kg group. These findings were concluded to be the result of endocrine-mediated effects of the chemical on thyroid function. In addition, increased liver weight, abnormal histological findings in the liver, and abnormal biochemical parameters related to liver function were observed in male and/or female rats in 5 mg/kg group and higher dose groups. The no-observed-effect level for 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) was concluded to be <5 mg/kg/day. In the uterotrophic assay of 3-(dibutylamino)phenol, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay of 3-(dibutylamino)phenol, the test chemical was orally administered at doses of 0, 50, 200, and 400 mg/kg/day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when this test chemical was orally administered at doses 0, 30, 100, and 300 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid weight increased in the male rats in the 300 mg/kg group, thyroid follicular epithelial cell hypertrophy was observed in a small number of male rats in the 300 mg/kg group, serum T3-values decreased in the female rats in the 300 mg/kg group, and a tendency for TSH-values to increase was observed in the male and female rats in the 300 mg/kg group. Therefore, 3-(dibutylamino)phenol was also concluded to have slight anti-thyroid acting effects as the endocrine-mediated effects. On the other hand, increased hemosiderin deposition in the spleen, increased spleen weight, hematological abnormalities, and squamous epithelial hyperplasia of the forestomach were detected in male and/or female rats in the 100 and/or 300 mg/kg groups, and thus the no-observed-effect level for 3-(dibutylamino)phenol was concluded to be 30 mg/kg/day.

Gonadal hormones-mediated effects on the stimulation of dopamine turnover in mesolimbic and nigrostriatal systems by cocaine- and amphetamine-regulated transcript (CART) peptide in male rats

Estradiol and testosterone modulated behavioral and neurochemical activities in the mesolimbic and nigrostriatal dopaminergic systems have been reported. We examined whether estradiol and testosterone affect stimulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the mesolimbic and nigrostriatal dopaminergic systems in this study. Intracerebroventricular administration of CART peptide increased dopamine turnover in the nucleus accumbens and striatum in male rats. Stimulation of dopamine turnover in nucleus accumbens and striatum by CART peptide were found in intact male rats, but not in castrated male rats. This stimulation was restored in castrated male rats by testosterone or estradiol priming, or by treatment with the water-soluble form of estradiol, but not by treatment with the membrane-impermeable form of estradiol. Estradiol and testosterone antagonists blocked testosterone's effects, but only estradiol antagonist blocked estradiol's effects. Moreover, treatment of dihydrotestosterone also restored the stimulation in castrated male rats. This dihydrotestosterone's effect was blocked by a testosterone antagonist, but not by an estradiol antagonist. All of these findings indicate that gonadal hormones play a regulatory role in stimulation of CART peptide in mesolimbic and nigrostriatal dopaminergic systems, and suggest that acts through intracellular rather than extracellular mechanisms.

Hershberger Assay for Antiandrogenic Effects of Phthalates

The antiandrogenic effects of seven phthalates, di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di-isononyl phthalate (DINP), di-isodecyl phthalate (DIDP), di-n-heptyl phthalate (DnHP), and mono-2-ethyhexyl phthalate (MEHP), were investigated by Hershberger assay in castrated male SD rats. An androgen agonist, testosterone (0.4 mg/kg/d), was administered for 10 consecutive days by subcutaneous (sc) injection as a positive control. Additionally, 20, 100, or 500 mg/kg body weight (bw)/d of 6 phthalates (DEHP, DBP, BBP, DINP, DIDP, or DnHP) or 10, 50, or 250 mg/kg bw/d of MEHP, the primary metabolite of DEHP, were also administered orally in combination with testosterone (0.4 mg/kg/d, sc) for 10 consecutive days, respectively. In the testosterone-treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani/bulbocavernosus muscles (LABC) weights were found to be significantly increased. Ventral prostate weights were significantly decreased in animals treated with DEHP or DBP at doses of 20 mg/kg bw/d or above, 500 mg/kg bw/d DIDP, and 250 mg/kg bw/d MEHP. Seminal vesicles weights were also significantly decreased by DEHP at > 100 mg/kg bw/d, DINP at > 20 mg/kg bw/d, DIDP at 500 mg/kg bw/d, or MEHP at 50 or 250 mg/kg bw/d, respectively. In addition, LABC weights were decreased by DEHP at 500 mg/kg bw/d, DINP at 500 mg/kg bw/d, and MEHP at 50 or 100 mg/kg bw/d. These data suggest that some phthalates possess antiandrogenic activity, and that multiple cross-talk between androgen, estrogen, and steroid hormone receptors occurs.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4´-[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols

We performed an uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) of 4,4 -[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and the uterine weight of rats given the 1,000 mg/kg dose of the test chemical plus ethinyl estradiol decreased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 100, 300, and 1,000 mg/kg day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when the test chemical was orally administered at doses 0, 100, 300, and 1,000 mg/kg day for at least 28 days, a decrease in LH values in rats of both sexes and a decrease in FSH and estradiol values in female rats were detected in the 1,000 mg/kg group, and abnormal estrous cycles, uterine glandular atrophy, persistence of ovarian corpora lutea, vaginal epithelial mucification, and mammary glandular hyperplasia were also observed in one female rat in the 1,000 mg/kg group. Therefore, the uterotrophic assay used in this study showed that the chemical has the estrogen-antagonist properties, and some potentially endocrine-mediated effects were detected in growing rats based on the results of the enhanced OECD test guideline No. 407. However, the changes were observed in rats given a high dose of the chemical, 1,000 mg/kg day.

The effects of nitric oxide–cGMP pathway stimulation on dopamine in the medial preoptic area and copulation in DHT-treated castrated male rats

Dopamine (DA) in the medial preoptic area (MPOA) provides important facilitative influence on male rat copulation. We have shown that the nitric oxide–cGMP (NO–cGMP) pathway modulates MPOA DA levels and copulation. We have also shown that systemic estradiol (E 2) maintains neuronal NO synthase (nNOS) immunoreactivity in the MPOA of castrates, as well as relatively normal DA levels. This effect of E 2 on nNOS probably accounts for at least some of the previously demonstrated behavioral facilitation by intra-MPOA E 2 administration in castrates. Therefore, we hypothesized that stimulation of the MPOA NO–cGMP pathway in dihydrotestosterone (DHT)-treated castrates should restore DA levels and copulatory behaviors. Reverse-dialysis of a NO donor, sodium nitroprusside (SNP), increased extracellular DA in the MPOA of DHT-treated castrates and restored the ability to copulate to ejaculation in half of the animals. A cGMP analog, 8-Br-cGMP, also increased extracellular DA, though not as robustly, but did not restore copulatory ability. The effectiveness of the NO donor in restoring copulation and MPOA DA levels is consistent with our hypothesis. However, the lack of behavioral effects of 8-Br-cGMP, despite its increase in MPOA DA, suggests that NO may have additional mediators in the MPOA in the regulation of copulation. Furthermore, the suboptimal copulation seen in the NO donor-treated animals suggests the importance of extra-MPOA systems in the regulation of copulation.

Effect of Testosterone on Intracavernous Pressure Elicited With Electrical Stimulation of the Medial Preoptic Area and Cavernous Nerve in Male Rats

We studied the effects of castration and testosterone (T) replacement on intracavernous pressure (ICP) elicited with electrical stimulation of the medial preoptic area (MPOA) and cavernous nerve (CN) in male rats. We measured the ICP during electrical stimulation of the MPOA and CN in castrated male rats with and without testosterone replacement. The experimental group consisted of 20-week-old male rats at 2 weeks (n=8), 4 weeks (n=8) and 8 weeks (n=8) after castration, and at 8 weeks after castration with T replacement (n=4). Intact 20-week-old rats (n=8) served as controls. The erectile response was expressed as the ICP/blood pressure (BP) ratio. The ICP/BP ratios during CN stimulation of the animals at 2, 4, and 8 weeks after castration were significantly lower than those of the intact animals. However, the erectile responses were not eliminated. In contrast to these peripherally evoked responses, erectile responses elicited by electrical stimulation of the MPOA were eliminated following castration. After testosterone replacement, both erectile responses were restored. Testosterone plays important roles in both the central and peripheral neural pathways for the maintenance and restoration of erectile capacity. The central control of erection shows more extensive changes following testosterone replacement than the peripheral control.

Sex hormones affect bone marrow dysfunction after trauma and hemorrhagic shock

Bone marrow (BM) dysfunction after trauma and hemorrhagic shock (T/HS) results in a decrease in clonogenic growth of BM progenitors through a plasma-mediated process. Although sex hormones have been shown to modulate some end-organ injury after shock, post-T/HS BM dysfunction has only been studied in male animals. Therefore, the present study examines the effects of sex hormones on post-T/HS BM dysfunction by measuring clonogenic growth of BM progenitors in castrated male rats and in ovariectomized and proestrus female rats. Laboratory experiment. University surgical research laboratory. Castrated and noncastrated male and ovariectomized and proestrus female Sprague-Dawley rats. All rats were subjected to either T/HS or sham shock with laparotomy (n = 3-5 per group). At 3 hrs after resuscitation, the rats were killed and plasma and BM mononuclear cells from bilateral femurs were harvested. BM mononuclear cells were cultured for erythroid burst-forming unit and granulocyte-macrophage colony-forming unit colonies to assess the extent of progenitor BM dysfunction. BM from noncastrated male rats subjected to T/HS demonstrated a significant decrease in granulocyte-macrophage colony-forming unit and erythroid burst-forming unit colony formation compared with BM of all the sham shock groups and with the castrated male and both female rat groups subjected to T/HS. In addition, plasma from noncastrated shocked male rats incubated in vitro with BM cells from unmanipulated male rats caused a significant suppression of BM granulocyte-macrophage colony-forming unit and erythroid burst-forming unit colonies compared with plasma from castrated rats subjected to either sham shock with laparotomy or T/HS. The profound BM dysfunction observed in noncastrated male rats after T/HS is not observed in proestrus female rats and castrated male rats. In addition, the in vitro plasma-mediated BM suppression present in male rats after T/HS is also lost in castrated male rats. Sex hormones seem to play a significant role in BM dysfunction after T/HS.

Effects of Androgens and Estradiol on Spine Synapse Formation in the Prefrontal Cortex of Normal and Testicular Feminization Mutant Male Rats

Recent studies suggest that, in female monkeys and rats, estrogens elicit dendritic spine synapse formation in the prefrontal cortex, an area that, similar to the hippocampus, plays a critical role in cognition. However, whether gonadal hormones induce synaptic remodeling in the male prefrontal cortex remains unknown. Here we report that gonadectomy reduced, whereas administration of 5alpha-dihydrotestosterone or estradiol-benzoate to castrated male rats increased, the number of medial prefrontal cortical (mPFC) spine synapses, with estradiol-benzoate being less effective than 5alpha-dihydrotestosterone. To investigate whether the androgen receptor contributes to the mediation of these changes, we compared the response of testicular feminization mutant (Tfm) male rats to that of wild-type animals. The number of mPFC spine synapses in gonadally intact Tfm rats and 5alpha-dihydrotestosterone-treated castrated Tfm males was considerably reduced compared to intact wild-type animals, whereas the synaptogenic effect of estradiol-benzoate was surprisingly enhanced in Tfm rats. These data are consistent with the hypothesis that remodeling of spine synapses in the prefrontal cortex may contribute to the cognitive effect of gonadal steroids. Our findings in Tfm animals indicate that androgen receptors may mediate a large part of the synaptogenic action of androgens in the mPFC of adult males. However, because this effect of 5alpha-dihydrotestosterone is not completely lost in Tfm rats, additional mechanisms may also be involved.

Pharmacological and X-Ray Structural Characterization of a Novel Selective Androgen Receptor Modulator: Potent Hyperanabolic Stimulation of Skeletal Muscle with Hypostimulation of Prostate in Rats

A novel, highly potent, orally active, nonsteroidal tissue selective androgen receptor (AR) modulator (BMS-564929) has been identified, and this compound has been advanced to clinical trials for the treatment of age-related functional decline. BMS-564929 is a subnanomolar AR agonist in vitro, is highly selective for the AR vs. other steroid hormone receptors, and exhibits no significant interactions with SHBG or aromatase. Dose response studies in castrated male rats show that BMS-564929 is substantially more potent than testosterone (T) in stimulating the growth of the levator ani muscle, and unlike T, highly selective for muscle vs. prostate. Key differences in the binding interactions of BMS-564929 with the AR relative to the native hormones were revealed through x-ray crystallography, including several unique contacts located in specific helices of the ligand binding domain important for coregulatory protein recruitment. Results from additional pharmacological studies effectively exclude alternative mechanistic contributions to the observed tissue selectivity of this unique, orally active androgen. Because concerns regarding the potential hyperstimulatory effects on prostate and an inconvenient route of administration are major drawbacks that limit the clinical use of T, the potent oral activity and tissue selectivity exhibited by BMS-564929 are expected to yield a clinical profile that provides the demonstrated beneficial effects of T in muscle and other tissues with a more favorable safety window.

A Histochemical Investigation of Different Distribution of TSH Cells in Adenohypophsis of Female and Male Rats

The aim of the present study was to investigate histochemically the localization of thyroid-stimulating hormone (TSH) cells and the number of TSH cells in adenohypophysis of ovariectomized adult females, intact females at estrous and diestrous phase of sexual cycles and castrated and non-castrated adult male rats. TSH cells, distributed throughout the pars distalis in all groups, were observed more frequent in areas near to the pars intermedia than other regions. The cells were round, ovoid, stellar-shaped and had contacts with sinusoids. Their nuclei were large and round-shaped. The cells were found mostly single or occasionally in groups of double and triple. The number of TSH cells was higher in estrous than diestrous phase in intact rats. In adenohypophysis of ovariectomized rats, their numbers were less than that of intacts in estrous, but higher than that of found in diestrous phases. Furthermore, the numbers of TSH cells in intact females during estrous phase was higher than those found in intact and castrated male rats. On the other hand, castration reduced the number of TSH cells while ovariectomy had no effect in this respect.

Estrogen modulates in vitro atrial bradycardia induced by Indian red scorpion venom via G-protein coupled mechanisms

Role of estrogen on cardiac dysrhythmia produced by Indian red scorpion ( Mesobuthus tamulus) venom was examined using rat right atrial preparations in vitro. In females, the M. tamulus venom produced an increase, a decrease and an increase in rate at 0.03, 0.3 and 3 μg/ml of venom, respectively, producing N-shaped response curve, whereas no such response pattern was observed in males. Force of contraction in females was increased at all the concentrations of the venom, while in males the increase was seen only at 3 μg/ml. Castration of male rats did not alter the venom response to female type, while ‘estrogenisation of castrated male rats’ (pseudofemales) produced a response similar to females. Tamoxifen reversed the venom-induced responses both in females and pseudofemales. Further in females, the venom action at 0.3 μg/ml was blocked by atropine. Response at this concentration was also blocked by pertussis toxin and methylene blue. Results suggest that the cholinergic component of venom response is modulated by estrogen receptors via G i-protein-guanylyl cyclase mechanism.

Effects of continuous intravenous administration of human metastin 45–54 on the activity in the hypothalamic-pituitary-gonadal axis of intact adult male rhesus monkeys ( Macaca mulatta)

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional study of GPR54 revealed that the receptor plays an essential role to modulate sex-hormones including GnRH. Thus, antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases such as prostate cancer or endometriosis. We recently reported 2-acylamino-4,6-diphenylpyridines as the first small molecule GPR54 antagonists with high potency. However, the representative compound 1 showed low brain exposure, where GPR54 acts as a modulator of gonadotropins by binding with its endogenous ligand, metastin. In order to discover compounds that have not only potent GPR54 antagonistic activity but also good brain permeability, we focused on converting the primary amine on the side chain to a secondary or tertiary amine, and finally we identified 15a containing a piperazine group. This compound exhibited high affinity to human and rat GPR54, apparent antagonistic activity, and high brain exposure. In addition, intravenous administration of 15a to castrated male rat suppressed plasma LH level, which indicates the possibility of a small molecule GPR54 antagonist as a novel drug for sex-hormone dependent diseases.

Testosterone and Renal Renin Angiotensin System in salt sensitive hypertension.

Sex hormones are important blood pressure (BP) regulators. The aim of this study was to test the hypothesis that castration attenuated salt sensitive hypertension in male Dahl (DS) and to determine if changes in Renin Angiotensin System (RAS) are involved. Intact and castrated male DS rats were placed on low sodium diet (0.3%, LS) or 4 weeks of high sodium diet (8%, HS). Mean arterial pressure (MAP) was monitored by telemetry. Kidney levels of angiotensinogen (AGTN), renin and AT-1R mRNA were measured by real time RT-PCR in LS and HS diet. Plasma renin activity (PRA) was determined by radioimmunoassay. Placement of DS rats on HS for 4 weeks caused a progressive increase in MAP in males (from 121±2 to 168±2 mm Hg; LS vs. HS; p <0.05). Castration in males attenuated hypertension (168±2 vs. 145±3 mmHg; male vs.castrate; p <0.05). Development of salt sensitive hypertension in male DS rats was associated with 3-fold increase of renal AGTN mRNA (p<0.05) and this effect was abolished by castration. Renin mRNA expression and PRA decreased with salt (40% decrease; p< 0.05) and was not affected by gonadectomy. AT-1R mRNA in kidney was not altered by either castration or salt. Our data suggest that testosterone augments the hypertensive response to HS in male DS. Intrarenal RAS may be involved in salt sensitive hypertension and testosterone could be an important modulator of this system in DS.