Early-life exposure to sex hormones promotes voluntary ethanol intake in adulthood. A vulnerability factor to drug addiction.

While there is extensive research on alcohol dependence, the factors that make an individual vulnerable to developing alcoholism haven't been explored much. In this study, we aim to investigate how neonatal exposure to sex hormones affects alcohol intake and the regulation of the mesolimbic pathway in adulthood. The study aimed to investigate the impact of neonatal exposure to a single dose of testosterone propionate (TP) or estradiol valerate (EV) on ethanol consumption in adult rats. The rats were subjected to a two-bottle free-choice paradigm, and the content of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens (NAcc) was measured using HPLC-ED. The expression of critical DA-related proteins in the mesolimbic pathway was evaluated through RT-qPCR and western blot analysis. Supraphysiological neonatal exposure to EV or TP resulted in increased ethanol intake over four weeks in adulthood. In addition, the DA and DOPAC content was reduced and increased in the NAcc of EV and TP-treated rats, and ß-endorphin content in the hypothalamus decreased in EV-treated rats. The VTA µ receptor and DA type 2 form short receptor (D2S) expression were significantly reduced in EV and TP male rats. Finally, in an extended 6-week protocol, the increase in ethanol consumption induced by EV was mitigated during the initial two hours post-naloxone injection. Neonatal exposure to sex hormones is a detrimental stimulus for the brain, which can facilitate the development of addictive behaviors, including alcohol use disorder.

Testosterone Propionate Promotes Proliferation and Viability of Bone Marrow Mesenchymal Stem Cells while Preserving Their Characteristics and Inducing Their Anti-Cancer Efficacy

Background: Various studies have reported the effects of testosterone on different cell types, yet bone marrow-derived mesenchymal stem cells' cellular responses to testosterone remain unknown. Aims: To investigate the effects of testosterone propionate, an oil-soluble short-acting form of testosterone, on human bone marrow-derived mesenchymal stem cells' proliferation and viability after 24 hours of incubation. We also investigated the impact of testosterone propionate on bone marrow-derived mesenchymal stem cell's polarization and cytotoxicity on K562 leukemia cell line. Study Design: In vitro study. Methods: We expanded commercially available bone marrow derived mesenchymal stem cells in vitro and treated them with testosterone propionate at concentrations ranging from 10-6-10-10 M for 24 hours. Ideal concentration was determined by evaluating cellular viability and proliferation with Annexin V/Propidium Iodide assay and carboxyfluorescein succinimidyl ester staining. The characteristic features of bone marrow-derived mesenchymal stem cells were evaluated by immunophenotyping and investigating their differentiation capacities. Bone marrow-derived mesenchymal stem cells' cytotoxic properties upon testosterone propionate treatment were determined by co-culturing the cells with K562 cells and with confocal imaging investigating polarization. Results: Testosterone propionate promoted proliferation and maintained the viability of bone marrow-derived mesenchymal stem at 10-8 M concentration. Further evaluations were conducted with the determined dose. The results showed that, apart from promoting mesenchymal stem cells' polarization and increasing their cytotoxicity on K562 cells, testosterone propionate did not alter differentiation capacities of bone marrow-derived mesenchymal stem cells and certain cell surface markers, but led to a significant increase in HLA-DR expression. Conclusion: The findings reveal that testosterone propionate promotes the proliferation and survival of bone marrow-derived mesenchymal stem cells in a dose-dependent manner without hampering their differentiation capacities, induces their polarization to the pro-inflammatory phenotype, and increases their cytotoxicity on the K562 cell line.

Modeling of Benign Prostatic Hyperplasia in Rats with a High Dose of Testosterone.

In order to optimize the testosterone model of benign prostatic hyperplasia, we studied the effect of castration and different doses of testosterone on the induction of the proliferative process in the prostate of Wistar rats. It was shown that 4-week subcutaneous administration of testosterone propionate in a dose of 20 mg/kg causes pronounced proliferative and hemodynamic disorders in the dorsolateral gland morphologically similar in castrated and non-castrated males. Administration of testosterone in a dose of 3 mg/kg had no significant effect on the dynamics of the pathological process in non-operated rats and normalized the structure of the gland in castrated animals. Morphological study showed that castration of males provides no visible advantages in reproducing the testosterone model of benign prostatic hyperplasia. The proposed non-traumatic modification of the model with a high dose of testosterone has good reproducibility and sensitivity to therapeutic agents, as shown by the example of finasteride.

SIRT3 affects mitochondrial metabolic reprogramming via the AMPK-PGC-1α axis in the development of benign prostatic hyperplasia.

BACKGROUND: Sirtuin 3 (SIRT3) has been reported to share an association with mitochondrial metabolic reprogramming. However, the molecular mechanism underlying is not well understood, especially in benign prostatic hyperplasia (BPH). Therefore, the purpose of this study was to research whether SIRT3 can affect the progression of BPH via the regulation of mitochondrial metabolic reprogramming. METHODS: Following the development of a rat model of BPH using testosterone propionate (TP), we extracted prostate tissues from sham-operated and BPH rats. Subsequently, bioinformatics prediction was used to screen the genes differentially expressed in BPH. To verify the role played by SIRT3 in BPH, we injected AAV9-SIRT3 into rats, followed by TP treatment. Prostate epithelial cells (PEC) were treated with TP to assess the mitochondrial morphology, mitochondrial membrane potential, and expression of enzymes related to the oxidative phosphorylation pathway after SIRT3 expression alteration. Finally, we examined the expression of AMPK-PGC-1α pathway in tissues and cells. RESULTS: SIRT3 was reduced in the prostate tissues of BPH rats. After overexpression of SIRT3, mitochondrial morphology was more stable in prostate tissues of BPH rats and in TP-treated PEC, with significant increases in mitochondrial membrane potential and in the expression of oxidative phosphorylation-related enzymes in the cytoplasm. Moreover, SIRT3 significantly activated the AMPK-PGC-1α signaling pathway, which maintained the stability of mitochondrial membrane potential as well as mitochondrial structure, thus alleviating the symptoms of BPH. CONCLUSION: SIRT3 maintained the stability of mitochondrial membrane potential as well as mitochondrial structure by activating the AMPK-PGC-1α pathway, thereby alleviating the symptoms of BPH.

Heat-Killed and Live Enterococcus faecalis Attenuates Enlarged Prostate in an Animal Model of Benign Prostatic Hyperplasia.

In the present study, we investigated the inhibitory effect of heat-killed Enterococcus faecalis (E. faecalis) and live E. faecalis on benign prostatic hyperplasia (BPH). The BPH rat model was established by administering male rats with testosterone propionate (TP, 5 mg/kg, in corn oil) via subcutaneous injections daily for four weeks after castration. The rats were divided into five groups: Con, corn oil-injected (s.c.) + DW administration; BPH, TP (5 mg/kg, s.c.) + DW administration; BPH+K_EF, TP (5 mg/kg, s.c.) + heat-killed E. faecalis (7.5 × 1012 CFU/g, 2.21 mg/kg) administration; BPH+L_EF, TP (5 mg/kg, s.c.) + live E. faecalis (1 × 1011 CFU/g, 166 mg/kg) administration; BPH+Fi, TP (5 mg/kg, s.c.) + finasteride (1 mg/kg) administration. In both of BPH+K_EF and BPH+L_EF groups, the prostate weight decreased and histological changes due to TP treatment recovered to the level of the Con group. Both of these groups also showed regulation of androgen-signaling factors, growth factors, and apoptosis-related factors in prostate tissue. E. faecalis exhibited an inhibitory effect on benign prostatic hyperplasia, and even heat-killed E. faecalis showed similar efficacy on the live cells in the BPH rat model. As the first investigation into the effect of heat-killed and live E. faecalis on BPH, our study suggests that heat-killed E. faecalis might be a food additive candidate for use in various foods, regardless of heat processing.

Neonatal programming with sex hormones: Effect on expression of dopamine D1 receptor and neurotransmitters release in nucleus accumbens in adult male and female rats.

Steroid sex hormones produce physiological effects in reproductive and non-reproductive tissues, such as the brain. In the brain, sex hormones receptors are expressed in cortical, limbic and midbrain areas modulating memory, arousal, fear and motivation between other behaviors. One neurotransmitters system regulated by sex hormones is dopamine (DA), where during adulthood, sex hormones promote neurophysiological and behavioral effects on DA systems such as tuberoinfundibular (prolactin secretion), nigrostriatal (motor circuit regulation) and mesocorticolimbic (driving of motivated behavior). However, the long-term effects induced by neonatal exposure to sex hormones on DA release induced by D1 receptor activation and its expression in nucleus accumbens (NAcc) have not been fully studied. To answer this question, neurochemical, cellular and molecular techniques were used. The data show sex differences in NAcc DA extracellular levels induced by D1 receptor activation and protein content of this receptor in male and female control rats. In addition, neonatal programming with a single dose of TP increases the NAcc protein content of D1 receptors of adult male and female rats. Our results show new evidence related with sex differences that could explain the dependence to drug of abuse in males and females, which may be associated with increased reinforcing effects of drugs of abuse.

Stromal-AR influences the growth of epithelial cells in the development of benign prostate hyperplasia.

Activation of epithelial-AR signaling is identified as the major cause of hyperproliferation of the cells during benign and malignant prostate conditions. However, the contribution of stromal-AR is also precarious due to its secretory actions that contribute to the progression of benign and malignant tumors. The present study was aimed to understand the influence of stromal-AR mediated actions on epithelial cells during BPH condition. The secretome (conditioned media-CM) was collected from AR agonist (testosterone-propionate-TP) and antagonist (Nilutamide-Nil) treated BPH patient-derived stromal cells and exposed to BPH epithelial cells. Epithelial cells exhibited increased cell proliferation with the treatment of CM derived from TP-treated stromal cells (TP-CM) but did not support the clonogenic growth of BPH epithelial cells. However, CM derived from Nil-treated stromal cells (Nil-CM) depicted delayed and aggressive BPH epithelial cell proliferation with increased clonogenicity of BPH epithelial cells. Further, decreased AR levels with increased cMyc transcripts and pAkt levels also validated the clonogenic transformation under the paracrine influence of inhibition of stromal-AR. Moreover, the CM of stromal-AR activation imparted positive regulation of basal/progenitor pool through LGR4, ß-Catenin, and ΔNP63α expression. Hence, the present study highlighted the restricted disease progression and retains the basal/progenitor state of BPH epithelial cells through the activation of stromal-AR. On the contrary, AR-independent aggressive BPH epithelial cell growth due to paracrine action of loss stromal-AR directs us to reform AR pertaining treatment regimes for better clinical outcomes.

Testosterone improves muscle function of the extensor digitorum longus in rats with sepsis.

Among patients with intensive care unit-acquired weakness (ICUAW), skeletal muscle strength often decreases significantly. The present study aimed to explore the effects of testosterone propotionate on skeletal muscle using rat model of sepsis. Male SD rats were randomly divided into experimental group, model control group, sham operation group and blank control group. Rats in experimental group were given testosterone propionate two times a week, 10 mg/kg for 3 weeks. Maximal contraction force, fatigue index and cross-sectional area of the extensor digitorum longus (EDL) were measured. Myosin, IGF-1, p-AKT and p-mTOR levels in EDL were detected by Western blot. Histological changes of the testis and prostate were detected by hematoxylin and eosin staining. We found that maximal contraction force and fatigue index of EDL in experimental group were significantly higher than in model control group. Cross-sectional area of fast MHC muscle fiber of EDL in group was significantly higher than in model control group. The levels of myosin, IGF-1, p-AKT and p-mTOR of EDL in experimental group were significantly higher than in model control group. In addition, no testicle atrophy and prostate hyperplasia were detected in experimental group. In conclusion, these results suggest that testosterone propionate can significantly improve skeletal muscle strength, endurance and volume of septic rats, and the mechanism may be related to the activation of IGF-1/AKT pathway. Moreover, testosterone propionate with short duration does not cause testicular atrophy and prostate hyperplasia in septic rats. Therefore, testosterone propionate is a potential treatment for muscle malfunction in ICUAW patients.

Androgenic regulation of sexually dimorphic expression of RNA binding motif protein 48 in the developing mouse cortex and hippocampus.

To further reveal the molecular mechanism underlying sexual differentiation of the mouse cerebral cortex and hippocampus, we reanalyzed our previous microarray study with Gene Ontology (GO) term enrichment and found that the GO term "RNA binding" was over-represented among the 89 sexually dimorphic candidate genes. Thus, we selected 16 autosomal genes annotated to the term RNA binding and profiled their mRNA expression in the developing male and female mouse cortex/hippocampus. During the first three weeks after birth, sex differences in mRNA levels of Khdrbs2, Nanos2, Rbm48, and Tdrd3 were observed in the mouse cortex/hippocampus. Of these genes, only the female-biased expression of Rbm48 in neonates was abolished by prenatal exposure to testosterone propionate (TP), while postnatal treatment of TP three weeks after birth increased Rbm48 and Tdrd3 mRNA levels in both sexes. Regardless of sex, the postnatal cortex/hippocampus also showed a marked increase in the content of androgen receptor (Ar) and estrogen receptor ß (Esr2), but a decrease in estrogen receptor α (Esr1) and aromatase (Cyp19a1), which might confer the different responses of Rbm48 to prenatal and postnatal TP. Our results suggest that androgen-regulated, sexually dimorphic Rbm48 expression might present a novel molecular mechanism by which perinatal androgens control development of sexual dimorphism in cortical and hippocampal structure and function.

Testosterone propionate can promote effects of acellular nerve allograft-seeded bone marrow mesenchymal stem cells on repairing canine sciatic nerve.

Peripheral human nerves fail to regenerate across long tube implants (>2 cm), and tissue-engineered nerve grafts represent a promising treatment alternative. The present study aims to investigate the testosterone propionate (TP) repair effect of acellular nerve allograft (ANA) seeded with allogeneic bone marrow mesenchymal stem cells (BMSCs) on 3-cm canine sciatic nerve defect. ANA cellularized with allogeneic BMSCs was implanted to the defect, and TP was injected into the lateral crus of the defected leg. The normal group, the autograft group, the ANA + BMSCs group, the ANA group, and the nongrafted group were used as control. Five months postoperatively, dogs in the TP + ANA + BMSCs group were capable of load bearing, normal walking, and skipping, the autograft group and the ANA + BMSCs group demonstrated nearly the same despite a slight limp. The compound muscle action potentials (CMAPs) on the injured side to the uninjured site in the TP + ANA + BMSCs group were significantly higher than that in the ANA + BMSCs group [CMAPs ratio at A: F(3, 20) = 191.40; 0.02, CMAPs ratio at B: F(3, 20) = 43.27; 0.01]. Masson trichrome staining revealed that in the TP + ANA + BMSCs group, both the diameter ratio of the myelinated nerve and the thickness ratio of regenerated myelin sheath were significantly larger than that in the other groups [the diameter of myelinated nerve fibers: F(3, 56) = 13.45; P < .01, the thickness ratio of regenerated myelin sheath: F(3, 56) = 51.25; P < .01]. In conclusion, TP could significantly increase the repairing effects of the ANA + BMSCs group, and their combination was able to repair 3-cm canine sciatic nerve defect. It therefore represents a promising therapeutic approach.

Gonadal hormones affect alcohol drinking, but not cue+yohimbine-induced alcohol seeking, in male and female rats.

Alcohol use disorder (AUD) is a chronic, relapsing disease characterized by maladaptive patterns of alcohol drinking and seeking. Though sex differences exist in the etiology of AUD, much remains to be elucidated concerning the mechanisms underlying sex-related vulnerability to developing excessive alcohol-motivated behavior. While a large body of evidence points to an important role of circulating gonadal hormones in mediating cocaine reinforcement, findings are less consistent with respect to ethanol. Critically, the effects of gonadal hormones on the reinstatement of ethanol seeking, a model of "craving"-like behavior that reveals pronounced sex differences, has not yet been examined. Thus, the goal of the present experiment was to directly compare manipulations of gonadal hormones in male and female rats on ethanol-motivated behavior. Rats received sham or gonadectomy surgery with or without hormone replacement prior to and throughout three weeks of operant ethanol self-administration to determine the effects of chronically high or low gonadal hormone levels on ethanol drinking. Hormone treatment ceased during extinction training, and the effects of an acute injection of either testosterone (in males) or estradiol (in females) on cue+yohimbine-induced reinstatement of ethanol seeking was determined. Separate groups of gonadally-intact female rats went through similar training, but the effects of either the antiestrogen, fulvestrant, the selective estrogen receptor modulator, clomiphene, or the estrogen receptor ß antagonist, PHTPP, on the reinstatement of ethanol seeking were determined. Chronic estradiol replacement produced significant increases in ethanol drinking in female rats, while chronic testosterone significantly decreased ethanol drinking in male rats. Gonadectomy alone only produced modest shifts in drinking towards the opposite-sex pattern, and did not eliminate the robust sex differences that persisted regardless of hormone manipulations. Neither prior chronic nor acute hormone manipulations altered cue+yohimbine-induced reinstatement of ethanol seeking, though blockade of estrogen receptors tended to reduce reinstatement in gonadally-intact females. Overall, our findings indicate that gonadal hormones at least partially mediate, but do not totally account for the sex differences evident in ethanol self-administration, and circulating gonadal hormones have little effect on the reinstatement of ethanol seeking. These results provide a foundation for future studies examining the neuronal mechanisms underlying sex differences in ethanol drinking and seeking.

Effect of low androgen levels on the sulphur dioxide signalling pathway in rat penile corpus cavernosum.

The aim of this study was to investigate the relationship between sulphur dioxide (SO2 ) signalling pathway and the changes in erectile function under low androgen levels. Thirty-six healthy male Sprague Dawley (SD) rats aged eight weeks were randomly divided into androgen replacement group, castration group and sham group. Rats in the androgen replacement group were subcutaneously injected with testosterone propionate at 3 mg/kg every other day postcastration. The maximum intracavernous pressure/mean arterial pressure (ICPmax /MAP) and the relative content of SO2 in the penile corpus cavernosum were measured. The mRNA and protein expressions of aspartate aminotransferase (AAT1 and AAT2), cysteine oxidase (CDO), endothelial nitric oxide synthase (eNOS) and phosphorylation of endothelial nitric oxide synthase (P-eNOS) were detected. ICPmax /MAP, P-eNOS/eNOS and the level of SO2 decreased significantly in the castration group compared to the other groups (p < 0.05). The expressions of mRNA and protein decreased significantly in the castration group compared to the androgen replacement group and the sham group (p < 0.05), while there was no significant difference between the androgen replacement group and sham group. Low androgen levels can inhibit erectile function by downregulating the SO2 signalling pathway.

Evaluation of triclosan in the Hershberger and H295R steroidogenesis assays.

Triclosan (TCS) is an antibacterial widely used in personal care products that exhibits endocrine disrupting activity in several species, with reports of altered thyroid, estrogen and androgen signaling pathways. To evaluate the androgenic mode of action, TCS was evaluated for androgen receptor mediated effects in the Hershberger assay and for altered androgen synthesis in the H295R steroidogenesis assay. In the Hershberger assay, castrated males were dosed by oral gavage for 10 days with corn oil (vehicle) or TCS (50 or 200 mg/kg/day) in the presence or absence of testosterone proprionate (TP, 0.2 mg/kg/day) prior to assessing accessory sex tissues (ASTs) weights. TCS alone or in combination with TP did not alter androgen dependent AST weights. Assessment of serum thyroxine (T4) demonstrated a significant dose-dependent decrease by TCS (50 or 200 mg/kg/day) co-administered with TP and TCS (200 mg/kg) without TP, but no differences in liver or thyroid weights. In the H295R assay, TCS from 0.01 to 10 µM had no effect on testosterone production but TCS at 3 µM and above did induce a significant increase in estrogen production. At 10 µM, TCS produced significant cytotoxicity which confounded the interpretation of the estrogenic effect at that concentration. Thus, TCS had no effect on androgen synthesis or activity in the models used, but did enhance estrogen production and suppress serum T4.

The impact of sex steroid agonists and antagonists on folliculogenesis in the neonatal porcine ovary via cell proliferation and apoptosis.

The objective of the study was to examine the effects of androgen and estrogen agonists or antagonists on the follicle formation, ovarian cell proliferation and apoptosis as well as plasma steroid concentration in neonatal pigs. Piglets were injected with testosterone propionate (TP, 20 mg/kg bw), flutamide (FLU, 50 mg/kg bw), 4-tert-octylphenol (OP, 100 mg/kg bw), ICI 182,780 (ICI, 400 µg/kg bw), methoxychlor (MXC, 100 mg/kg bw) or corn oil (CTR, controls) between postnatal Days 1 and 10 (n = 4/group). Heart blood was collected and ovaries were excised from the 11-day-old piglets. The lower percentage of oocytes within an egg nest and higher ovarian expression of active caspase 3 were found in TP (androgen excess) piglets compared to controls. FLU-induced androgen deficiency decreased the percentage of primordial follicles, increased that of early primary follicles and diminished ovarian cell proliferation. OP-induced estrogen action increased the percentage of primordial and developing follicles as well as cell proliferation. ICI-induced estrogen deficiency decreased the percentage of transitional follicles and ovarian cell proliferation, while increased the percentage of primordial follicles and the abundance of active caspase 3. Treatment with MXC, exhibiting estrogenic, antiestrogenic, and antiandrogenic activities, declined the percentage of developing follicles and cell proliferation. Moreover, the investigated compounds differentially affected plasma steroid level. In conclusion, the present study demonstrated clear effects of TP and FLU during the earliest stages of folliculogenesis in pigs (nest breakdown and follicle assembly), whereas OP and ICI influenced also the subsequent stages of follicle initial recruitment and growth. Therefore, the androgen and estrogen seems to be important for the follicle assembly and follicle growth in neonatal porcine ovaries.

Neonatal programming with testosterone propionate reduces dopamine transporter expression in nucleus accumbens and methylphenidate-induced locomotor activity in adult female rats.

Research in programming is focused on the study of stimuli that alters sensitive periods in development, such as prenatal and neonatal stages, that can produce long-term deleterious effects. These effects can occur in various organs or tissues such as the brain, affecting brain circuits and related behaviors. Our laboratory has demonstrated that neonatal programming with sex hormones affects the mesocorticolimbic circuitry, increasing the synthesis and release of dopamine (DA) in striatum and nucleus accumbens (NAcc). However, the behavioral response to psychostimulant drugs such as methylphenidate and the possible mechanism(s) involved have not been studied in adult rats exposed to sex hormones during the first hours of life. Thus, the aim of this study was to examine the locomotor activity induced by methylphenidate (5mg/kg i.p.) and the expression of the DA transporter (DAT) in NAcc of adult rats exposed to a single dose of testosterone propionate (TP: 1mg/50µLs.c.) or estradiol valerate (EV: 0.1mg/50µLs.c.) at postnatal day 1. Our results demonstrated that adult female rats treated with TP have a lower methylphenidate-induced locomotor activity compared to control and EV-treated adult female rats. This reduction in locomotor activity is related with a lower NAcc DAT expression. However, neither methylphenidate-induced locomotor activity nor NAcc DAT expression was affected in EV or TP-treated adult male rats. Our results suggest that early exposure to sex hormones affects long-term dopaminergic brain areas involved in the response to psychostimulants, which could be a vulnerability factor to favor the escalating doses of drugs of abuse.

Facilitation of sexual behavior in ovariectomized rats by estradiol and testosterone: A preclinical model of androgen effects on female sexual desire.

In the United States and Canada, there are no approved treatments for hypoactive sexual desire disorder in postmenopausal women. Testosterone improves female sexual desire in naturally- and surgically-menopausal women maintained on estrogen replacement therapy, and long-term safety data from randomized placebo-controlled clinical trials has yielded promising results. However, the mechanisms associated with its efficacy are not known, and could be addressed using preclinical rodent models; yet there is no systematic evaluation of the effects of estradiol and testosterone on female rat sexual behavior. The aim of these studies was to assess whether testosterone propionate (TP) facilitates sexual behaviors, particularly appetitive sexual behaviors, in Long-Evans and Wistar ovariectomized (OVX) rats primed with estradiol benzoate (EB). In Experiment 1, Long-Evans OVX rats were treated with Oil (O), 10µg EB+O, O+200µg TP, 10µg EB+500µg progesterone (P), or 10µg EB+200µg TP. In Experiment 2a, Wistar OVX rats were treated with varying doses of EB (2.5, 5, or 10µg) 48h prior, and TP (0, 200, or 400µg) 4h prior to testing in a Latin-Square design. A subset of animals was used in Experiment 2b and treated sequentially with EB (0, 2.5, 5, or 10µg) followed by TP (0, 200, or 400µg, in a Latin-Square design) 48h prior to sexual behavior testing. All tests occurred in the bilevel pacing chamber. Frequencies of female appetitive (hops/darts, solicitations, level changes) and consummatory (lordosis quotient and magnitude) sexual behaviors as well as the number of defensive behaviors towards males were scored. Number of mounts, intromissions and ejaculations from males were also scored. In EB-primed OVX Long-Evans rats, 200µg TP administered 4h prior to testing facilitated hops/darts and lordosis ratings beyond EB alone, and to levels equivalent to EB+P. In contrast, that regimen was not successful in EB-primed OVX Wistar rats. When EB and TP were co-administered 48h prior to testing, 10µg EB+200µg TP significantly increased hops/darts and level changes beyond that observed by 10µg EB alone. In summary, the administration of EB and TP to OVX Long-Evans and Wistar rats facilitates appetitive measures of sexual behavior. Strain differences exist that likely reflect underlying differences in sensitivities to EB, and the EB-primed OVX Long-Evans rat may be useful for studying mechanisms of TP-facilitation of desire due to higher baseline sexual inhibition.

Increasing estrus expression in the lactating dairy cow.

Using an activity monitoring system (AMS) equipped with an accelerometer, 2 experiments were conducted to test the hypotheses that (1) enhancing progesterone before inducing luteolysis or (2) exposing cows to estradiol cypionate (ECP) or testosterone propionate (TP) after luteolysis would increase occurrence and intensity of estrus. Our goal was to determine if more cows could be detected in estrus by an AMS compared with other estrus-detection aids. In experiment 1, cows (n=154) were fitted with both an AMS collar and a pressure-sensitive, rump-mounted device (HeatWatch; HW) and assigned to 3 treatments: (1) no CL + progesterone insert (CIDR) for 5d, (2) CL only, or (3) CL + 2 CIDR inserts for 5d to achieve a range in concentrations of progesterone. Prostaglandin F2α was administered to all cows upon CIDR insert removal or its equivalent. Progesterone concentration up to 72h posttreatment was greatest in CL + 2 CIDR, followed by CL only, and no CL + CIDR cows. Estrus occurred 14 to 28h earlier in no CL + CIDR compared with CL-bearing cows. Estrus intensity was greater for CL + 2 CIDR than for CL-only cows. The AMS and HW detected 70 and 59% of cows defined to be in estrus, respectively. In experiment 2, cows (n=203) were equipped with both an AMS and a friction-activated, rump-mounted patch (Estrotect patch) and assigned to receive 1mg of ECP, 2mg of TP, or control 24h after PGF2α. Concentrations of estradiol 24 and 48h after treatment were greater in ECP cows compared with controls. Estrus expression detected by AMS or patches in cows defined to be in estrus tended to be greater or was greater for ECP compared with controls, respectively. Compared with controls and in response to TP or ECP, estrus occurred 8 to 18h earlier and was of greater intensity for ECP cows, respectively. The AMS and patches determined 73 and 76% of cows defined to be in estrus, respectively. Of cows exposed to the AMS, HW, or patches, 70, 61, and 75%, respectively, were detected in estrus and more than 93% of these subsequently ovulated. In contrast, of the residual cows not detected in estrus, 62 to 77% ovulated in the absence of detected estrus. Only ECP was successful in inducing more expression and intensity of estrus, and proportions of cows detected in estrus exceeded 80%. Given the large proportion of cows equipped with AMS collars ovulating in the absence of estrus, further research is warranted to determine if more pregnancies can be achieved by inseminating those cows not detected in estrus at an appropriate time when PGF2α is administered to induce luteolysis.

Influence of testosterone on phase II metabolism and availability of soy isoflavones in male Wistar rats.

Genistein and daidzein are the main isoflavones in soy. Their potential beneficial or adverse effects in males like the prevention of prostate cancer or the impact on reproductive functions are controversially discussed. Major determinants of their bioactivity are the absorption and biotransformation of isoflavones. In this study, we focused on the influence of testosterone on plasma availability and phase II metabolism of isoflavones. Male Wistar rats, receiving an isoflavones rich diet, were randomized into three groups: Two groups were orchiectomized (ORX) at postnatal day (PND) 80 and treated for 11 days with testosterone propionate (TP) (ORX TP group) or a vehicle (ORX group) after a 7 days lasting hormonal decline. The third group served as control and remained intact. Rats were sacrificed at PND 98. ORX rats had reduced isoflavones plasma levels. Differently regulated mRNA expressions of transporters relevant for transport of phase II metabolites in liver and kidney may be responsible for this reduction, more precisely Slc10a1 and Slc21a1 in kidney as well as Slc22a8 in liver. While main phase II metabolites in intact rats were disulfates and sulfoglucuronides, the amount of sulfate conjugates was significantly diminished by ORX. In accordance with that, mRNA expression of different sulfotransferases was reduced in liver by ORX. The observed effects could be almost restored by TP treatment. In conclusion, testosterone, and likely further androgens, has a huge impact on phase II metabolism and availability of isoflavones by influencing the expression of different sulfotransferases and transporters.

Induction of renal senescence marker protein-30 (SMP30) expression by testosterone and its contribution to urinary calcium absorption in male rats.

The aim of this study was to investigate the involvement of androgen, mainly testosterone, in the expression of renal senescence marker protein-30 (SMP30) in male rats. We found that the renal SMP30 expression was up-regulated by endogenous testosterone stimulation during puberty. Interestingly, androgen-deficient orchidectomized (ORX) rats exhibited lower SMP30 mRNA and protein expression in the kidney, and that was restored by testosterone propionate (TP) replacement. Abrogation of androgen receptor (AR) activity by co-treatment with flutamide abolished testosterone-induced SMP30 expression in the kidney as well as in the NRK52E cells. However, SMP30 expression was unaltered in the liver of ORX rats. We also showed a positive correlation between renal SMP30 expression and plasma testosterone level during the aging process. TP-induced SMP30 expression in ovariectomized (OVX) rats was observed and was an evidence to explain the gender difference of SMP30 levels. Immunofluorescence assay showed that renal SMP30 was specifically expressed in the proximal tubular segments of the kidney. The urinary Ca(2+) level was increased in both ORX and male aging rats. Taken together, our results indicate a novel role of testosterone in regulating SMP30 expression specifically in the kidney to contribute to urinary calcium absorption.

Aromatized testosterone attenuates contextual generalization of fear in male rats.

Generalization is a common symptom of many anxiety disorders, and females are 60% more likely to suffer from an anxiety disorder than males. We have previously demonstrated that female rats display significantly accelerated rates of contextual fear generalization compared to male rats; a process driven, in part, by activation of ERß. The current study was designed to determine the impact of estrogens on contextual fear generalization in male rats. For experiment 1, adult male rats were gonadectomized (GDX) and implanted with a capsule containing testosterone proprionate, estradiol, dihydrotestosterone proprionate (DHT), or an empty capsule. Treatment with testosterone or estradiol maintained memory precision when rats were tested in a different (neutral) context 1day after training. However, male rats treated with DHT or empty capsules displayed significant levels of fear generalization, exhibiting high levels of fear in the neutral context. In Experiment 2, we used acute injections of gonadal hormones at a time known to elicit fear generalization in female rats (e.g. 24h before testing). Injection treatment followed the same pattern of results seen in Experiment 1. Finally, animals given daily injections of the aromatase inhibitor, Fadrozole, displayed significant fear generalization. These data suggest that testosterone attenuates fear generalization likely through the aromatization testosterone into estradiol as animals treated with the non-aromatizable androgen, DHT, or animals treated with Fadrozole, displayed significant generalized fear. Overall, these results demonstrate a sex-dependent effect of estradiol on the generalization of contextual fear.