Effects of testosterone in the VMN on copulation,partner preference,and vocalizations in male rats

The present study tested whether testosterone propionate (TP) implanted in the ventromedial nucleus (VMN) of the hypothalamus could initiate performance, motivational, or sociosexual components of sexual behavior in castrated male rats. Twenty-seven intact male Long Evans rats were pretested for copulation, partner preference, and 50-kHz vocalization and were subsequently castrated. Approximately 3 weeks after castration, males were retested to confirm that these behaviors had declined, and groups were assigned. Groups 1 and 2 were implanted with bilateral stainless steel cannulae directed at the VMN that were either filled with TP (TVMN group) or remained empty (Blank group). A third group (TSC) was implanted subcutaneously with two 10-mm Silastic capsules filled with testosterone. Restoration of behavior was measured for 2 weeks after implants. We found that copulation and 50-kHz vocalization were not restored by TP in the VMN alone. However, partner preference returned to preoperative levels in both the TVMN and TSC groups, indicating that TP in the VMN was sufficient to restore sexual motivation. Following behavioral testing, prostate glands and seminal vesicles were weighed and confirmed that TP did not leak into the periphery in the TVMN group. Immunostaining for androgen receptors also verified that TP spread was confined to the immediate area surrounding the cannula tip. These results suggest that androgen activation at the VMN is sufficient to induce the motivational components of male sexual behavior, whereas activation of other brain sites is required for copulation and ultrasonic vocalization.     

Comparing the relative amount of testosterone required to restore sexual arousal, motivation, and performance in male rats

It is well established that male rat reproductive behaviors including sexual arousal, motivation, and performance are dependent on circulating levels of testosterone (T). The present study was designed to (1) compare the relative amount of T required to restore these different aspects of behavior in castrated rats, and (2) create an animal model for clinical populations with sexual impairments. Twenty-nine male Long–Evans rats were tested before and after castration for sexual performance (copulation), motivation (partner preference), and arousal (50 kHz ultrasonic vocalizations; measured together with scent marking). Sexual arousal was also inferred from copulation data. Rats were then assigned to one of four groups, and T was re-introduced via Silastic capsule implants varying in length and content: No T (empty capsules), Low T (2 mm capsules), Medium T (5 mm capsules), or High T (two 10 mm capsules). The highest dose was intended to restore physiological levels. Results indicate that High T is required for 50 kHz vocalizations, while Medium T was sufficient for the restoration of copulation, partner preference, and scent marking. These data suggest that sexual arousal may be most sensitive to reductions in testosterone. The role of T levels in measures of generalized and specific (sexual) arousal is discussed in the context of other reproductive behaviors. Furthermore, because the Low T group showed impairments across all behaviors during post-implant tests, we propose that these animals may provide a good animal model for studying clinical conditions marked by reduced motivation and arousal, including Hypoactive Sexual Desire Disorder.     

Anabolic androgenic steroid affects social aggression and fear-related behaviors in male pair-housed rats

This study examines the effect of chronic administration of the anabolic androgenic steroid nandrolone decanoate (ND) on dominant and subordinate male rats in a pair-housed condition. Pair-housed rats were assessed for dominance status based on their behavior and alterations in body weights. Throughout the study the rats were allowed limited social interactions on a daily basis. At all other times, a Plexiglas divider kept the rats separated, allowing olfactory and visual contact between the cage mates while preventing significant physical contact. One week into the study all subjects were subcutaneously implanted with a pellet that continuously infused either ND (15 mg/kg/day) or placebo for 21 days. Following the pellet implant, behavioral tests including reassessment for dominance status, and a conditioned fear test were conducted over a period of approximately 2 months to investigate possible long-term changes. The main finding is that during the allowed social interactions, the dominant ND-pretreated rats spent more time on highly aggressive behaviors than the dominant placebo-treated rats. In addition, the probability for highly aggressive behaviors was maintained for the ND-treated rats throughout the study, whereas it was decreased for the placebo-treated rats. The ND-treated subordinate rats showed less fear in a potential threatening situation compared to placebo-treated controls. These findings support the relatively long-term behavioral changes that have been seen in humans after abuse of ND and other anabolic androgenic steroid compounds.     

Evidence for mobile phone radiation exposure effects on reproductive pattern of male rats: Role of ROS

The relationship between radiofrequency electromagnetic fields emitted from mobile phone and infertility is a matter of continuing debate. It is postulated that these radiations may affect the reproduction pattern spell by targeting biochemistry of sperm. In an attempt to expedite the issue, 70 days old Wistar rats (n = 6) were exposed to mobile phone radiofrequency (RF) radiation for 2 h per day for 45 days and data compared with sham exposed (n = 6) group. A significant decrease (P < 0.05) in the level of testosterone and an increase in caspase-3 activity were found in the RF-exposed animals. Distortions in sperm head and mid piece of sperm mitochondrial sheath were also observed as captured by Transmission Electron Microscope (TEM). In addition, progeny from RF-exposed rats showed significant decreases in number and weight as compared with that of sham-exposed animals. A reduction in testosterone, an increase in caspase-3, and distortion in spermatozoa could be caused by overproduction of reactive oxygen species (ROS) in animals under mobile phone radiation exposure. Our findings on these biomarkers are clear indications of possible health implications of repeated exposure to mobile phone radiation.     

Spilanthes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects on sexual behavior in male rats

According to Indian Systems of Medicine, Spilanthes acmella (L.) Murr. (Family - Asteraceae), is considered effective in the treatment of sexual deficiencies especially due to ageing. In the present study, characterization of ethanolic extracts of the Spilanthes acmella flower and its effect on general mating pattern, penile erection and serum hormone levels of normal male Wistar albino rats were investigated and compared with sildenafil citrate. In vitro nitric oxide release was also investigated in human corpus cavernosum cell line. As N-alkylamides are a promising group, their profiling was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS¹ and MS² fragmentation data were used for identification purposes. For assessment of sexual behavior, animals were divided into five groups of eight male rats. The extracts (50, 100 and 150 mg/kg body weight/day) and sildenafil citrate (5 mg/kg body weight/day) (positive control) were administered orally for 28 days. The behavioral and sexual parameters were observed at days 0, 15, 28 and after a lapse of 7 and 14 days of discontinuance of drug treatment. Five N-isobutylamides, one 2-methylbutylamide and one 2-phenylethylamide were identified. The orally administered extract had a dose dependent positive effect on mounting frequency, intromission frequency and ejaculation frequency and the most significant effects (p < 0.05) were observed at 150 mg/kg treatment, even after a lapse of 7 and 14 days of discontinuance of drug treatment. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. With 150 mg/kg of ethanolic extract the values for FSH, LH and testosterone were 3.10 ± 0.25 mlU/ml, 6.87 ± 0.18 mlU/ml and 3.72 ± 0.12 ng/ml, respectively. In vitro nitric oxide release was 21.7 ± 2.9 μM, which was significantly higher compared to the control group (p < 0.01). Sildenafil citrate exhibited also a significant effect on NO release, but no effect on hormone levels of rats was observed. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in vitro and in vivo. N-Alkylamides might attribute to the improved sexual potential. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent.     

Effects of testosterone metabolites on copulation, medial preoptic dopamine, and NOS-immunoreactivity in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. In a previous study, we identified 17beta-estradiol (E(2)) as the metabolite of testosterone (T) that maintains MPOA basal extracellular DA levels. However, the presence of dihydrotestosterone (DHT), an androgenic metabolite of T, is required for the female-induced increase in MPOA DA observed during copulation. Recently, we reported that assays of MPOA tissue DA content showed that castrates actually had more stored DA than did gonadally intact males. Therefore, the reduction in extracellular levels in castrates was not due to decreased availability of DA; most likely it was due to decreased release. Furthermore, T upregulates neuronal nitric oxide synthase (nNOS) in the MPOA. NO has been implicated in the regulation of DA release in the MPOA. It is not known, however, which metabolite(s) of T regulate(s) tissue stores of DA and/or nNOS in the MPOA of male rats. The present experiments were designed to test the following: (1) whether E(2), DHT, or the combination of the two influences MPOA DA tissue levels, an indication of stored DA, in male rat castrates; and (2) whether E(2), DHT, or the combination of the two influences NOS-ir in the MPOA of castrated male rats. The results indicate that E(2) up-regulates nNOS-ir in the MPOA and maintains tissue content of DA at levels similar to those in T-treated rats. DHT did not influence nNOS-ir, while attenuating the effect of castration on tissue DA content.     

Effects of dietary selenium and vitamin E concentrations on phospholipid hydroperoxide glutathione peroxidase expression in reproductive tissues of pubertal maturing male rats

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is the second intracellular selenium (Se)-dependent glutathione peroxidase (GSH-Px) identified in mammals. Our objectives were to determine the effect of dietary vitamin E and Se levels on PHGPX activity expression in testis, epididymis, and seminal vesicles of pubertal maturing rats, and the relationship of PHGPX expression with testicular development and sperm quality. Forty Sprague-Dawley male weanling rats (21-d old), were initially fed for 3 wk a torula yeast basal diet (containing 0.05 mg Se/kg) supplemented with marginal levels of Se (0.1 mg/kg as Na₂SeO₃) and vitamin E (25 IU/kg as all-rac-α-tocopheryl acetate). Then, rats were fed the basal diets supplemented with 0 or 0.2 mg Se/kg and 0 or 100 IU vitamin E/kg diet during the 3-wk period of pubertal maturing. Compared with the Se-supplemented rats, those fed the Se-deficient diets retained 31, 88, 67, and 50% of Se-dependent GSH-Px activities in liver, testis, epididymis, and seminal vesicles, respectively. Testes and seminal vesicles had substantially higher (5-to 20-fold) PHGPX activity than liver. Dietary Se deficiency did not affect PHGPX activities in the reproductive tissues, but reduced PHGPX activity in liver by 28% (P < 0.0001). Dietary vitamin E supplementation did not affect PHGPX activity in liver, whereas it raised PHGPX activity in seminal vesicles by 43% (P < 0.005). Neither dietary vitamin E nor Se levels affected body weight gains, reproductive organ weights, or sperm counts and morphology. In conclusion, expression of PHGPX activity in testis and seminal vesicles was high and regulated by dietary Se and vitamin E differently from that in liver.     

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.     

Short- and long-term effects of immunization against gonadotropin-releasing hormone, using Improvac, on sexual maturity, reproductive organs and sperm morphology in male pigs

The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac™ Pfizer Ltd.), on sexual maturity, development of the reproductive organs, and the morphology of caudal epididymal spermatozoa in non-castrated male pigs. The pigs were slaughtered 4, 16 or 22 weeks after the second Improvac™ vaccination. A total of 80 crossbred non-castrated male pigs were included in this study comprising two experiments, a short-effect (Experiment 1) and a long-effect (Experiment 2). The first experiment included 56 pigs, 24 of them were maintained as controls and 32 were vaccinated twice, and slaughtered 4 weeks after the second vaccination. The second experiment included 24 pigs, 12 controls and 12 vaccinated twice, and slaughtered either 16 weeks (n = 6) or 22 weeks (n = 6) after the second vaccination. None of the immunized pigs was sexually mature at slaughter, i.e. 4, 16 or 22 weeks after second vaccination. Corresponding results of the control pigs showed that 50% had reached sexual maturity at the age corresponding to 4 weeks after the second vaccination, and 100% at slaughter 16, respectively, 22 weeks after vaccination. At 4, 16 and 22 weeks after second vaccination both testes weight and bulbourethral length were significantly reduced (p < 0.001). The percentages of proximal droplets and abnormal heads were significantly lower in the control pigs than in the immunized pigs at slaughter 4 weeks after vaccination, whereas distal droplets were higher. For the other morphological parameters no significant differences were seen, but all mean values except for acrosome defects were numerically lower in the control pigs compared with the immunized pigs. For pigs slaughtered 16 or 22 weeks after vaccination, the vaccination effect was significant for percentages of proximal droplets, distal droplets, acrosome defects, acrosome abnormality and abnormal heads (p = 0.017-0.001). The immunization clearly disrupted the number and morphology of the interstitial Leydig cells, lasting throughout the study period (4-22 weeks after vaccination). Spermatogenesis was also clearly affected in the immunized pigs, to various degrees, from mild disruption (spermatocyte loss, decrease of the normal number of layers of germ cells) to severe loss of germ cells including tubuli with Sertoli cells-only (complete disappearance of germ cells), also covering the entire study period. The results indicated that the effect of immunization persisted for at least 22 weeks after the second vaccination.     

Low level lead (Pb) exposure during gestation and lactation: assessment of effects on pubertal development in Fisher 344 and Sprague-Dawley female rats

Studies using both Fisher 344 and Sprague-Dawley (SD) rat lines have shown that gestational and/or lactational maternal lead (Pb) exposure causes delayed reproductive maturation in their respective female offspring. Because these studies utilized different experimental regimens for dosing and for monitoring Pb levels, it has not been possible to determine which rat line provides the best model for low level Pb toxicity studies. This study was designed to address this issue. Adult Fisher and SD female rats were dosed with either a solution of PbAc containing 12 mg of Pb/ml or sodium acetate (NaAc) for controls. Dosing began 30 days prior to breeding and continued until their pups were weaned at 21 days of age. At the time of breeding and through weaning the blood lead (BPb) levels in the Fisher dams averaged 37.3 microg/dl and the SD dams averaged 29.9 microg/dl. Pb delayed the timing of puberty (p < 0.01) in Fisher offspring, and suppressed serum levels of luteinizing hormone (LH, p < 0.001) and estradiol (E2, p < 0.01). These effects did not occur in the SD offspring. Doubling the dose given to the SD rats increased their BPb levels to 62.6 microg/dl, yet there were still no effects noted. These results indicate that Fisher offspring are more sensitive to maternal Pb exposure with regard to puberty related insults than are SD rats, suggesting that the Fisher line may be a more reliable rodent model to study the effects of low level Pb toxicity.     

Projection from the ventral bed nucleus of the stria terminalis to the retrorubral field in rats and the effects of cells in these areas on mating in male rats versus gerbils

This research identified the rat counterpart of the lateral cell group of the sexually dimorphic area (SDA) found in medial preoptic area (MPOA) gerbil of gerbils. The lateral SDA (lSDA) is critical for mating in male gerbils and contains most of the SDA cells projecting to the retrorubral field (RRF), a projection that is also important for mating. Therefore, to locate the counterpart of the lateral SDA, we traced the inputs to the rat RRF, which were dense in the ventral part of the bed nucleus of the stria terminalis (BST). To determine if the ventral BST or its projection to the RRF affects mating in male rats, we disrupted them bilaterally by placing cell-body lesions bilaterally in the ventral BST or unilaterally there and in the contralateral RRF. We also studied the effects of RRF lesions in both rats and gerbils. Bilateral ventral BST lesions, which left the medial preoptic nucleus intact, produced persistent and severe mating deficits. Disconnecting the ventral BST from the RRF also had long-lasting, but less severe, consequences. RRF lesions produced only temporary mating deficits in rats, but virtually eliminated mating in gerbils. The recovery of mating in rats after RRF, but not ventral BST, lesions, and the intermediate effects of disconnecting these areas from each other suggest that the ventral BST may contain mating-related projection neurons other than those projecting to the RRF or that its RRF-projecting cells send collaterals to another site. In either case, the pedunculopontine tegmental nucleus or raphe nuclei may be involved.