Russell D. Romeo

Puberty and the maturation of the male brain and sexual behavior: recasting a behavioral potential.

The pubertal transition from the juvenile to adult state requires significant changes in behavior to meet the demands for success and survival in adulthood. These behavioral changes during puberty must be mediated by changes in the structure and/or function of the central nervous system. Despite the profound consequences of puberty on an animals behavioral repertoire, the mechanisms underlying pubertal maturation of the nervous system remain largely unknown. In this review, we provide a synthesis of neural development during puberty as it relates to maturation of male reproductive behavior. We first outline neuroendocrine events associated with puberty and review work from our laboratory that identifies pubertal changes in the neural substrate controlling male reproduction by comparing the neural responses of prepubertal and adult males to steroids and female chemosensory cues. We then raise the question of whether puberty is a sensitive period in which gonadal hormones influence the structural and functional organization of neural circuits underlying male reproductive behavior. The central thesis of this review is that the development of the nervous system during puberty alters the way in which the male responds to social stimuli, involving the restructuring of neural circuits that integrate steroidal and sensory information and ultimately mediate steroid-dependent social behaviors in adulthood.

Actions of Testosterone in Prepubertal and Postpubertal Male Hamsters: Dissociation of Effects on Reproductive Behavior and Brain Androgen Receptor Immunoreactivity☆

This study was conducted to determine whether there is a increase in responsiveness to the activating effects of testosterone on male reproductive behavior during puberty in male golden hamsters and whether responsiveness to behavioral actions of testosterone is correlated with the ability of testosterone to upregulate brain androgen receptor immunoreactivity (AR-ir). Sexually naive male hamsters were castrated at 21 or 42 days of age and implanted subcutaneously with a pellet containing 0, 2.5, or 5 mg of testosterone. One week later, males were given a 10-min mating test with a receptive female. Animals were euthanized 1 hr after the behavioral test, and blood samples and brains were collected. Plasma testosterone levels were equivalent in prepubertal and adult males that had been administered the same dose of testosterone. However, adult males exhibited more mounts, intromissions, and ejaculations than prepubertal males, demonstrating that postpubertal males are more responsive than prepubertal males to the effects of testosterone on sexual behavior. In both age groups, testosterone increased the number of AR-ir cells per unit area in several brain regions involved in male sexual behavior, including the medial preoptic nucleus (MPN), medial amygdala, posteromedial bed nucleus of the stria terminalis, and magnocellular preoptic nucleus (MPNmag). Surprisingly, testosterone increased AR-ir in the latter three regions to a greater extent in prepubertal males than in adults. Thus, prepubertal males are more responsive to the effects of testosterone on AR-ir in these regions. In a separate experiment, a pubertal increase in the number of AR-ir cells per unit area was found in both the MPN and MPNmag of intact male hamsters. These results indicate that a testosterone-dependent increase in brain AR during puberty may be necessary, but is not sufficient, to induce an increase in behavioral responsiveness to testosterone.

Pubertal and seasonal plasticity in the amygdala

The present experiments investigated the effects of pubertal maturation and photoperiod on the size of brain regions that mediate mating behavior in the male Syrian hamster. We hypothesized that the low levels of reproductive behavior exhibited by prepubertal and photoinhibited males would be correlated with morphological changes in the neural circuit that mediates mating behavior. We found that the Nissl-stained cross-sectional area of the posterodorsal subdivision of the medial amygdala was significantly smaller in prepubertal and photoinhibited males compared to photostimulated adult males. These differences appear to be caused by a decrease in somal size of individual cells in the ventral aspect of this nucleus. We also found that prepubertal males have a larger anterior subdivision of the medial amygdala (MeA) compared to adults. This difference in the MeA does not appear to be caused by alteration in somal size since somal size did not differ significantly between juveniles and adults. It is concluded that the neural circuit that mediates male mating behavior in this species is capable of significant morphological plasticity during both pubertal development and in adulthood. Furthermore, these alterations may reflect underlying mechanisms of the deficits in sexual behavior exhibited by prepubertal and photoinhibited males.

Pheromones Elicit Equivalent Levels of Fos-Immunoreactivity in Prepubertal and Adult Male Syrian Hamsters ☆

Male reproductive behavior in the Syrian hamster is dependent on both pheromones from the female and the presence of gonadal steroid hormones. The pheromones are contained within female hamster vaginal secretions (FHVS) and stimulate anogenital investigation and mounting by the male. Administration of testosterone to castrated male hamsters facilitates anogenital investigation, mounts, and intromissions in adults, but elicits only anogenital investigation in prepubertal males. One hypothesis for why the full complement of reproductive behaviors is not activated by testosterone in prepubertal males is that the neural processing of pheromonal cues encountered during anogenital investigation is different in juveniles and adults. In the present experiment, we investigated the influence of sexual maturity on Fos expression in response to FHVS in the male Syrian hamster. We predicted a greater increase in Fos-immunoreactivity after exposure to FHVS within the neural circuit mediating male reproductive behaviors in adult compared to prepubertal males. Intact adult and prepubertal males were exposed to either a clean cotton swab or a swab containing FHVS. We found that, compared to animals exposed to a clean cotton swab, both prepubertal and adult males exposed to FHVS have a greater amount of Fos-immunoreactivity within several brain nuclei comprising the neural circuit mediating male reproductive behavior. Furthermore, this Fos response was equivalent in the two age groups. These results suggest that the inability of the prepubertal male hamster to perform the full repertoire of male reproductive behaviors is not due to a lack of a neuronal activation in response to the pheromonal cues present in FHVS.

Effects of gonadal steroids during pubertal development on androgen and estrogen receptor-α immunoreactivity in the hypothalamus and amygdala

Perinatal development is often viewed as the major window of time for organization of steroid-sensitive neural circuits by steroid hormones. Behavioral and neuroendocrine responses to steroids are dramatically different before and after puberty, suggesting that puberty is another window of time during which gonadal steroids affect neural development. In the present study, we investigated whether the presence of gonadal hormones during pubertal development affects the number of androgen receptor and estrogen receptor alpha-immunoreactive (AR-ir and ER alpha-ir, respectively) cells in limbic regions. Male Syrian hamsters were castrated either before or after pubertal development, and 4 weeks later they received a single injection of testosterone or oil vehicle 4 h prior to tissue collection. Immunocytochemistry for AR and ER alpha was performed on brain sections from testosterone-treated and oil-treated males, respectively. Adult males that had been castrated before puberty had a greater number of AR-ir cells in the medial preoptic nucleus than adult males that had been castrated after puberty. There were no significant differences in ER alpha-ir cell number in any of the brain regions examined. The demonstration that exposure to gonadal hormones during pubertal development is associated with reduced AR-ir in the medial preoptic nucleus indicates that puberty is a period of neural development during which hormones shape steroid-sensitive neural circuits.

Medial preoptic area dopaminergic responses to female pheromones develop during puberty in the male Syrian hamster.

Chemosensory cues from receptive females do not elicit similar reactions before and after puberty in male hamsters. While pheromones facilitate a complex display of reproductive behavior in adults, prepubertal males do not engage in these same behaviors. Dopamine (DA) released from the medial preoptic area (MPOA) in response to a receptive female or her odors is an important component of the neural events underlying adult male rat sexual behavior. The current experiment investigated whether increased dopaminergic activity occurs in the adult male hamster MPOA in response to female pheromones, and if so, whether this response is absent in prepubertal males, which do not mate. Sexually nai;ve prepubertal and adult male hamsters were exposed to cotton swabs with or without pheromone from an estrous female for 0, 5, 15, or 25 min, after which brains were collected and frozen on dry ice. The MPOA was micropunched from frozen coronal sections (500 microm), and concentrations of DA and its primary metabolite DOPAC were determined by high-performance liquid chromatography-electrochemical detection. DOPAC was used as an index of dopaminergic activity. DOPAC levels significantly increased in adults after 15 min exposure to pheromone. In contrast, MPOA DOPAC concentrations did not increase in prepubertal males exposed to pheromone. These data demonstrate that the neural processing of sexually relevant chemosensory stimuli matures during puberty. The absence of a DA response to female pheromones prior to puberty may contribute to the inability of prepubertal males to display reproductive behavior.

Dihydrotestosterone activates sexual behavior in adult male hamsters but not in juveniles

The effect of an androgenic metabolite of testosterone, dihydrotestosterone (DHT), on reproductive behavior and brain androgen receptor (AR) immunoreactivity was compared in juvenile and adult male Syrian hamsters. Prepubertal and adult animals were castrated and treated with 0, 500, or 1000 microg of DHT daily for 1 week and then tested for their ability to engage in mating behavior. The 1000-microg dose of DHT activated intromissions in adult but not prepubertal males. Brains were collected immediately after the behavioral test to investigate whether the lack of a behavioral response to DHT prior to puberty is associated with fewer AR-immunoreactive (AR-ir) cells in the forebrain nuclei that mediate male sexual behavior. In four of the five nuclei within the behavioral circuit that were examined, the number of AR-containing cells was similar in prepubertal and adult males treated with 1000 microg of DHT. Only in the anterior medial amygdala (MeA) was there a greater number of AR-ir cells in adults. These data indicate that (1) DHT does not activate components of male reproductive behavior prior to puberty and (2) the lack of behavioral responsiveness to DHT in prepubertal males is most likely not related to an overall reduction in ARs within the forebrain circuit that mediates mating behavior.

Estradiol Induces Hypothalamic Progesterone Receptors but Does Not Activate Mating Behavior in Male Hamsters (Mesocricetus auratus) Before Puberty

This study investigated pubertal changes in neural and behavioral responses to estradiol. Gonadectomized pre- and postpubertal male hamsters (Mesocricetus auratus) were treated with 0.00, 0.05, 0.10, or 0.25 mg estradiol and tested 1 week later for sexual behavior with a receptive female. Estradiol activated behavior in postpubertal, but not prepubertal, males. In contrast, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) immunoreactivity in forebrain nuclei that mediate mating behavior was similar in pre- and postpubertal males. Thus, absence of a behavioral response before puberty is not associated with reduced levels of steroid receptors. Because estradiol induced PR in prepubertal males, these data also suggest that ERa is functional before puberty. Therefore, gonadal steroids facilitate male reproductive behavior only after as-yet-unidentified developmental processes occur during puberty.

GnRH mRNA Increases with Puberty in the Male Syrian Hamster Brain

Puberty in the male Syrian hamster (Mesocricetus auratus) is characterized by decreased responsiveness to testosterone mediated negative feedback, but the neural mechanism for this change remains elusive. We hypothesized that decreased inhibition of the gonadotropin‐releasing hormone (GnRH) system results in increased neurosecretory activity, which includes an increase in GnRH gene expression. This study examined GnRH mRNA in male hamsters before and after puberty, and sought to determine if any increase in mRNA was specific to particular subpopulations of GnRH neurones. Brains were collected from 21‐day‐old prepubertal males (n=5) and 56‐day‐old postpubertal males (n=5). Alternate 10 μm coronal sections from fresh‐frozen brains were collected throughout the septo‐hypothalamic region, and 25% of those sections were processed for in‐situ hybridization histochemistry using an 35S‐riboprobe complementary to hamster GnRH. No differences were observed in the number of GnRH mRNA expressing cells in any region, but in the diagonal band of Broca (DBB)/organum vasculosum of the lamina terminalis (OVLT) there was a significant increase in labelling intensity (defined as area of the cell occupied by silver grains) in postpubertal males. A second analysis compared the frequency distributions of cells based on labelling intensity between prepubertal and postpubertal males. This analysis revealed significant differences between the two frequency distributions in all areas analysed (DBB/OVLT, medial septum (MS), and preoptic area (POA)). Furthermore, examining the distribution of cells in these regions revealed a shift to the right in the postpubertal population of cells, which indicated an increased number of GnRH neurones with greater labelling intensity. These data clearly demonstrate increased GnRH mRNA during puberty. Furthermore, they suggest that the previous observation of brain region specific pubertal decreases in GnRH‐immunoreactivity only within the DBB/OVLT and MS but not the POA are not due to differential levels of GnRH gene expression, but could indicate increased release from these neurones during puberty.

Increased expression of forebrain GnRH mRNA and changes in testosterone negative feedback following pubertal maturation.

Pubertal development is associated with increased activity of the gonadotropin releasing hormone (GnRH) neuronal system and rising gonadal steroid levels. The purpose of this study was to determine whether different circulating levels of testosterone affect GnRH mRNA and luteinizing hormone (LH) to the same degree prior to and following pubertal maturation. Pre- and post-pubertal male Syrian hamsters were gonadectomized and treated with timed-release testosterone pellets (0, 0.5, 1.5, or 2.5mg) for one week. Following treatment, three separate brain tissue dissections containing the majority of GnRH cell bodies, tenia tecta and medial septum (TT/MS), diagonal band of Broca/organum vasculosum of the lamina terminalis (DBB/OVLT), and preoptic area (POA), were analyzed for GnRH mRNA levels by RNase protection assay and terminal plasma luteinizing hormone concentrations were determined by radioimmunoassay. Pre-pubertal animals were more sensitive to testosterone negative feedback on LH. Conversely, the ability of testosterone to reduce GnRH mRNA was much greater after pubertal development. Specifically, GnRH mRNA in the TT/MS was considerably higher in adults, and testosterone reduced GnRH mRNA in a dose-dependent manner only in adults. These data indicate that although testosterone is a powerful suppressor of LH release before puberty, it does not have appreciable control over GnRH mRNA until after puberty. Furthermore, the pubertal increase in GnRH mRNA appears to occur via steroid feedback-independent mechanisms in the male Syrian hamster.