Sexually dimorphic estrogen receptor alpha mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards.

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERalpha) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERalpha mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.

Sex and seasonal differences in morphology of limbic forebrain nuclei in the green anole lizard.

Sex and seasonal differences in the brain occur in many species and are often related to behavioral expression. For example, morphology of limbic regions involved in male sex behavior are larger in males than in females, and sometimes are larger in the breeding than non-breeding season. Morphology can often be altered in adulthood by manipulating levels of steroid hormones. In untreated green anole lizards, previous work indicated that neuron soma size and density did not differ between the sexes in the preoptic area (POA) or ventromedial nucleus of the amygdala (AMY), two brain regions involved in the control of male reproductive behaviors [O'Bryant, E.L., Wade, J., 2002. Seasonal and sexual dimorphisms in the green anole forebrain. Horm. Behav. 41, 384-395.]. However, soma size was larger in both areas in breeding than non-breeding animals. The current study examined sex and seasonal differences in estimated brain region volume and total neuron number in the POA, AMY, and the ventromedial hypothalamus (VMH), a region typically involved in female reproductive behaviors. The volume of the POA was larger in males, and the POA and VMH of breeding animals were larger than those of non-breeding individuals. Differences in cell number did not exist in either of these two regions. In contrast, neuron counts in the AMY were greater in non-breeding than breeding animals, but the volume did not differ between the seasons. These data suggest that the structure of limbic brain regions is dynamic in adulthood and that parallels between morphology and the expression of masculine behavior exist for the POA, whereas other relationships are more complicated.

Steroid receptor expression in the developing copulatory system of the green anole lizard (Anolis carolinensis).

In adulthood, the copulatory system in male green anole lizards is characterized by the presence of two hemipenes, each controlled by ipsilateral muscles. These structures are present in both sexes early in development, but prior to hatching regress completely in females. Embryonic treatment with steroid hormones alters the morphology of the copulatory system, suggesting active roles for both androgens and estrogens in sexual differentiation. To elucidate the timing and sites of steroid hormone action in the embryonic copulatory system, the distributions of androgen receptor (AR) and estrogen receptor-alpha (ER alpha) mRNA expression were examined. In situ hybridization was conducted on the rostral tail of anoles at three stages spanning differentiation of the copulatory structures: embryonic days (E) 13, 18, and 24 (hatching occurs at approximately E34). At E13, males expressed significantly higher levels of AR mRNA in both hemipenes and muscles than did females, while females at the same age tended to express higher levels of ER alpha mRNA in these structures. By E18, hemipenes and copulatory muscles were regressed in most females, and were not present in any females at E24. In males, no effect of age was detected on the expression of either AR or ER alpha. These data suggest that peripheral copulatory structures in the embryonic anole are direct targets for the actions of both androgens and estrogens in sexual differentiation, consistent with the idea that estradiol facilitates regression in females and androgen promotes survival in males. However, the issue of whether or not a critical period exists remains open.

Androgen-dependent regulation of brain-derived neurotrophic factor and tyrosine kinase B in the sexually dimorphic spinal nucleus of the bulbocavernosus.

Castration of adult male rats causes the dendrites of androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) to retract. Brain-derived neurotrophic factor (BDNF), via activation of tyrosine receptor kinase B (trkB), has been implicated in mediating androgen effects on SNB dendrites. We used in situ hybridization to demonstrate that SNB motoneurons in gonadally intact adult male rats contain mRNA for both BDNF and trkB. Two weeks after gonadectomy, both transcripts were significantly decreased in SNB motoneurons but not in the non-androgen-responsive motoneurons of the adjacent retrodorsolateral nucleus (RDLN). In a second experiment, target perineal and foot muscles of SNB and RDLN motoneurons, respectively, were injected with the retrograde tracer Fluorogold, and then immunocytochemistry was performed to examine the distribution of BDNF and trkB proteins in SNB and RDLN motoneurons and their glutamatergic afferents. Confocal analysis revealed that gonadectomy induces a loss of BDNF protein in SNB dendrites but not in RDLN dendrites. Testosterone treatment of castrates prevented the loss of BDNF from SNB dendrites. Confocal analysis also revealed trkB protein in SNB and RDLN dendrites and in their glutamatergic afferents. Gonadectomy had no discernable effect on trkB protein in SNB or RDLN motoneurons or in their glutamatergic afferents. These results suggest that androgen maintains a BDNF-signaling pathway in SNB motoneurons that may underlie the maintenance of dendritic structure and synaptic signaling.