Tomohiro Hamada

Estrogen Receptor α Gene Promoter 0/B Usage in the Rat Sexually Dimorphic Nucleus of the Preoptic Area

The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) is two to four times larger in male rats than in females; however, the mechanism for the establishment of sexual dimorphism and the function of this nucleus is almost unknown. Perinatal estrogen can cause sexual dimorphism via the estrogen receptor alpha (ERalpha). Recently, transgenic rats were generated that express enhanced green fluorescent protein (EGFP) under the control of the ERalpha gene promoter 0/B to tag ERalpha-positive neurons in the brain. In the present study, we examined whether this EGFP expression could be a marker for the SDN-POA in adults. EGFP-labeled cells were distributed in the core of the SDN-POA (0/B-SDN) of male and female transgenic rats, in accordance with the Nissl staining and immunoreactivity for the SDN marker, calbindin. They were also immunoreactive for ERalpha. The core was bigger in volume and contained more 0/B-SDN neurons in males than in females. The EGFP-tagged cells were packed more densely in the female core than that in males. Subcutaneous injection of 100 mug 17beta-estradiol to females on the day of birth, or orchidectomy of male neonates, reversed the sexually dimorphic phenotype of the volume of the 0/B-SDN, despite not affecting the cell number. We suggest that this EGFP expression in the SDN-POA could be a useful marker to clarify the sexual differentiation and function of the SDN-POA. Moreover, the ERalpha gene promoter 0/B plays a key role in the organization of the sexual differentiation of the SDN-POA.

Site-specific regulation of gene expression by estrogen in the hypothalamus of adult female rats.

Estrogen plays critical roles in the neuroendocrine system of adult female rats through separate actions, respectively, in the preoptic area (POA) and the ventromedial nucleus of the hypothalamus (VMH). Seven-week-old rats were treated with/without estrogen after they were ovariectomized, and four estrogen-responsive, neuronal system-related genes, encoding alpha4 neuronal nicotinic acetylcholine receptor (Chrna4), GABA(A) receptor delta (Gabrd), serotonin receptor 6 (Htr6), and GABA transporter 2 (Slc6a13), were investigated by real-time RT-PCR and Western blot analyses to examine their differential regulation by estrogen between the anterior part containing POA and the posterior part containing VMH. We further examined Bax, Bcl2, and Prkce, the former two genes to be involved in the gene expression network of Chrna4 and the latter gene, that of Gabrd. The regulation of Bax and Bcl2 by estrogen differed between the anterior and posterior parts. The results demonstrated differential regulation of these neuronal system-related genes by estrogen between the anterior and posterior parts of the hypothalamus and suggested the roles of gene expression networks for the respective genes in the neuroendocrine system of adult female rats.

Estrogen-induced cell signaling in the sexually dimorphic nucleus of the rat preoptic area: Potential involvement of cofilin in actin dynamics for cell migration

Estrogen is a key factor to induce the sexually dimorphic nucleus (SDN) in the preoptic area (POA) of the rat brain. Identification of estrogen-dependent signaling pathways at SDN in POA during the critical period is a prerequisite for elucidating the mechanism. In the present study, we treated female rats with/without 17β-estradiol (E2) at birth, designated as postnatal day 1 (P1), and prepared total RNA from brain slices containing SDN for DNA microarray analysis. Among the estrogen-responsive genes identified, protein kinase C-delta (PKC-δ) was significantly up-regulated by E2 at P5. We examined the downstream effectors of PKC-δ protein by Western blotting and found an E2-induced PKC-δ/Rac1/PAK1/LIMK1/cofilin pathway. In the pathway, E2 suppressed the phosphorylation (inactive form) of cofilin. This result was supported by immunohistochemistry, where the phosphorylation/dephosphorylation of cofilin occurred at SDN, which suggests that cell migration is a cue to create sexual dimorphism in POA.

Estrogen modulates voltage dependent potassium currents in the EGFP-tagged neurons in the hippocampus of estrogen receptor gene promoter transgenic rats

Gamma-1 ( -1) is a protein identified as a small subunit of voltagedependent calcium channel of skeletal muscles. At least seven other homologous proteins are expressed in mammalian brain. Gamma-2 (stargazin), -3, -4 and -8 were shown to associate with AMPA receptors and designated as transmembrane AMPA receptor regulatory proteins, however, it has not been identified which proteins are associated with -5 and -7. We generated specific antibodies to -5 and -7 and studied their biochemical properties and expression in rodent brain. We found that both -5 and -7 proteins were N-glycosylated and were highly phosphorylated in brain. AMPA receptors were coimmunoprecipitated both with -5 and with -7 from synaptosomal proteins solubilized with either CHAPS or digitonin. No specific association was observed in Triton X-100. These results suggested that -5 and -7 may also play some regulatory roles in expression or function of AMPA receptors.