| ObjectiveTheories concerning the etiogenesis of sexual orientation that focus on the sexual differentiation of the brain have enjoyed growing support. Quite a number of structural and functional sex differences have been reported in the human hypothalamus that may be related to sexual orientation. One of the popular focuses of interest in this respect is the relation between the morphology of sexually dimorphic nucleus of the preoptic area of hypothalamus (SDN-POA) and sexual orientation. Previous findings showed that the neuronal number and neuronal size of the SDN-POA of male rats are greater than that of female rats. Moreover, prenatal or perinatal administration of androgen to female rats would lead SDN-POA to masculinisation (i.e., increase in volume), and therefore reorientated the sexual behaviours and sexual preferences of adult rats.The aim of the present study was to determine the effect of perinatal androgenon the morphology of SDN-POA and distribution of hormone receptor in such nucleus of adult female rats.Materials and Methods1 AnimalsFemale and male Sprague-Dawley rats2 Methods*2.1 ExperimentaTproceduresPostnatal Sprague-Dawley rats were divided into 3 groups, 10 rats in each group. The female treatment group at postnatal day 1 to day 3 was injected (i.p.) with testosterone propionate (TP) (500u,g/50ul/rat);while the female controls and male controls were injected with aseptic oil (50uJ/rat). Then all animals were housed for 90 days in plastic cage and maintained on a 12h:12h lightdark cycle with free access to food and tap water. After 90 days, brains of all animals were obtained at autopsy. Brain were removed from the skull and weighed, followed by about 1 month of fixation in 4% formaldehyde at 5°C. By referring to brain atlas, the hypothalamic area including SDN-POA was dissected using a self-made cutting apparatus, dehydrated and embedded in paraffin. Serial 5-um coronal sections were cut on a Microm microtome and mounted onto sequentially numbered labeled APES-coated slides. Only 4 sections, namely section A, B, C, and D, would be elected from every 10 serial sections. All sections were deparaffined, rehydrated, underwent Nissl staining or immunohistochemical staining (SABC), and cover-slipped. Serial sections were examined on a Nikon ECLIPSE microscope integrated with Nikon DXM digital camera and a Nikon ACT-1 image analysis system. Images obtainedwould be further analyzed using Image Pro-plus software (Mediacybernetics;Carlsbad, CA). The perimeter of SDN-POA was digitized throughout all chosen sections. Cross-sectional area of the SDN-POA was measured and Image Pro-plus software would calculate the positive staining spots within each AOL 2.2 Measurementsa) Neuronal density: each purple spot on slide C represented one neuron;the sum of purple spots within the AOI would give the neuronal number of SDN-POA, and then the neuronal density of SDN-POA would be given by dividing tEe neuronal number to the cross-sectional area of AOI.b) Expression of hormone receptor: hormone receptors on slide A, B, and D were represented by different-graded brown spots;calculating the number of positive cells and total cell number would give the rate of expression of hormone receptor within each AOI.3 StatisticsStatistical significance was assessed using the SPSS 11.0 for Windows. Differences between groups were tested using ANOVA (LCD). A 5% level of significance was used in all statistical tests.Results1 Comparison of neuronal density of SDN-POA of rats among threegroupsThe neuronal density of SDN-POA of controlled male rats was significantly greater than that of controlled female rats (P<0.0l). Compared to controlled female rats, the neuronal density of SDN-POA of treated female rats was significantlygreater (PO.001);while compared to controlled controlled male rats, the neuronal density of SDN-POA of treated female rats was also greater (PO.05).2 Comparison of expression of estrogen receptor in SDN-POA of ratsamong three groupsThe expression of estrogen receptor alpha in SDN-POA of controlled female rats was significantly higher than that of controlled male rats (PO.05). Compared to controlled female rats, the expression of estrogen receptor alpha in SDN-POA of treated female rats was significantly lower (P<0.001);while compared to controlled male rats, the expression of estrogen receptor alpha in SDN-POA of treated female rats was also lower, but no significant difference was found (P>0.05).The expression of estrogen receptor beta in SDN-POA of treated female rats was higher than that of controlled male rats, but lower than that of controlled female rats. However, no significant difference was found among this three groups (P>0.05).3 Comparison of expression of androgen receptor in SDN-POA of ratsamong three groupsThe expression of androgen receptor in SDN-POA of controlled male rats was significantly higher than that of controlled female rats (PO.001). The expression of androgen receptor in SDN-POA of treated female rats was significantly lower, when compared to both male and female controls (PO.001, PO.01).Conclusion1 The present study proved that neurobiological basis, namely brain tissues, isfundamental to the manifestation of male sexual behaviour of homosexual female rats, and the biological variations of the brain can be due to perinatal administration of exogenous androgen. The results of this study have further confirmed the reliability of the present homosexual animal model.2 Our study showed that, the neuronal density of sexually dimorphic nucleus ofthe preoptic area of hypothalamus of homosexual female rats decreased- At the same time, the expression of estrogen receptor alpha was downregulated, but no change was found in the expression of estrogen receptor beta;while the expression of androgen, due to several reasons, also showed downregulation.
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