| Homosexuality is a sexual orientation that is characterized by sexual attraction by individuals of the same sex. In recent years, more and more reports on homosexuality, according to statistics, about4%of the world population is homosexual in a life, about3%-6%of Chinese population is homosexual, thus homosexual population is about36million at present in China. Regarding to mechanism underlying human homosexuality, in addition to the social, family, cultural and psychological factors, more profound biological factors are involved in this behavior, but the biological mechanism underlying this behavior is not entirely clear. In addition to humans with homosexual behavior, fruit flies, monkeys, sheep, mice, cows and other animals also show a homosexual or bisexual behavior, and the percentage of homosexual individuals in these species is much higher than humans. Elucidating the mechanism underlying animals homosexual behavior can shed lights on and provide important information to evolutionary biology, animal physiology, genetics and other fields, and the research can help us to understand the formation of human homosexuality, thus and treated homosexuality correctly.The sexual orientation of animal is associated with genetic factors, hormones, neurotransmitters, receptors and the environment. From a neuroendocrine perspective, the levels of hormone in early life play a crucial role in development of the brain. The estrogen transformed from androgen by aromatase, shapes male brain structure under normal physiological levels associated with male sexual orientation. On the contrary, the low levels of estrogen possibly induce feminization brain associated with female sexual orientation. In previous studies concerning homosexual orientation only non-social animals were used as models. However, it remains unclear whether early hormone levels have a certain effect on sexual-orientation of social animals. Socially monogamous mandarin voles (Microtus mandarinus) show more complex social behavior and close social attachments than other traditional laboratory animals. Mandarin voles, in general, show a significant heterosexual partner preference and typical male or female sexual behavior. The abnormal levels of gonadal hormone during early development definitely affect adult sexual orientation and sexual behavior, so the mandarin vole is a ideal model animal for investigating the mechanism of occurrence of the same sexual orientation behavior. Using this species, a richer neurobiology information will be provided. By knocking-out study of estrogen a, P receptors (ERa, ERβ genes, it is established that ERa mediates male sexual behavior and sexual orientation, ERβ shapes the feminization of behavior. By studying the congenital androgen receptor (AR) anomaly of animal models, it is obtained that AR impacts male sexual behavior and sexual orientation. ERa, ERβ, AR, neuropeptide oxytocin (OT) and vasopressin (AVP) interacte each other and their distribution pattern in the brain regions is sexual dimorphic and region-specific. However, the association between distribution properties of these hormone receptors and neuropeptides, neural activation in social interaction and sexual orientation also remain unclear. Thus, this study can help us to clarify the relationship between these hormone, hormone receptors and the sexual orientation. In addition, whether individuals with homosexual or opposite sexual orientation display different levels of depression, anxiety ability in spatial learning and memory has not been investigated. Effects of human social and cultural factors on the behavior of homosexual orientation can be excluded via using animal model and differences in the emotion, learning and memory between different sexual orientation individuals can also be found by current study. Based on the above propositions, the current study aimed to answer the following questions using socially monogamous mandarin vole:1) whether perinatal estrogen and estrogen receptor antagonist treatment can induce homosexual orientation in adulthood;2) whether the female individuals with the homosexual or heterosexual-orientation show different distributions of ERa, ERβ and OT in the relevant brain regions;3) whether the male individuals with the homosexual or opposite-sexual orientation show different distribution of AR and AVP in the relevant brain regions;4) whether the individuals with the homosexual or opposite-sexual orientation display different levels of depression and anxiety;5) and whether the individuals of the homosexual or opposite-sexual orientation have different ability in spatial learning and memory;6) the whether the expression of OT and Fos, AVP and Fos in brains of individuals with the homosexual or opposite-sexual orientation are different. Following social interaction. Pups in the prenatal7days to postnatal21days were respectively treated:①subcutaneous injection of sesame oil.②subcutaneously injected clomiphene citrate (Clomiphene citrate).③subcutaneous injection of estrogen (P-Estradiol). Treated male and female animals were divided into three groups. Main results are as follows: 1. Female sexual orientation experiments.①female control group (FC)②females treated with clomiphene citrate (FT1)③female treated with estrogen (FT2). The sexual orientation test was conducted until the female at adulthood. The results showed that:the animals from FT2group spent significantly longer time and more frequently sniffing estrous females and anogenital region and flanks of anesthezed female, as well as engaged in more mounting and affiliation with male for longer times, and investigated males for less time. Differently, the animals from FC group spent longer time and more frequently sniffing sexually mature male, and anogenital region and flanks of anesthezed male, and display more lordosis and affiliation with male, but engaged in less mounting. The levels of exploratory behavior of male and female animal from FT1were lower. They also did not showed significant sexual orientation. It is suggested that early estrogen exposure to female contributed to masculinization of the brain and homosexual orientation at adulthood.2. Immunohistochemical results of female with diverse sexual orientation:in FT2, the numbers of estrogen receptor a immune reaction neurons (ERα-IRs) in the preoptic area (mPOA), anterior hypothalamic area (AHA), the bed nucleus of stria terminalis (BNST), the medial amygdaloid nucleus (MeA) were significantly fewer than the animals from FC group, both the animals from FC and FT2groups were significantly more than those from FT1group. ERa-IRs and Immune dual-labeled of ERa and oxytocin (OT) cells (OT/ERa dual-labeled cells) in the nucleus (PVN) and supraoptic nucleus (SON) were significantly fewer than those from FC and FT1. Whereas, the numbers of estrogen β immune reaction neurons (ERβ-IRs) in the AHA, mPOA, BNST and MeA were significantly more than those from FC and FT1groups, the numbers of ERβ-IRs and OT/ERβ dual-labeled cells in the PVN and SON were not significantly different among three groups. Taken together, the various sexual orientation in the females induced by early manipulation may be closely associated with the changes in expression of ERa, ERβ and OT in specific brain regions.3. Male sexual orientation experiments.①male control group (MC)②males treated with clomiphene citrate (MT1)③male treated with estrogen (MT2). Sexual orientation test was conducted until the male at adulthood. The results indicated that:the animals from MT1group spent significantly longer time and more frequently sniffing sexually mature male and anogenital region and flanks of anesthezed male, displayed more lordosis and affiliation, and less mounting. The animals from MC and MT2 groups spent significantly more time and more frequently sniffing estrous and anogenital region and flanks of anesthezed females, showed more mounting and affiliation with female, and less lordosis. It is indicated that early treatment with clomiphene citrate facilitated the feminization of male brain, promoted homosexual orientation.4. Immunohistochemistry results of male with different sexual orientation:In the animals from MT1group, the numbers of immune androgen receptor neurons (AR-IRs) in the preoptic area (mPOA), anterior hypothalamic area (AHA), the bed nucleus of stria terminalis (BNST) in the medial amygdaloid nucleus (MeA), the AR and vasopressin (AVP) immune double-labeled neurons, namely AR/AVP in the nucleus (PVN) and supraoptic nucleus (SON) were significantly fewer than the animals from MC and MT2groups. Therefore, changes in male sexual orientation induced by early treatments may be closely associated with the variation of AR and AVP.5. In the open field test (OF), the animals from FT2engaged in less staying in central area, shorter locomotor distance and the fewer total transitions. We hypothesize that prenatal and postnatal estrogen treatment increased the levels of anxiety-like behavior. Similarly, the animal from MT1group also spent less time in central area, moved shorter distance, crossed lines fewer than the animal from MC group. We hypothesize that prenatal and postnatal treatment with estrogen receptor antagonist affected the normal development of masculine brain and increased the levels of anxiety-like behavior.6. In the tail suspension experiments (TST), the animals from FT2group exhibited significantly longer total duration and more frequency in immobility than the animals from FC group. Similarly, the animals from MT1group also engaged in more immobility than the animals from MC group. These results indicated that male and female with different sexual orientation showed different levels of depression.7. Water maze test:the animals from FC and MT1groups spend significantly more time finding the platform than animals from MC and FT2groups. The animals from FT2and MC groups did not showed significant difference in latency to platform. The results showed that animals with different sexual orientation have different ability in spatial memory.8. Social interaction behavior①The females were divided into six groups:FC-intact male, FC-estrus female, FT1-intact male, FT1-estrus female, FT2-intact male, FT2-estrus female. FC, FT1and FT2were observed respectively in the social interaction test. Compared with other four groups, the subject animals from FC-intact male interaction and FT2-estrus female interaction had more sniffing and affiliation behaviors, and less aggression. The subject animals from FT2-intact male interaction and FT2-estrus female interaction had more mounting and less lordosis behavior than the subject animals from other four groups. The result demonstrated that female with different sexual orientation showed different levels of masculine and defeminization behavior in the social interaction.②The males were divided into six groups:MC-intact male, MC-estrus female, MT1-intact male, MT1-estrus female, MT2-intact male, MT2-estrus female interaction. The animals from MC, MT1and MT2groups were observed respectively in the social interaction test. Compared with other three groups, the subject animals from MC-intact male, MTl-estrus female and MT2-intact male interaction had more aggression, less sniffing and affiliation. The subject animals from MT1-intact male interaction and MT1-estrus female interaction had less mounting behavior and more lordosis than the subject animals from other four groups. The result showed that male with different sexual orientation displayed different levels of feminization and demasculination behavior in the social interaction.9. Changes in serum testosterone (T) and17β-estradiol (E2) concentrations following the social interaction①The levels of serum17β-estradiol (E2) were the highest in the subject animals from FC-intact male interaction, followed by FC-estrus female interaction, FT2-intact male interaction and FT2-estrus female interaction, the lowest was in those from FT1-intact male and FTl-estrus female interaction. The serum testosterone (T) concentrations were the highest in the subject animals from FT2-estrous female interaction, followed by FT2-intact male interaction, those were the lowest from FT1-intact male, FT1-estrus female interaction, FC-intact male and FC-estrus female interaction.②The levels of serum E2were the highest in the subject animals from MT1-intact male interaction, followed by MC-intact male, MT1-estrus female and MT2-intact male interaction, those were the lowest from MC-estrous female and MT2-estrous female. The serum T concentrations were higher in the subject animals from MC-estrous female and MT2-estrous female than other four groups.10. The changes in AVP-IRs, OT-IRs, Fos-IRs in the brain in the social interaction ①The levels of Fos immune reaction neurons (Fos-IRs) in the subject animals following FC-intact male and FT2-estrus female interaction in the AHA, mPOA, BNST, PVN and SON were significantly higher than other four groups. The levels of Fos-IRs in the subject animals following FC-intact male, FC-estrous female, FT2-intact male and FT2-estrus female interaction in the MeA were significantly higher than the subject animals in other two groups including FT1-intact male and FT1-estrous female groups. The levels of AVP immune reaction neurons (AVP-IRs) and AVP/Fos dual-labeled cells were the highest in the subject animals from FT2-estrous female interaction, followed by FT2-intact male interaction, the lowest was in those from FT1-intact male, FT1-estrus female interaction, FC-intact male and FC-estrus female interaction. The levels of OT immune reaction neurons (OT-IRs) and OT/Fos dual-labeled cells were the highest in the subject animals from FC-intact male interaction, followed by FC-estrus female interaction, FT2-intact male interaction and FT2-estrus female interaction, the lowest was in those from FT1-intact male and FT1-estrus female interaction.②The levels of Fos-IRs in the subject animals following MC-intact male, MT1-estrous female and MT2-intact male interaction in the mPOA, PVN and SON were significantly fewer than other three groups. The levels of Fos-IRs in the subject animals following MC-intact male, MT1-estrous female and MT2-intact male interaction in the AHA were significantly higher than other three groups. Fos-IRs in the BNST and MeA had no difference among six groups, Fos-IRs in all six group were expressed in high level in the AHA. The levels of AVP-IRs and AVP/Fos dual-labeled cells in the subject animals following MC-estrous female and MT2-estrous female interaction were higher than those following other four groups interaction. The levels of OT-IRs and OT/Fos dual-labeled cells were the highest in the subject animals from MT1-intact male interaction, followed by MTl-estrus female, those were the lowest from MC-intact male, MC-estrous female, MT2-intact male and MT2-estrous female. These results suggest that male with different sexual orientation had different patterns of neuronal activity possibly induced by the early estrogen receptor antagonists treatments.In summary, the present study concludes that early gonadal hormone and anti-estrogen treatment can alter sexual orientation of the animals at adulthood. The adult female individuals with homosexual orientation show a male pattern in the distribution of ERa, ERβ and OT. On the contrary, the adult male individuals with homosexual orientation show a female pattern in the distribution of AR and neuropeptide AVP. The distribution differences of these hormone receptor and neuropeptide between individuals with different sexual orientation might affect emotion, learning and memory, and also affect neuron Fos, neuropeptides AVP and OT expression after social interaction. Finally, homosexual behaviors were mediated by integration of gonadal hormone, neuropeptites and central nervous system.
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