The Effects Of Pubertal Exposure To Bisphenol A On Social Behaviour In Female Mice

Bisphenol A(BPA),one of ubiquitous environmental Endocrine disrupting chemicals(EDCs),is widely used in the production of daily chemical products and food packaging materials due to its ability to increase the plasticity,transparency and flexibility of plastic products.Because of its similar chemical structure to diethylstilbestrol,BPA has estrogen-like activity,anti-estrogenic activity and anti-androgenic activity.BPA affects brain development and synaptic plasticity in adult brain,as well as behavioral development and sex differentiation.Social behavior is considered to be any behavior caused by,or affecting,another individual,usually of the same species,including communication,hierarchy,aggression,courtship,mating,maternal care,etc.,which is crucial to the survival and reproduction of animals.Previous studies in our lab found that puberty exposure to BPA reduced the ability of sociosexual behavior,social investigation and aggression between adult male mice and conspecifics in opposite sex,and increased the ability of affiliation.Given the fact that there are sexual dimorphisms in brain development and behavior,however,whether pubertal exposure to BPA exerts different effects on female social behavior compared to males.BPA is assumed to have estrogen-like effect and antiestrogenic effect in terms of existing literature reported,and estrogen is proposed to regulate neuropeptide oxytocin(OT)in female mice and thus affect social behavior.Then,the hypothesis that BPA affects social behavior by interfering with estrogen which in turn regulates neuropeptide OT system in the brain is needed to be considered.In this study,female mice is chosen as experimental animals,and experiments are performed by examining alteration of social behavior after pubertal BPA exposure,followed by detection of the number of mitral cells and granular cells,the length of mitral cell layer and granular cell layer.ELISA assay is conducted to measure the levels of estradiol and OT in brain and serum of female mice,then immunofluorescence analysis is performed to detect the number of OTimmunoreactive(OT-ir)neurons in brain areas related to social behavior.Immunohistochemical studies is further designed to measure the positive expression of oxytocin receptorimmunoreactive(OTR-ir)cells,estrogen receptor alpha-immunoreactive(ER?-ir)and estrogen receptor beta-immunoreactive(ER?-ir)in brain areas related to social behavior.Taken together,the aim of all measurements above is to explore the mechanism of social behavior in female mice caused by BPA acting on estrogen and estrogen receptor as well as neuropeptide OT system.The research methodsAfter 1 week of accustomation,3-week-age ICR female mice in clean grade were randomly divided into three groups.They were given vehicle or BPA(0.4,4 mg/kg/day)exposure,respectively.From the 4th week,BPA was continuously administrated for 21 days(PND 28-PND49).Social play behaviors between adolescent female mice or sexually different mice were detected from the 7th and a half week(PND 52),followed by examining social interaction and general sniffing behavior between adult female mice measured by adult same-sex social interaction behavior or receptivity tested by adult sexually different social interaction behavior from the 12 th week.And female receptivity is further subject to sexual behavior in an enriched environment,then maternal care behavior was designed to define care giving behavior towards pups by dam,followed by maternal aggression against intruders,mostly male mice with sexual experience,to test the defense state of dam.The morphology of accessory olfactory bulb was detected by methylene blue staining in paraffin section.And the levels of oxytocin(OT)and estradiol(E₂)in serum and brain were detected by ELISA assays.Immunofluorescence via frozen section was used to detect the positive expression of OT-ir neurons in brain regions linked to social behavior including OB,v BNST,LS,m POA,PVN,SON and Me A.The positive expression of OTR-ir,ER?-ir and ER?-ir cells in brain regions related to social behavior including v BNST,mPOA,PVN,SON,MeA and VMH were detected by immunohistochemistry.The results of the study1 behavior1.1 Adolescent Social PlayThe time of allogrooming between adolescent female mice in social play after pubertal exposure to BPA decreased pronouncedly compared with the control group(P<0.05,P<0.01),but did not affect the time of sniffing,chasing and general social play.BPA did not affect the social play behavior between male and female mouse.1.2 Adult social behaviorAlthough BPA exposure did not affect other indicators of same-sex behavior and overall social interaction between female mice,exposure to BPA decreased the overall duration(P<0.01)and the sniffing time(P<0.01),increased allogrooming time(P<0.01)in social interaction of the opposite sex.Pubertal BPA exposure did not affect the duration,frequency and latency of female mice to accept the mounting behavior from male mice.In maternal behavior,compared with the control group,pubertal exposure to 4 mg/kg/d BPA significantly prolonged the latency of dam carrying all 3 pups back to the nest(P<0.01)and increased the duration of maternal aggression against intruders by dam(P<0.05),but did not affect the nesting time of dam.It is suggested that pubertal exposure to BPA disrupted social interaction of opposite sex and higher dosage of BPA exposure may suppress maternal behavior,enhance the defense status of dam,however,not affect receptive behavior caused by male.2 Morphological structure of accessory olfactory bulbMethylene blue staining of paraffin section showed that the number of mitral cells and granule cells,the length of mitral cell layer and granulocyte layer in female accessory olfactory bulb were not significantly affected by BPA exposure during puberty,suggesting that BPA exposure had no significant effect on the structure of accessory olfactory bulb of female mice.3 OT and OTR systemsELISA detection of brain and serum OT levels showed that BPA exposure increased OT level in serum(P<0.01),but did not affect OT level in brain,suggesting that BPA may promote OT level in serum,but has no effect on OT level in the brain.Froze-section immunofluorescence showed that OT-ir positive neurons were mainly located in the paraventricular nucleus(PVN)and supraoptic nucleus(SON)of the hypothalamus as its source,while OT expression in other brain regions was relatively lower and there was no significant difference after BPA exposure.Compared with the control group,the number of OT positive neurons in the PVN brain region was significantly decreased in the 4 mg/kg/d BPA exposed group(P<0.01),and the number of OT-ir positive neurons in SON brain region was significantly increased in 0.4 mg/kg/d BPA dosage group(P<0.01).It is suggested that higher dosage of BPA exposure may inhibit the positive expression of OT-ir in PVN brain region,while lower dosage of BPA exposure may promote the positive expression of OT-ir in SON brain region.Immunohistochemistry showed that the positive expression of OTR-ir in the medial preoptic region(m POA)and medial amygdala(MeA)was significantly decreased in female mice exposed to BPA(P<0.05,P<0.0001;P<0.01,P<0.05),while the expression of OTR positive cells in other social brain regions showed no difference.It is suggested that BPA exposure may inhibit the expression of OTR-ir in m POA and Me A brain region.4 Estrogen and estrogen receptorsELISA assays showed that BPA exposure did not affect estradiol levels in brain and serum.Immunohistochemical assay of estrogen receptor showed that BPA exposure significantly reduced the positive expression of ER?-ir in m POA and Me A brain region(P<0.01,P<0.01;P<0.001,P<0.001),but had no significant effect on the positive expression of ER?-ir in brain regions related to social behavior.Conclusion:Pubertal BPA exposure mainly affected adult social behavior.BPA can reduce the sniffing ability between female and opposite sex conspecifics and interfere with maternal behavior and maternal aggression.These interfering effects of BPA may be mediated by decreasing the levels of OT-ir in PVN and the levels of OTR-ir and ER?-ir in m POA and Me A brain areas.