| Pest rodents usually cause huge damages to many aspects in humans.Strong reproductive ability is one of the main reasons for rodent breakouts,but it is still not well known about its regulatory mechanisms,which limits the development of theories and technologies for rodent control.Seasonal breeding is a common reproductive strategy for most rodent animals and their gonadal gland displayed the shift between inflation and atrophy in annual cycle.Thereinto,gonadal inhibition in non-breeding season was called“natural sterile mode”and has aroused much research concern.Photoperiod and melatonin are the key factors to regulate seasonal changes of gonadal gland.In our previous studies,it has been found that as a strict seasonal breeder,gonadal growth of newborn Brandt’s voles was stimulated under increasing long-photoperiod and inhibited by decreasing short-photoperiod;meanwhile,short-photoperiod and exogenous melatonin can upregulate the expression of Dio3 gene in whole hypothalamus.These data indicated that the photoperiod and melatonin play key roles in the regulation of gonad development in Brandt’s vole,but we still do not know the mechanisms of molecular regulatory.Therefore,in present study,three issues about male Brandt’s vole were studied:1)the distribution of seasonal reproduction-related genes in hypothalamus,2)the effects of melatonin on gonadal development and gene expression in hypothalamus,and 3)comparison of methylation level in potential promoter region of hypothalamic Dio3 gene under between long-and short-photoperiod.The main results are as follows:1.Hypothalamic zones were separated after freezing microtome section and gene expression was detected by qPCR in adult voles at 6,8,11 weeks age.Results showed that as the decreasing order,the abundance of gene expression was from the arcuate nucleus（ARC）,the anteroventral periventricular arcuate（AVPV）,the 3rd ventricle（3V）and the dorsomedial hypothalamus（DMH）in Dio2 gene（typeⅡiodothyronine deiodinases）,was from AVPV,ARC,3V,and DMH in Dio3 gene,was from ARC,3V,AVPV,and DMH in Kisspeption gene（Kiss-1）;was from 3V,DMH,ARC and AVPV in gonadotropin-inhibitory hormone gene（Rfrp-3）;and was from AVPV,ARC,3V,and DMH in gonadotropin-releasing hormone gene（GnRH）,respectively.2.After successive daily melatonin subcutaneous injection as 6μg and 50μg per day from 4 weeks age,male voles were sampled after 2,4,7 weeks later to record the weight of body and gonadal glands,as well as expression of hypothalamic genes related to reproduction.Results showed that exogenous melatonin significantly inhibited the somatic and gonadal development of male Brandt’s vole,which was the most significant in the group treated with 50μg/day after treated 4 weeks later.Meanwhile,melatonin injection significantly inhibited the expression of Dio3 gene in 3V of hypothalamus,rather than other genes.These results implied that there are time and dose effects of the inhibition of melatonin to gonadal development,and the Dio3 gene maybe plays a key role in photoperiodic regulations of male gonadal development in Brandt’s vole.3.After the analysis of proximal promoter region of Dio3 gene,the methylation level were measured and compared between voles under long-or short-photoperiod from pregnant period at 4,8,12 weeks age,as well as gene expression.Results showed that,compared with long-photoperiod,no significant difference was detected in the methylation of the proximal promoter region of the Dio3 gene between two photoperiodic conditions,though short-photoperiod significantly up-regulated the gene expression.Therefore,there is no evidences to support the role of methylation in photoperiod regulation of gonadal development in Brandt’s vole.In conclusion,these results indicated that melatonin maybe plays a key role to mediate the photoperiodic pathway for the gonadal development in Brandt’s vole,the Dio3 gene in the 3V region should be important to decode photoperiod signals.However,the current evidences do not support the role of DNA methylation in the regulation of hypothalamic Dio3.Further studies should be designed in further research. |
