The Role Of Eya3 In The Reproductive Activities Regulated By Photoperiod In Cricetulus Barabensis

Seasonal breeding animals regulate their physiological state and behavior by sensing seasonal changes of environmental factors changed with seasons,and then adapt to the external environment conditions for survival and reproduction.Photoperiod is one of the main environmental factors regulating the seasonal reproduction of animals.The seasonal reproductive activities of mammals are mainly regulated by the hypothalamus-pituitary-gonadal axis,and complex neuroendocrine regulatory networks exist between the external ecological factors and the internal hypothalamic gonadotropin-releasing hormone(GnRH).TSH-DIO2/DIO3 system is one of the important pathways regulating the seasonal reproductive activity of mammals by photoperiod.TSHB(Thyroid stimulating hormone ?-subunit),induced in the pars tuberalis(PT)of the pituitary gland,plays a key role in the TSH-DIO2/DIO3 pathway.As an upstream inducers of TSHB transcription,Eya3(eyes absent 3)also participated in this regulatory pathway.The Cricetulus barabensis is the main farmland rodent in the northern plains of China,which has strong reproductive capacity and harmfulness.C.barabensis is a typical seasonal breeding animal,and its population fluctuation is influenced by seasonal reproduction activity.In order to explore the molecular mechanism of Eya3 in the reproductive activities regulated by photoperiod in C.barabensis through the TSH-DIO2/DIO3 pathway,we investigate the expression pattern of Eya3 using methods of molecular biology and immunohistochemistry in this study.In this study,the cDNA sequence of Eya3 was cloned for the first time by RT-PCR in C.barabensis.The sequence is 2064 bp in length,containing 1719 bp complete CDS and part of the 5' and 3' non-coding region(Gen Bank accession number: MF289978).Bioinformatics analysis demonstrated that Eya3 encodes a total of 572 amino acids,its amino acid sequence includes a C-terminal Eya domain composed of 271 amino acids and an N-terminal transcriptional activity region rich in proline,serine and threonine.The Eya3 protein has tyrosine kinase activity.The amino acid sequences encoded by Eya3 in C.barabensis,Mesocricetus,Rattus norvegicus,Mus musculus and other species were compared in multiple ways and the phylogenetic tree was constructed.The result was consistent with the traditional classification of species,indicating that Eya3 was conserved in the process of species evolution.The relative expression patterns of Eya3 mRNA and protein in pituitary gland of C.barabensis in different ages were detected by real-time fluorescence quantitative PCR and immunohistochemistry technology.The results showed that:(1)In females,the expression level of Eya3 was highest in the subadult stage at the mRNA level and the protein level(P<0.05),which was in accordance with that the pattern of expression in the age stage of DIO2 and DIO3 in hypothalamus of C.barabensis.(2)In males,the expression of Eya3 was highest in the adulthood stage at the mRNA level and the protein level(P<0.05),which was also consistent with the pattern of expression in the age stage of DIO2 and DIO3 in hypothalamus of C.barabensis.(3)The enzyme-linked immunosorbent assay was adopted to detect the serum concentrations of FSH,LH and TSH in different ages.The results showed that the concentrations of these hormones were all affected by the developmental ages and basically showed a trend of decreasing with age,but the concentration of TSH in male and female individuals was the lowest in subadult stage(P<0.01).The results showed that the expression of Eya3 was gender-specific and age-specific dependent,which suggested that Eya3 was involved in regulating the expression of DIO2 and DIO3 in hypothalamus of C.barabensis.The relative expression patterns of Eya3 mRNA and protein in pituitary gland of C.barabensis in different photoperiods were detected by using real-time fluorescence quantitative PCR and immunohistochemistry technology.The results showed that:(1)At the mRNA level and protein level,the expression level of Eya3 was higher under long day(LD)than under short day(SD)condition(P<0.05),which was consistent with the photoperiodic expression pattern of TSHB in the pituitary and DIO2 in the hypothalamus,but was opposite to the photoperiodic expression pattern of DIO3 in the hypothalamus of C.barabensis.(2)The C.barabensis had significantly higher mean final body weights and organ coefficient of the reproductive organs exposed to the LD photoperiods than the individuals in SD condition(P<0.05).(3)The enzyme-linked immunosorbent assay was used to detect the serum concentrations of FSH,LH and TSH under different photoperiod conditions.The results showed that the concentrations of these hormones were higher under LD than SD condition,suggesting that the reproductive activity of C.barabensis was more active in LD condition than SD condition.All results suggest that LD could promote the expression of Eya3 in pituitary of C.barabensis,and the photoperiod may regulate the expression of Eya3 and TSHB in the pituitary and DIO2/DIO3 in the hypothalamus to further regulate the reproductive hormone,as a result,regulate the seasonal reproductive activity of the C.barabensis.In summary,this study explored the differential expression patterns of Eya3 in the pituitary of C.barabensis in different ages and different photoperiod conditions which revealed the molecular mechanism of Eya3 involved in the photoperiod-regulated seasonal reproduction of C.barabensis.The result of this study is helpful to further explore the complex regulative mechanism of seasonal reproduction of C.barabensis,and provides a theoretical basis for the mechanisms about the outbreak of population abundance and the seasonal fluctuation as also provides new means to control the rodent pests.