| Seasonal reproduction is a phenomenon that mammals reproduce only in specific seasons in order to adapt to seasonally changing environmental conditions.In seasonal breeding animals,ovaries,as an important reproductive organ of female mammals,usually show periodic variation with the changes of seasons.Follicle development is the basic event in the reproductive process and the ovum released by the mature follicle is the key factor to produce offspring.TGF-β1/SMAD signal pathway is involved in the cell proliferation and differentiation,and plays an important role in the process of follicle development through functional paracrine/autocrine of the ovary to regulate the reproductive activities of mammals.The TGF-β1/SMAD signal pathway transfers extracellular signals into the cell and regulates the expression of specific genes,thereby regulating follicle development.The Djungarian hamster(Phodopus sungorus)is a typical seasonal breeding animal,and its breeding activity varies with seasonal changes.In this study,the genetic effects of TGF-β1/SMAD signal pathway on the process of photoperiod regulating the follicular development in Djungarian hamster were investigated,which provides a theoretical basis for understanding the reproductive mechanism of female mammals.In this study,36 adult female Djungarian hamsters were randomly divided into three groups according to light duration: long photoperiod group(16 light: 8 dark,Long Photoperiod,LP)and medium photoperiod group(12 light: 12 dark,Medium Photoperiod,MP)and short photoperiod group(8 light: 16 dark,Short Photoperiod,SP).After 6 weeks of treatment,the ovaries of the Djungarian hamsters were taken to be used in the following research.The body weight and the mass of the ovarian of Djungarian hamsters were weighed from different photoperiod conditions;the contents of E2 and AMH were determined by enzyme-linked immunosorbent assay(ELISA);the difference of the ovarian structure and the number of growing follicles were detected by paraffin section and HE staining;real-time fluorescence quantitative PCR and Western Blot were used to detect the differential expression levels of genes involved in the TGF-β1/SMAD signal pathway at m RNA and protein levels;the correlation of the expression levels of each factor involved in the TGF-β1/SMAD signal pathway with follicle number and FSHR with follicle number were analyzed;the correlation between the expression levels of each factor involved in the TGF-β1/SMAD signal pathway and between the expression levels of each factor and FSHR were analyzed.The specific results are as follows:(1)The results of body weight and ovarian weight of Djungarian hamsters showed that under different photoperiod conditions,there was no significant difference in body weight of Djungarian hamsters(P > 0.05),but there was an increasing trend in body weight of individuals in long photoperiod group;there were significant differences in ovarian weight among different photoperiod groups,and the long photoperiod group was significantly higher than the short light group(P < 0.05).The results suggest that photoperiod affects gonadal development in mammals.(2)Under different photoperiod conditions,the results of E2 concentration in feces and AMH content in serum showed that the concentration of E2 increased with the extension of light time,and the concentration of E2 in long photoperiod group was significantly higher than that in short photoperiod group(P < 0.05).AMH content under short photoperiod condition was significantly higher than that under long photoperiod condition(P < 0.05).These results indicated that E2 and AMH were involved in the regulation of follicular development of Djungarian hamsters.The increase of AMH content in serum levels and the decrease of E2 concentration in faecal levels can be used as the early predictor of follicular atresia and the index of ovarian follicular reserve capacity of female Djungarian hamsters.(3)The results of HE test showed that the number of primary follicles of Djungarian hamsters under long photoperiod condition was significantly higher than that under short photoperiod condition(P < 0.05),but there was no significant difference between medium and short photoperiod groups(P > 0.05).The number of secondary follicles under long and medium photoperiod conditions was significantly higher than that in short photoperiod group(P < 0.05).The number of antral follicles under long and medium photoperiod conditions was significantly higher than that in short photoperiod group(P < 0.05).There was no significant difference in the diameters of primary oocytes and secondary oocytes under different photoperiod conditions(P >0.05),but the diameters of the oocytes under short photoperiod condition were smaller than those under long and medium photoperiod conditions.The diameters of primary and secondary follicles were significantly different under different photoperiod conditions,that is,the diameters of primary and secondary follicles under long and medium photoperiod conditions were significantly higher than those under short photoperiod conditions(P < 0.05).The results indicated that long photoperiod conditions were more conducive to follicle development.(4)The differential analysis of the expression levels of TGF-β1,TGF-βRI,TGF-βRII,SMAD2,SMAD3,SMAD4 and FSHR at the m RNA level in ovarian showed that the m RNA expression levels of TGF-β1,TGF-βRI,TGF-βRII,SMAD2 and SMAD3 decreased with the increase of light duration,and the expression levels of individuals under long photoperiod condition were significantly lower than those under short photoperiod condition(P < 0.05).The expression levels of TGF-β1,TGF-βRI,TGF-βRII and SMAD3 under medium photoperiod condition were significantly lower than those under short photoperiod condition(P < 0.05).The m RNA expression levels of SMAD4 and FSHR increased with the extension of light duration,and the expression levels of SMAD4 and FSHR under long photoperiod condition were significantly higher than those under short photoperiod condition(P < 0.05).(5)The differential analysis of the expression levels of TGF-β1,TGF-βRI,TGF-βRII,SMAD2,SMAD3,SMAD4 and FSHR at the protein level in ovarian showed that the protein expression levels of TGF-β1,TGF-βRI,TGF-βRII,SMAD2 and SMAD3 decreased with the extension of light duration,and the expression levels of individuals under long photoperiod condition were significantly lower than those under short photoperiod condition(P < 0.05).The protein expression levels of TGF-βRI,SMAD2 and SMAD3 under medium photoperiod condition were significantly lower than those under short photoperiod condition(P < 0.05).The protein expression levels of SMAD4 and FSHR increased with the extension of light duration,and the expression levels of individuals under long photoperiod condition were significantly higher than those under medium and short photoperiod conditions(P < 0.05).(6)The correlation analysis between the expression levels of each factor of the TGF-β1/SMAD signal pathway and the number of follicles and the expression levels of FSHR and the number of follicles in the ovary of Djungarian hamsters showed that a highly significant correlation exist between the expression level of TGF-β1 at the protein level and the number of follicles.The expression quantity of TGF-βRI,SMAD2,SMAD4 and FSHR at the protein level was significantly correlated with the number of follicles.The expression quantity of TGF-βRII at the m RNA level was significantly negatively correlated with the number of follicles.These results indicated that the TGF-β1/SMAD signal pathway and FSHR play an important role in the regulation of follicular development.(7)The correlation analysis between the expression levels of each factor involved in the TGF-β1/SMAD signal pathway and between the expression of each factor and FSHR showed that FSHR was significantly negatively correlated with the expression quantity of TGF-β1,TGF-βRI,TGF-βRII,SMAD2 and SMAD3 at the protein level and significantly positively correlated with that of SMAD4 at the protein level.The expression quantity of TGF-β1 was significantly positively correlated with that of TGF-βRI,TGF-βRII and SMAD3 at the m RNA level,significantly negatively correlated with that of SMAD4 at the m RNA level,significantly positively correlated with that of SMAD2 at the protein level and highly significantly correlated with that of TGF-βRI at the protein level.The expression quantity of TGF-βRI was significantly correlated with that of TGF-βRII at both m RNA and protein levels.The expression quantity of TGF-βRI was significantly correlated with those of SMAD2 and SMAD3 at m RNA level,highly significantly correlated with those of SMAD2 and SMAD3 at protein level,and highly significantly negatively correlated with that of SMAD4 at m RNA level.The expression quantity of TGF-βRII was significantly correlated with those of SMAD2 and SMAD4 at the m RNA level,highly significantly correlated with that of SMAD2 at the protein level,and highly significantly correlated with that of SMAD3 at both the m RNA and protein levels.The expression quantity of SMAD2 was significantly correlated with those of SMAD3 and SMAD4 at the protein level.The expression quantity of SMAD3 was significantly correlated with that of SMAD4 at the protein level.The results suggest that the various factors act synergistically to regulate the complex process that regulates follicle development in the female Djungarian hamsters.In conclusion,photoperiod is an important environmental factor affecting follicular development in Djungarian hamsters,and TGF-β1/SMAD signal pathway plays an important role in the process of photoperiod regulating reproductive activities of the Djungarian hamsters.The results of this study provide a theoretical basis for further studying the follicular development,oocyte maturation and molecular mechanism of mammalian seasonal reproduction in Djungarian hamsters. |
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