The Dynamic Variation Of TH In Plasma And The Expression And Distribution Of THRs And TH Synthase In HPO Axis During The Estrus Cycle Of Ganjia Tibetan Sheep

As an important endocrine gland of the body,Thyroid hormones(THs)secreted by the thyroid gland not only participates in the growth,development and metabolism of the body,but also plays a vital role in regulating the reproductive function of animals.THs include T3 and T4.DIO2 and DIO3 are the key enzymes that catalyze the transformation of TH with different activities.Their activity changes can dynamically balance the relative balance of T3 and T4 concentrations.They mediate the regulation of photoperiodic signals on the concentration of TH in hypothalamus,which is very important in the seasonal reproductive state of mammals.TH can activate or inhibit the transcription of target genes and exert its biological function by binding with specific receptor THRs and acting on thyroid hormone response elements.Thyroid hormone receptors including THRα and THRβ,which are encoded by THRαand THRβ genes,respectively.In order to explore the regulation of thyroid hormone,thyroid hormone synthase and thyroid hormone receptor on reproductive activities of Ganjia Tibetan sheep,Ganjia Tibetan sheep were studied in estrus cycle(proestrus,estrus,metestrus and disestrus)and anestrus.The dynamic changes of T3 and T4 in plasma of Ganjia Tibetan sheep during estrus cycle and anestrus period were determined and analyzed by enzyme-linked immunosorbent method.Real-time fluorescent quantitative polymerase chain reactionand Western blottingwere used to detect the expression DIO2,DIO3 m RNA and protein in HPOA and thyroid tissue,the expression of m RNA and protein of THRαand THRβ in HPOA and the pineal tissue,The immunohistochemical staining were used to analyzed the distribution of immune positive cells in pineal gland and HPOA,The resultsare as follows:1.The contents of T3 and T4 in plasma of Ganjia Tibetan sheep during estrus cycle and anestrus period changeded alternately in a pulsing and fluctuating manner.T4 was significantly higher than that of T3(P<0.05).The average concentration of T3 reaches the maximum in the proestrus,the average concentration of T4 reaches the maximum in the estrus,suggesting that T3 and T4 are involved in estrus and reproductive activities,and the amount of T3 and T4 in estrus cycle is significantly higher than that in anestrus(P<0.05),indicating that TH plays a role in the seasonal reproductive conversion of Tibetan sheep.2.DIO2 and DIO3 m RNA and protein were expressed in HPOA and thyroid tissues of Ganjia Tibetan sheep during estrus cycle and anestrus period,and the differences were significantin each period(P<0.05).The relative expression of DIO2 m RNA and protein in hypothalamus was the highest in diestrus period,and the relative expression of DIO3 m RNA and protein were the highest in estrus period(P<0.05);The relative expression of DIO2 m RNA and protein in pituitary gland were the highest in proestrus(P<0.05),while the expression of DIO3 m RNA and protein wereboth the highest in metestrus;the relative expression of DIO2 m RNA and its proteinwere the highest in metestrus in ovary(P<0.05),(P<0.05),while the expression of DIO3 m RNA and its proteinwere the highest in in estrus(P<0.05);The expression of DIO2 m RNA and protein in thyroid were the highest in proestrus,while the expression of DIO3 m RNA and protein was the highest in metestrus(P<0.05).DIO2 m RNA and its proteins showed a trend of high expression mainly in disestrus and prostrus,while DIO3 m RNA and its proteins showed a trend of high expression mainly in estrus and misestrus,This difference in expression from the gene and protein levels suggests that DIO2 mainly plays its regulatory role in disestrus and proestrus,while DIO3 mainly plays its regulatory role in estrus and misestrus,which participate in the reproductive regulation of Tibetan sheep in different ways in different periods of estrus cycle.3.The differential expression and distribution of THRα,THRβ m RNA and protein were found in the pineal gland,hypothalamus,pituitary and ovarian tissues of Ganjia Tibetan sheep during estrus cycle and anestrus,and the immunopopositive cells varied in different tissues,both of which were expressed in the nucleus and cytoplasm.The positive cells of THRα in pineal glandwere mainly distributed in glial cells,the relative expression of its m RNAin pineal gland was the highest in metestrus and its proteinexpression were the highest in proestrus(P<0.05),while while the positive cells of THRβ were mainly distributed in pineal gland cells.the expression of its m RNA and protein were the highest in anestrus(P<0.05);the positive cells of THRαwere mainly distributed in the cytoplasm of nerve cells and vertebral cells in hypothalamus,the relative expression of its m RNA and protein in hypothalamus was the highest in disestrus,while the positive cells of THRβ were mainly distributed in glial cells,cell bodies of large neurons and cytoplasm of vertebral cells,the relative expression of its m RNA was the highest in metestrus and its protein expressionwas the highest in metestrus(P<0.05);the positive cells of THRα in the pituitary gland were mainly distributed inthe cytoplasm of basophil cells while the positive cells of THRβ were mainly distributed eosinophilic and basophil cells,the expression of THRα、THRβ m RNA and protein in pituitary gland wereboth the highest in disestrus(P<0.05);the positive cells of THRα and THRβ in ovary were all distributed in the inner membrane of follicle,granulosa cells and corpus luteum cells,the expression of THRα、THRβ m RNA and protein in ovary wereboth the highest in metestrus(P<0.05).In the four tissues,THRα and THRβshowed a high expression trend in misestrus and disestrus,suggesting that TH may influence its specific receptor THRαand THRβ,and regulate reproductive activity of Tibetan sheep.In conclusion,the plasma content of T3 and T4 in Tibetan sheep is significantly higher in the whole estrous cycle than anestrus,indicating that TH participates in the regulation of estrus and ovulation,and the m RNA and protein expression of DIO2,DIO3,THRα,THRβm RNA and its protein in HPO axis are different,indicating that thyroid hormone and thyroid synthase play an important role in regulating the HPO axis of Tibetan sheep organs and the estrus and ovulation of Tibetan sheep.