Effect Of Melatonin-mediated Circadian Clock Genes Cry On Estradiol Secretion Of Bactrian Camel

Bactrian camel,as an economic species peculiar to Northwest China,its seasonal reproduction severely limits the litter rate of bactrian camels.In addition to the melatonin-mediated regulation of the hypothalamic-pituitary-gonadal(HPG)axis,mammalian seasonal reproduction is also regulated by the circadian clock.More and more experimental data show that there is an inseparable relationship between the periodic secretion of melatonin and the circadian clock gene,but the specific molecular mechanism of melatonin action is not clear.Other studies have shown that the c AMP/PKA/CREB pathway is involved in the regulation of melatonin in many important physiological activities of the body,but whether it is involved in the regulation of circadian clock genes still needs to be confirmed by research.In this study,Bactrian camel was used as the research object.Bactrian camel ovarian granulosa cells were cultured in vitro.By using methods of overexpression and knockdown of the melatonin receptor gene(MT1 and MT2 genes)to detect the effect on the circadian clock gene.To detect the expression content and localization of MT1/2 and Cryptochrome(Cry)genes in the reproductive axis by q PCR and immunohistochemistry.The Elisa method was used to detect the effects of MT and Cry genes on the secretion of estradiol(E2),and the bioinformatics analysis of MT and Cry proteins was performed to explore the relationship between melatonin and the circadian clock genes and their effects on reproduction.The results showed that: 1.The results of c AMP detection by Elisa showed that the MT or Cry gene was overexpressed,the c AMP concentration was decreased,and the MT or Cry gene was silenced,and the c AMP concentration was increased.q PCR and Western blotting experiments were used to detect changes in other genes on the signal pathway after overexpressing the MT gene.The results showed: at 24 h and 36 h,in addition to the extremely high expression of the gene Cry1/2,other genes(GNB2,PKA,CREB,Per1/2/3,Clock)were significantly reduced,and feedback changes or tended to balance occur at 48 h.The test results after silenced MT are completely opposite to those of overexpressed MT.In order to further verify the gene correlation,the gene changes on the signal pathway were detected after overexpression and silencing of the gene Cry.The results showed that they were completely consistent with the results of overexpression and silencing of MT,suggesting that there was a link between MT and Cry,and provide reliable scientific theoretical basis for the analysis and study of c AMP/PKA/CREB pathway.It was also confirmed that the circadian clock on the ovarian granulosa cells,as the peripheral circadian clock,maintained a consistent rhythm with the master clock(the supraoptic nuclear clock).2.The q PCR and WB experiments showed that MT and Cry were expressed in the tissues of Bactrian camel HPG axis,indicating that both MT and Cry could affect animal reproduction through HPG axis.The difference is that MT is most expressed in the pineal gland,and CRY is highest in the hypothalamus.This is confirmed by the melatonin’s main secretion site is the pineal gland,and CRY mainly exists in the supraoptic nucleus of the hypothalamus(SCN).The experimental results also found that there was no difference on protein MT1 and MT2 expression in the same tissue,as did CRY1 and CRY2(except in the epididymis).In the pineal gland and hypothalamus,both MT and CRY proteins were diffusely expressed.The difference was that MT only had positive expression on the cell membrane and cytoplasm,while CRY protein had positive expression on the cell membrane,cytoplasm,and nucleus.MT and CRY were significantly expressed in both adenohypophysis and neurohypophysis.The expression of the four proteins were mainly located in capillary wall cells and glandular cells which around blood vessels.On the one hand,it indicated that the transport of melatonin in the pituitary was mainly blood transport;on the other hand,it implied that there was a connection between MT and CRY protein.The expression of the four proteins was very similar in the testis and ovary.They were mainly located in the basal membrane and leydig cells of the testis,and diffused in the ovary.The difference was that melatonin receptor was not expressed in the cell nucleus,while CRY protein was.In the epididymis,four proteins had different expressions.MT1,MT2,and CRY1 proteins were positively expressed on luminal epithelial cells,peritubular muscle-like cells,and luminal sperm,and the nuclei are negative.In addition to the positive expression of CRY2 protein in luminal epithelial cells,peritubular muscle-like cells and luminal sperm,the cilia showed strong positive expression,which indicated that the CRY2 protein played a greater role in sperm development and maturation during the sperm passing through the epididymis.3.Cell immunolocalization results showed that MT was localized on the cell membrane and cytoplasm,and CRY protein was mainly localized in the nucleus.Adding exogenous melatonin or overexpressing the MT gene increased the estradiol concentration(P <0.01),and the estradiol concentration decreased significantly after blocking or silencing MT(P <0.01).When MT was overexpressed and exogenous melatonin was added,the concentration of estradiol decreased(P<0.01),indicating that there was feedback inhibition.Overexpressing the Cry gene,the estradiol concentration increased significantly(P <0.01),and the silent Cry gene decreased the estradiol concentration(P <0.01).4.Prediction of the physicochemical properties,advanced structure and homology of MT and CRY proteins by bioinformatics methods,the results showed: The half-life of MT1/2 and CRY1/2were 30 h,the N-terminus of the peptide chain was methionine,and they were all basic proteins.Except MT1 was a stable protein,the rest were unstable proteins.MT1/2 was a hydrophobic protein with a transmembrane structure,and CRY1/2 was a hydrophilic protein without a transmembrane structure.The major secondary structural elements contained in these four proteins were alpha helices and random coils.Subcellular localization results showed that MT1/2 protein was mainly located in cell membrane and endoplasmic reticulum.CRY1/2 protein was mainly located in the cytoplasm and nucleus.The phylogenetic tree was constructed and found that the MT1 and CRY1 proteins of the Bactrian camel and the MT1 and CRY1 proteins of the Arabian camel had the closest molecular evolutionary distance,indicating that they were the closest.In this paper,melatonin was linked to the circadian clock gene,and it was determined that the c AMP/PKA/CREB pathway was involved in the regulation of the circadian clock by melatonin.This result will play a very important role in future research on the physiological regulation of reproductive mammals.