| Bactrian camel is an ancient and precious species of livestock that is unique resources exist in the desert and have important economic and scientific value.Bactrian camel is a strict seasonal estrus animal,which the slow growth,long reproductive cycle and the low reproductive performance,and the ovulation mechanism is not quite clear yet,therefore,the purpose is study the functions of differential expression proteins in Bactrian camels.Firstly,we selected the serum of the bactrian camel(male camel)from the breeding season and the non-breeding season to screening the differential proteins related to reproduction via iTRAQ.Furthermore,screening and identification of differential protein related to hormone synthesis and secretion-Retinol binding protein 4(RBP4)via bioinformatics and molecular biology.Verify the function of RBP4 protein via cell model in vitro;Next,screening differentially expressed proteins in ovarian tissues after ovulation induced by intramuscular injection of Seminal Plasm(SP)via iTRAQ.Finally,further screening and identification of functional protein-Calcium/calmodulin kinase II-γ(CAMK2G)involved in regulating ovulation induction by bioinformatics,endocrinology,histomorphology,imaging and molecular biology.The regulatory Mechanism of CAMK2 G on estrogen receptors was verified via cell model in vitro.The results are as follows:(1)A total of 359 serum proteins were identified via iTRAQ,based on fold change cutoffs of 1.2 and 0.8 and p <0.05,a total of 32 different proteins were found to be differentially expressed in Bactrian camels,of which 11 were up-regulated and 21 were down-regulated in estrus.Five differential expression proteins(PCGF5,H1.2,ANTXR2,FOLR1 and RBP4)related to reproduction was screened and identified.(2)RBP4 expression level was different on the gonad axis,and the testis expression level was the highest.Adding different concentrations of VitA can affect the expression levels of IGFs,RBP4 and AR in Sertoli cells in Bactrian camels,and with a dosedependent effect on RBP4(negative regulation)and AR(positive regulation).Overexpression(knock-down)of RBP4 inhibited(promoted)the expression of IGFs and down-regulated(upregulated)the expression level of AR.RBP4 can regulate androgen receptor AR expression through steroid synthesis pathway.(3)A total of 404 differential expression proteins from ovarian tissues,260 up-regulated proteins(SP group)and 144 down-regulated proteins were screened via iTRAQ,subsequently,Further,five differential proteins(CAMK2G,FST,NR5A1,PAK1,and PRL)that can be involved in inducing ovulation were screened and identified by bioinformatics.we found that 5 differential proteins have different expression levels in the gonad axis and reproductive organs after induction ovulation by RT-PCR,Western blot and tissue immunofluorescence.Our results suggestion that the gonad axis is involved in the induction ovulation.(4)Regulating the expression level of CAMK2 G has different effects on various horomone receptors;overexpression/knockdown of CAMK2 G can up/down regulate the ER expression level;Specific inhibitor KN-93 of CAMK2 G can down-regulate the ER expression level;CAMK2G can mediate the expression of 17βHSD and P450 scc to regulate the expression level of estrogen receptor(ER).This study revealed that RBP4 was closely related to breeding of male Bactrian camels,and it is mainly involved in regulating androgen synthesis.Therefore,it is suggested that RBP4 can be used as a regulatory factor to regulate hormone synthesis and secretion;CAMK2G regulates estrogen receptor expression via involved in steroid pathway.It is revealed that CAMK2 G may be a regulatory molecule for the ovulation induced by Bactrian camel. |
