Effects Of VEGFC-VEGFR3 Signialing On Regulating Proliferation And Apoptosis Of Spermatogonia And Associated Mechanism

Background and objective: For successful fertilization,males have to produce billions of spermatozooa per day.The balance between proliferation and apoptosis in spermatogonia is of great importance for enough amount of germ cells during the process of spermatogenesis.Despite there have been some reports about regulating this process by a range different of factors,an appropriate culture system had not been set up for spermatogonia to proliferate efficiently and survive consistently.So,a new factor: vascular endothelial growth factor C(VEGFC)was identified from our previous RNA sequencing data of the germ cells in every stage of spermatogenesis.It was found that the high expression of VEGFC/VEGFR3 transcript in spermatogonia was reversed by onset of meiosis and droped markedly to a lower level.It suggested that VEGFC may play a role in the maintenance or/and proliferation of spermatogonia in physiological condition.In order to verify this conjecture,It was firstly identified the expression of VEGFC and its receptor in testes,and then proved the dual role that VEGFC promoted proliferation and anti-apoptosis in mouse spermatogonial cell line(GC-1)in vitro and revealed its related regulatory mechanisms,and compared the production capacity between obstructive azoospermia and non-obstructive azoospermia.Methods:PART 1: Firstly,the mRNA and protein expression of VEGFC and its receptor in spermatogonia and its cell lines,Sertoli cells and testis were verified by RT-PCR,immunocytochemistry and Western blotting.Then,shRNA was used to silence the protein of vascular endothelial growth factor receptor 3(VEGFR3)in GC-1 cells.GC-1 cells were cultured in vitro with or without exogenous VEGFC factor to activate or block VEGFC-VEGFR3 signaling.Finally,the effects of VEGFC-VEGFR3 signal on the proliferation of GC-1 cells were detected by CCK-8 and EDU assays.The apoptosis of GC-1 cells was analyzed by flow cytometry with Annex V-PI fluorescent staining.The changes of proliferation and apoptosis related protein were also detected by Western blotting.PART 2: Immunofluorescent staining was used to detect the expression of VEGFR3 protein in human testis by co-staining the anti-VEGFR3 antibody with the marker antibody of germ cells in every stage of spermatogenesis.Sertoli cells were isolated from 12 cases of non-obstructive azoospermia testis in vitro,and their ability to secrete VEGFC was tested by enzyme linked immunosorbent assay(ELISA).Results:Part I Immunofluorescence staining showed that VEGFC was widely expressed in mouse testis,while VEGFR3 was specifically expressed in mouse spermatogonia.The results of CCK8 and EDU showed that,in vitro VEGFC factor could promote the proliferation of GC-1 cells,while the silencing of VEGFR3 protein inhibited the proliferation.The results of flow cytometry showed that the silencing of VEGFR3 resulted in more apoptosis of GC-1 cells.The results of QPCR assay showed that VEGFC factor up-regulated the mRNA levels of c-Fos and Egr1 transcription factors in GC-1 cells.Western blotting results showed that on the one hand VEGFC-VEGFR3 signal caused phosphorylation of AKT and MAPK protein in a short time,and then promote the expression of Cyclin D and PCNA protein;on the other hand,the deficiency of VEGFC-VEGFR3 signaling leaded to a descent of BCL-2 protein expression in GC-1 cells,and then activate the downstream Caspase protein which eventually lead to increased apoptosis.PART 2: VEGFR3 protein is mainly expressed in spermatogonia and spermatocytes.The concentration of VEGFC protein both in the sertoli cell culture and its supernatant of patients with obstructive azoospermia were significantly higher than those from non-obstructive azoospermia.Conclusions: Our study demonstrated that VEGFC and its receptor VEGFR3 were expressed in mouse spermatogonia and the dual roles of VEGFC in promoting proliferation and anti-apoptosis of GC-1 cells through its receptor VEGFR3.On the one hand,VEGFC signaling activate the AKT and MAPK pathway and then upregulated the expression of Cyclin D protein,thereby promoting cell proliferation;the other hand,it could hinder the activation of Caspase protein to inhibit apoptosis via maintaining the expression of BCL-2 protein.In addition,this paper also found that the ability that producing VEGFC protein in patients with spermatogenesis disorder was decreased,suggesting that it may have a certain relationship between them.