Molecular Mechanism Of ATF6 And Its Downstream Gene TSSK4 Regulating Spermatogenesis

Background:Testis specific serine/threonine protein kinase 4(TSSK4)plays an important role in male germ cell development and sperm maturation.It may participate in CRE/CREB pathway and stimulate CRE/CREB pathway by phosphorylating CREB on Ser-133.TSSK4 mainly exists in sperm,especially in the head and flagellum.In recent years,it has been found that TSSK4 is highly expressed in testis,but lowly expressed in lung,kidney,spleen,brain and thymus,etc.Endoplasmic reticulum plays an important role in regulating lipid synthesis,protein folding and calcium homeostasis in eukaryotic cells.When cells are stimulated by external environment,the uptake and release of Ca2+ in endoplasmic reticulum are disturbed,or the protein processing and transportation are impaired,which will lead to the increase of misfolded and unfolded proteins in the endoplasmic reticulum cavity,causing ER stress,and then activating UPR.ATF6 is the main signal transduction molecule in UPR.Under physiological state,ATF6 binds with GRP78/BiP protein to form a stable complex.When endoplasmic reticulum stress occurs,ATF6 will dissociate from GRP78/BiP protein,and then transport to Golgi apparatus.They are hydrolyzed by Golgi membrane proteins SIP and S2P to form activated ATF6(p50 ATF6).These fragments enter into the nucleus and bind to the endoplasmic reticulum stress response element(ERSE)in the promoter region,and promote the transcription of transcription factors containing the ERSE and downstream genes of UPR target molecules.They also promote the production of XBP1 and other proteins induced by IRE1,promoting the correct folding and transport of unfolded proteins in the endoplasmic reticulum,and maintain the homeostasis of the endoplasmic reticulum.If ERS persisted and UPR could not restore endoplasmic reticulum homeostasis,it would lead to apoptosis.The occurrence of male reproductive system diseases is affected by many factors,such as environment,heredity,living habits and so on.Studies have shown that endoplasmic reticulum stress caused by environmental toxicants can lead to changes in testis and epididymis function,spermatogenic cell apoptosis,sperm number and motility decline,leading to male sterility in the end.ATF6 may be closely related to male reproductive system diseases as a receptor of endoplasmic reticulum stress.Our study further explore the role of ATF6 in male reproductive process,and provide a new idea for the research and treatment of male reproductive system diseases.Methods:Semen samples of normal people and infertile patients are collected and analyzed.The expression of p50 ATF6 in semen of normal people and infertile patients is detected by Western blotting;the ATF6 knockout mice model is constructed,and the effects of ATF6 knockout on spermatogenesis of male mice are explored through six phenotypic experiments,including testicular tissue section hematoxylin-eosin staining,sperm count and sperm motility detection,seminiferous tubule area,testicular weight ratio and fertility test;the transcriptome sequencing information is used to screen the downstream gene of ATF6,and is verified by qPCR;whether p50 ATF6 binds to the downstream gene and the possible binding site are determined by dual luciferase reporter assay(DLR)and chromatin immunoprecipitation(ChIP)assay.The effect of overexpression of p50 ATF6 on TSSK4 in 293T cell line is detected by Western blotting;the expression of CREB and phospho-CREB in testicular tissue sections of both ATF6 knockout mice and wildtype mice are detected through immunohistochemistry.Results:Compared with the control group,the expression of p50 ATF6 in semen of some patients with clinical infertility is significantly decreased or even not expressed.The reproductive cycle of ATF6 knockout mice is significantly prolonged,the number of mice per fetus is reduced,and there is a phenomenon of feeding offspring.ATF6 knockout affects the spermatogenesis of male mice.The specific performance is that compared with the control group,the testicular development of ATF6 knockout male mice is delayed,sperm count and sperm motility are decreased,seminiferous tubule area and testicular weight ratio are decreased.TSSK4,a downstream gene of ATF6,is screened by transcriptome sequencing and qPCR,then verified at both mRNA and protein levels.To explore the molecular mechanism of how p50 ATF6 regulating TSSK4 in 293T cells,we find that p50 ATF6 is able to promote the activity of TSSK4 promoter,and p50 ATF6 protein can combine with the DNA sequence of TSSK4 through dual luciferase reporter assay and chromatin Immunoprecipitation.At the same time,immunohistochemical results show that there is no significant difference in the expression of CREB in testis of ATF6 knockout mice,but the expression of phospho-CREB decreases significantly.Conclusions:ATF6 can affect the fertility of mice by regulating TSSK4.On the other hand,ATF6 can indirectly affect the expression level of TSSK4's downstream genes.