The Effect Of Regulating H3k9me3 On The Proliferation Of Bovine Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs)are the basis for the continuous production of sperm,and their special stemness has promising applications in the treatment of male infertility and species preservation,etc.In rodents,the in vitro culture and specific molecular markers of SSCs have been extensively reported,but its progress in domestic animals such as cattle and pigs is very slow due to the inconvenience in obtaining the material and long growth period.Further validation and active exploration of new specific markers are still needed in cattle due to the great differences between species.Based on the above issues,we established a bovine feeder-free in vitro culture system.The calves testis SSCs were isolated and purified.On the 7th day of in vitro culture,SSCs began to proliferate and appeared as round cell clones,and on the 20th day,the SSCs clones were radial.Alkaline phosphatase(AKP)staining showed that the cell clones were strongly positive for AKP.Immunofluorescence staining of cells expressd pluripotency protein SRY-box transcription factor 2(SOX2)and SSCs-specific molecular marker ubiquitin C-terminal hydrolase 1(UCHL1).q RT-PCR analysis expressed the pluripotency gene octamer binding transcription factor 4(OCT4)and the SSCs-specific marker gene promyelocytic leukaemia zinc finger(PLZF).Therefore,the above results indicate that the cells cultured in vitro in a feeder-free system SSCs clones.It has been reported that the glial cell line-derived neurotrophic factor(GDNF)family receptor alpha-1(GFRα1)and phospholipase D family member 6(PLD6)can be used as marker proteins of mouse SSCs.However,their expression in bovine testis is unclear.Logically,whether GFRα1 and PLD6 can be used as specific markers of bovine SSCs were verified in this study.The hematoxylin-eosin(HE)staining of calf testiscular tissues showed that the gonocycytes/SSCs are located in the center of the eminiferous cords.Both immunohistochemistry and free-floating immunofluorescence staining showed that the GFRα1~+cells are in the same position as SSCs in HE staining.The immunofluorescence staining showed that GFRα1~+cells are NANOG~+(pluripotency protein)cells.The expression of NANOG and GFRA1 in calf eminiferous cords is further verified at the m RNA level.The above results indicate that GFRα1 can be used as a specific marker protein for bovine SSCs.In the validation of PLD6,immunohistochemistry indicated that PLD6 is distributed in SSCs of the pre-sexually mature bovine testis tissues,as well the spermatogonia in adult bovine testis tissues.Immunofluorescence double-labeling staining showed that PLD6~+cells completely overlapped with germ cell marker protein DEAD-box helicase 4(DDX4,also known as VASA)positive cells,and partially overlapped with the UCHL1~+cells.The immunofluorescence staining of SSCs showed that the cell clones were positive for PLD6.The q RT-PCR results demonstrated that the m RNA expression level of PLD6 in adult bovine testis tissue was significantly higher than that in calves,and the expression level in SSCs was significantly higher than that in Sertoli cells.The above results indicate that PLD6 is expressed in SSCs and can be used to mark pre-sexually mature bovine SSCs,but does not have SSCs specificity in adult cattles.It’s demonstrated that Histone H3 lyine 9(K9)trimethylation(H3K9me3)is closely related to the development of mouse SSCs.Finally,the relationship between H3K9me3 and SSC proliferation was explored.Treatment of SSCs with methyltransferase inhibitor Chaetocin and transfection of SSCs with si RNA of suppressor of variegation 3-9 homolog 1(SUV39H1)reduced the level of H3K9me3,and EDU cell proliferation assay detected that the cell proliferation was decreased;while transfection with demethylase lysine demethylase 4D(KDM4D)si RNA into SSCs increased the level of H3K9me3 in SSCs,and EDU cell proliferation assays showed that the cell proliferation was promoted.In conclusion,bovine SSCs were obtained by establishing a culture system without feeding layer,and whether GFRα1 and PLD6 could be used as marker proteins of bovine SSCs was verified.Finally,the effects of regulating H3K9me3 level on the proliferation of bovine SSCs were investigated.These results lay a basic foundation for elucidating the regulation mechanism of histone methylation in the proliferation and differentiation of bovine SSCs.