Identification Of PLD6 As A Molecular Marker For Porcine Undifferentiated Spermatogonia

Undifferentiated spermatogonia as adult stem cells existed in the testis,have a functional ability to maintain self-renewal and sustain production of spermatozoa throughout the reproductive lifespan of a male.Studies on the biological characteristics of undifferentiated spermatogonia can facilitate better understandings of spermatogenesis and unravel the mechanisms for self-renewal and differentiation of male germline stem cells.However,the rarity of undifferentiated spermatogonia in the testis and the lack of reliable surface markers obstruct the related studies and applications.Studies have reported that the phospholipase D family member 6(PLD6)expressed in undifferentiated spermatogonia of mice.In order to identify whether PLD6 can be used as a molecular marker for porcine undifferentiated spermatogonia,this study aimed to explore the distribution characteristics of PLD6 in porcine undifferentiated spermatogonia,so as to optimize the enrichment method and improve the purification efficiency of porcine undifferentiated spermatogonia.At the same time,this study attempted to establish a feeder-free culture system of porcine undifferentiated spermatogonia,which laid a foundation for the follow-up research of male germline stem cells.The main research results are as follows:(1)Collection of testicular tissue at neonatal,puberty and adulthood from different species,then the expression and distribution of PLD6 in testicular tissue were detected by immunohistochemistry.The results showed that PLD6 was specifically expressed in spermatogonia of pre-pubertal porcine testes and the expression patterns of PLD6 in the testicles of mice,goats and bulls were consistent with that in pig.(2)To determine the constitution of the PLD6~+cell fraction in pre-pubertal porcine testes,double immunofluorescent analysis was conducted to check the co-staining of PLD6 with markers for testicular cells.The results showed that PLD6 was specifically expressed in porcine undifferentiated spermatogonia.(3)Two-step enzymatic digestion and differential plating were used to enrich the testicular cells from pre-pubertal porcine testes.The collected cells consisted of 95%viable cells as determined by trypan blue dye exclusion.In order to further improve the purity of germ cells,Magnetic-activated cell sorting was conducted using PLD6 as a surface marker.The sorted-and unsorted-cell groups were identified by Western Blot,qRT-PCR and flow cytometric analysis(FCA).The results of FCA showed that PLD6~+cells represented about8.5-fold enrichment for porcine undifferentiated spermatogonia.Furthermore,the results of Western Blot and qRT-PCR both revealed that the expression of porcine undifferentiated spermatogonia markers in the PLD6~+cell fraction were significantly higher than that in the unselected total testicular cells and PLD6~-cells,whereas the expression of testis somatic cell markers was substantially lower than that in the unselected total testicular cells and PLD6~-cells.These proved that PLD6 can be used as a surface marker to enrich undifferentiated spermatogonia from the pre-pubertal porcine testis.(4)We carried out spermatogonial transplantation to identify the biological functions of PLD6~+cells and compared the enrichment efficiency.The sorted PLD6~+cell fraction and unsorted total testicular cell population were pre-labeled with a red fluorescent dye PKH26before transplanted into the seminiferous tubules of the busulfan-treated recipient mouse testis tissue,respectively.After 8 weeks,the numbers of fluorescently labeled porcine germ cell colonies were evaluated in dispersed seminiferous tubules of recipient testis tissue.The results showed that PLD6~+cells could colonize and proliferate in seminiferous tubules of recipient mice.Quantification of germ cell colonies showed that the PLD6~+cells generated over 9-fold more colonies than the unselected total testicular cell population,suggesting that PLD6~+population was significant enriched porcine spermatogonial stem cells.(5)The PLD6~+cells were cultured without feeder in vitro,and the cell types were identified by immunofluorescence.By establishing a feeder-free culture system,PLD6~+cells can be cultured for 4 weeks in vitro without differentiation,and expressed the molecular markers of undifferentiated spermatogonia throughout the culture period.This indicated that PLD6~+cells still had the characteristics of germ stem cells.In conclusion,this study demonstrated that PLD6 is a surface molecular marker of undifferentiated spermatogonia in testes of pre-pubertal pigs,and could be utilized to unprecedentedly enrich porcine undifferentiated spermatogonia.This research provide the basis for further studies on the cultivation of porcine undifferentiated spermatogonia,as well as provide a theoretical reference for future research of PLD6 in male germ cells of other species.