Isolation And Culture Of Bovine Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs), the postnatal male germ-line stem cells, self-renew throughout life and, after puberty, provide daughter cells that differentiate into spermatozoa. Up to now, spermatogonial stem cells and haemotopoietic stem cells are the only two types of adult stem cells which can be detected by their function, and moreover the former is the only type of stem cells which can be morphologically identified by intercellular bridges. The spermatogonial stem cell, therefore, represents a useful model for studies of biology of the adult stem cells. Recently, remarkable progress has been made in this field with the aid of SSCs transplantation technique and other stem cell related techniques. The ability to isolate/enrich, characterize, culture, cryopreserve, modify genetically, and transplant these unique cells provides a powerful tool for the studies of stem cell biology, therapy of male infertility, preservation of endangered animals, generation of transgenic animals, and breeding of farm animals with superior characters.At present, the research of SSCs has transferred from mice to livestock and economical animal and some progress had been made. However, the study of bovine SSCs is at its initial stage and there are a lot of questions need to be probed, such as a short survival time in vitro, limited capability of proliferation and division, lack of SSCs line with stem cell potential,etc. Nowdays, internal SSCs reseachs mainly focused on small sized experimental animals and human. SSCs reseaches of large domestic animal such as pig, cattle are not reported in our country until now.In this research, based on a systematic discussion of SSCs researches of lagrge domestic animals, the effects of three penetrating cryoprotectants, dimethyl sulfoxide(DMSO), propylene(PE) and ethylene(GE) on the cryopreservation of bovine testes tissue were examined firstly. Then the effects of concentration of newborn bull serum(NBS) and different thawing temperature were tested. Subsequently, adult bovine seminiferous epithelium were dispersed by collagenase-trypsin and cultured in varied conditions to investigate the effects of Sertoli cells and serum concentration on the survival and proliferation of bovine SSCs. The seminiferous epithelium of newborn bull were also disgested, cultured and their growth behavior were observed.From our experiment ,we can result that: the highest cell recovery rates of DMSO, PG and EG were 85.3%, 82% and 83.4% at concentrations of 10% respectively;The proportions of spermagonia after thawing were 61.1%, 54.3% and 55% res pectively; the statistics analysis showed that there was no significant differences between them(P>0.05);testicular tissue cryopreserved in freezing solution containing 10% DMSO+80%MEM+10%NBS were thawed at 4℃, 37℃and 97℃~100℃respectively, the cell recovery rates were 23.4%, 85.1% and 85% respectively;there was no significant difference between the last two groups but their cell recovery rates were significantly higher than that of under 4℃(P<0.01);the recovery rates of cells frozen in freezing solutions containing 0, 5%, 10%, and 20% of NBS were 84.6%, 84.2%, 85.5% and 83.4% respectively and no significant difference among them(P>0.05);in our co-culture system, bovine SSCs attached quickly and and they proliferated and divided obviously faster than that in solo culture system;when bovine SSCs were cultured in media without serum, they would soon be died. With 2.5% serum, SSCs could survive for a long time and proliferate conspicuously. But with 5% and 10% of serum, the preliferation of somatic cells was more obvious than SSCs;GDNFR PCR amplificated the reverse transcription of total RNA of cultured SSCs and an objective cDNA fragment of 342bp was obtained;CD9 immuocytochemistry results showed that the round spermatogonia were CD9+ while the cells in control group wereCD9-;when the calf seminiferous epithelium cells were cultured in vitro, gonocytes could survive and proliferate, but they didn't show some characteristic growth behavior similar to SSCs during culture.From our experimental result,we can conclude that : DMSO at concentration of 10%~15% was the best cryopretectant for cryopreservation of bovine seminiferous epithelium tissue;37℃~100℃water bath could be used to thaw cryopreserved bovine testis tissue; NBS concertration had no significant effects on the cell recovery rates of cryopreserved bovine testis tissue after thawing;in Sertoli cells-SSCs co-culture system, Sertoli cells could enhance the survival and proliferation of SSCs;MEM containing 2.5% serum provided an optimal condition for survival and proliferation of bovine SSCs, and it also avoid the rapid growth of testis somatic cells;the specific surface markers of CD9 and GFRalpha-1 could be used to identify bovine SSCs in vitro;bovine gonocytes couldn't differentiate into SSCs in vitro culture given no any other additives were used.