Proliferation And Differentiation Effects Of Spermatogenic Cells In Vitro And Micro-insemination

Objective: To investigate the proliferation and differentiation potential of newbornmouse testis spermatogenic cells in vitro by improved culture mediumin order topromote the survival, proliferative and differential rates as well as the survival time ofspermatogenic cells in vitro.Mthods: Mixed spermatogenic cells of7~8days mouses were cocultured in basicculture medium (group A), conditioned medium (group B)(basic culture medium+20ng/ml EGF+10ng/ml GDNF), basic culture medium contained with20%,40%and80%testicular abstract (TA)(group C1, C2and C3), respectively, and conditioned mediumcontained with20%,40%and80%TA (group D1, D2and D3), respectively. Theproliferative and differential effects of TA, GDNF and EGF on mouse spermatogeniccells in vitro were evaluaed by detecting growth behaviors, cell numbers and viability ofspermatogenic cells, chromosome ploidy distribution as well as expression ofpachytene-specific phosphoprotein gene (P19) and haploid sperm cell-specific transitionprotein gene (TP1).Results:①Morphological observation Cells division could be found after3～4days culture both in control and experimental groups. In group B, the cell colonies andsporadic round spermatids around colonies could be observed after7～8days culture,colony outbreak and elongating spermatids or spermatids with flagella were observedafter10days culture, and apoptosis of partial germ cells and sertoli cells appeared after20days culture. The growth velocity of spermatogenic cell in group C1, C2and C3aswell as in group D1, D2and D3fell in between group A and group B. A few coloniescontaining majority of dividing cells and few round spermatids could be observed after7～8days culture. The number of cell colonies increased obviously and spermatogeniccells of various stages could be observed after10days cultured, but the cell density waslower than group B. The whole bottom of culture dishes was filled with cell coloniesafter15days culture. Cells grew poorly in group A after passage, and cell growth stepdown significantly with dark colour and poor adhesion in group B after passage2～3generations, but the cell proliferation could still be observed in group C1, C2and C3aswell as group D1, D2and D3after4months subcultured.②Spermatogenic cell countThe spermatogenic cells of every culture stage in group B were significantly higher thanthe other groups (P＜0.05), while the spermatogenic cells in group A were significantlylower than other groups(P＜0.05). There were no significant difference among othergroups.③Spermatogenic cell survival rate Spermatogenic cell survival rate in groupA, significantly lower than group B after20days culture.showed no significantdifference from group B after5days,10days and15days culture. The spermatogeniccell survival rate of every culture stage in other groups showed no significant differencewith group A and B.④TP1/P19The TP1/P19in group A was significantly higher thangroup B on15th day culture(P＜0.05), which showed significantly higher than in groupB than other groups on20th day cultrue(P＜0.05). There was no significant differencein TP1/P19among the rest of groups.⑤The proportion of haploid sperm cells Theproportion of haploid sperm cells in group B was significantly higher than other groupson15th and20th day cultrue(P＜0.05), which showed no significant difference among other groups.Conclusion: Added appropriate amount GDNF and EGF into basic medium in mixedcells culture systems could significantly enhance proliferation and differentiation effects,but could not extend the duration of spermatogenic cells in vitro. The spermatogeniccell numbers and cultrue duration increased after added appropriate amount of TA intobasic medium. Added TA into the culture medium with appropriate concentrationcytokines such as EGF and GDNF could also significantly increased spermatogeniccells culture duration in vitro, but would decrease the proliferation and differentiationefforts of spermatogenic cells. Objective: To explore the fertilization potential of haploid spermatids andpostfertilization embryo development potential after micro-injection of round/elongated spermatids by observing the fertilization rate, cleavage rate and morula andblastula formation rate.Mthods: Mature sperms(A), round (RS)/elongated(ES) spermatids from adult mousetestis(group B) and mixed spermatogenic cells of7~8days mouse(group C) wereperformed intracytoplasmic microinjection, and the rates of fertilization, cleavage,morula and blastula formating were observed an compared.Results: Although the rates of4-8cell cleavage, morula and blastula formation ingroup B were higher than group C, but there was no significant difference in the ratesof fertilization, cleavage, morula and blastula formation between two groups,meanwhile the rates of fertilization, cleavage,4-8cell cleavage, morula and blastula formation in group A were significantly higher both than group B and group C.Conclusion: There was no significant difference in fertilizability between the haploidspermatides from adult mouse testis and spermatogenic cells in vitro culture, however,the former was superior in embryonic development potential to the latter. Themicro-fertilization potential and embryo development ability of mature sperms weresignificantly higher than haploid spermatides both from adult mouse testis andspermatogenic cells in vitro culture.