Abstract
A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals.
Highlights
In the adult mouse testis, spermatogenesis originates from spermatogonial stem cells (Asingle, As), that can self-renew or differentiate into committed paired (Ap) and aligned (Aal) spermatogonia
In this work we show that mouse spermatogonia, cultured under simulated microgravity (SM) conditions in rotary cell culture system (RCCS), undergo a spontaneous differentiation and entry into the early prophase of the first meiotic division
After 48 h of culture of isolated differentiating Kit-positive spermatogonia in RCCS, we found a dramatic increase in the number of meiotic nuclei, most of which were at the leptotene stage
Summary
In the adult mouse testis, spermatogenesis originates from spermatogonial stem cells (Asingle, As), that can self-renew or differentiate into committed paired (Ap) and aligned (Aal) spermatogonia. The heterogeneous population of germ cells, including stem cells and committed spermatogonia, is collectively called undifferentiated spermatogonial population, or Kit-negative spermatogonia. This population expresses different stem cell markers (such as Plzf, Oct, Nanos3) but does not express the Kit tyrosine-kinase receptor [1,2,3,4]. We recently demonstrated that, in postnatal testis, ATRA increases meiotic entry of differentiating spermatogonia in vitro by activating the Kit signalling pathway [10] and by stimulating a significant increase of Stra, a fundamental regulator of meiosis in both female and male mice [11,12]. To ATRA, addition of KL, a growth factor essential for survival and proliferation of Kit positive germ cells [5,13,14], increases the percentage of meiotic nuclei in cultured spermatogonia, concomitantly with an upregulation of Stra8 [10]
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