Divergent Vascular Endothelial Growth Factor a (VEGFA) Signaling Determines Spermatogonial Stem Cell Fate

The vascular endothelial growth factor A (Vegfa) gene can be spliced into divergent isoform family types- angiogenic and antiangiogenic. Our laboratory has been investigating the role of these sister isoform families in male fertility and the mechanisms by which they contribute to maintenance of spermatogonial stem cells (SCC). Our hypothesis is that there must be a balance of VEGFA angiogenic to antiangiogenic isoforms to maintain the spermatogonial stem cell niche. Specifically, angiogenic isoforms stimulate SSC renewal and proliferation while antiangiogenic VEGFA isoforms induce differentiation or apoptosis of male germ cells. Initial experiments involved postnatal mice (day 5-7) treated with either VEGFA165 angiogenic or VEGFA165b antiangiogenic isoforms whose germ cells were harvested at day 8 and then transplanted into recipient mice. Reduced colonization of SSCs was observed after 6 weeks in recipient males that received germ cells from donor mice testes that were treated with VEGFA165b. These data suggest that the antiangiogenic VEGFA isoforms may arrest SSC proliferation or renewal by causing apoptosis or by differentiating them into later-stage spermatogonia. Using conditional knockout mice we eliminated both VEGFA isoform types in Sertoli and germ cells (using Dmrt1-Cre) which resulted in reduced sperm counts despite compensatory increases in genes known to promote SSC maintenance (Ret, Sin3a, and Neurog3). Elimination of NRP1, a co-receptor that binds angiogenic VEGFA isoforms but not antiangiogenic isoforms, from Sertoli and Leydig cells (using Amhr2-Cre) reduced fertility, reduced angiogenic VEGFA signaling and downregulated genes important in SSC maintenance (Gdnf, Ret, Sin3a, Neurog3, Foxo1, and Kitl). Additionally, NRP1 loss reduced phosphorylation of RET, which is a receptor of GDNF a major initiator of SSC self-renewal. Interestingly, NRP1 also appeared to be co-localized with cells that were positive for ID4, a putative SSC marker, and there were reductions in ID4/NRP1 double positive cells following NRP1 elimination. Thus, elimination of NRP1 also appeared to accelerate putative SSC cell loss, possibly due to apoptosis or reduction of VEGFA angiogenic isoform survival functions. To compare the effects of all VEGFA isoforms to inhibiting just angiogenic isoform signaling, we used Sertoli cell-specific knockouts of both NRP1 and VEGFA (Sry-Cre). Males had reduced fertility in both cases. Preliminary findings from spermatogonial stem cell transplantation experiments indicated reduced SSC colonization in donor mice testes. Taken together, these findings further implicate the importance of regulating VEGFA signaling to ensure SSC maintenance and male fertility and suggest that VEGFA isoforms are regulators of the SSC niche.