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Divergent VEGFA signaling determines spermatogonial stem cell fate
The vascular endothelial growth factor A (Vegfa) gene can be spliced into angiogenic and antiangiogenic isoforms. Elimination of all isoforms from Sertoli and germ cells in mice altered expression of genes regulating spermatogonial stem cells (SSCs) and resulted in male subfertility. Treatment with antiangiogenic isoforms of VEGFA in donor mice reduced SSC colonization in recipient seminiferous tubules. Thus, we hypothesized antiangiogenic VEGFA isoforms reduce SSC numbers while angiogenic VEGFA isoforms contribute to SSC self-renewal. The objective of these studies was to determine how VEGFA signaling regulates SSC maintenance. Mouse models were utilized where all VEGFA isoforms or neuropilin-1 (NRP1), co-receptor that binds only angiogenic isoforms, was eliminated from the testis (KO). Each experiment had at least 10 animals, and data were analyzed using the Dunnett’s test in JMP. Elimination of NRP1 using Amhr2-Cre (Sertoli/Leydig) resulted in KO males siring fewer pups in all litters (P = 0.03) with a tendency for increased days to get females pregnant vs controls (P < 0.1). NRP1 KO males also tended to have reduced whole testis Gdnf mRNA, an effector of SSC self-renewal (P < 0.08) and increased Stra8, a pro-differentiation factor (P < 0.09) at 6 months. Their caput epididymides contained 36% fewer sperm (P < 0.05) compared to controls. Whole testis mRNA of SSC maintenance genes tended to be or were reduced in males from the same line at 3 months: Gdnf (P < 0.05); Ret (P < 0.09), Foxo1 (P = 0.003); Sin3a (P < 0.04), Kitl (P < 0.1), and Neurog3 (P < 0.01). Zbtb16 expression (P < 0.001) and ZBTB16+ undifferentiated spermatogonia per tubule were increased in KO males (P < 0.002). There were fewer spermatogonia that co-expressed ZBTB16 and ID4 (P < 0.05), a potential SSC marker, suggesting fewer viable SSCs. NRP1 loss also reduced phosphorylation of RET (P = 0.04); and pull down experiments demonstrated that RET and KDR, VEGFA receptor, co-immunopreciptate, suggesting they interact. Loss of either VEGFA or NRP1 isoforms via Sry-Cre (Sertoli) resulted in reduced male fertility vs controls and recipient tubules devoid of germ cells when used as SSC donors at postnatal day (P) 30 or 60. VEGFA loss caused a compensatory increase in SSC renewal genes Id4 (P < 0.009) and Bcl6b (P < 0.003) at P60, and NRP1 loss increased expression of the pro-differentiation gene, c-Kit (P< 0.04). Thus, VEGFA signaling is critical for regulating SSC maintenance and male fertility.
Keywords: testis, spermatogonial stem cell, VEGFA, NRP1