VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance

Abstract

Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo.

Keywords: Self-renewal; Spermatogonia; Testis; VEGFA164; VEGFA165b.

Fig. 1

Model of spermatogonial stem cell (SSC) homeostasis. SSCs are considered a subpopulation of the undifferentiated A spermatogonia and may divide into other SSCs, divide into progenitor cells more committed to differentiation, or undergo cell death. The various stages of A undifferentiated spermatogonia, even when chained, are also thought to separate and resume self-renewal capabilities to maintain a viable SSC pool (A_s [A_single] type A spermatogonia, A_pr [A_paired] type A spermatogonia, A_al [A_aligned] type A spermatogonia)

Fig. 2

Spermatogonial stem cell (SSC) niche comprised of Sertoli cells with contributions from blood vessels, peritubular myoid cells, and Leydig cells. Various factors that contribute either to self-renewal or to differentiation are depicted in the cells from which they are produced. The large inset SSC (right) shows the pro-renewal or pro-differentiation factors that are expressed by the germ cells

Fig. 3

Depiction of rodent VEGFA isoforms. Angiogenic isoforms include VEGFA205, 188, 164, and 120, whereas VEGFA189b, 165b, and 121b are antiangiogenic. Various growth factors activate serine-arginine protein kinase 1 (SRPK1), which phosphorylates serine/arginine-rich splicing factor 1 (SRSF1) to target the proximal splice site (PSS) of the Vegfa gene and generate angiogenic VEGFA isoforms. Known, biologically active, rodent, angiogenic VEGFA isoforms are 205, 188, 164, and 120. Inhibition of SRPK1 by specific inhibitors such as SRPIN340 favors distal splice site (DSS) selection and the generation of antiangiogenic isoforms. Antiangiogenic isoforms contain an 8b exon that is substituted for an 8a exon in angiogenic VEGFA isoforms. VEGFA206189b, 165b, and 121b are the most well-represented rodent antiangiogenic isoforms in the literature. Exons 6 and 7 encode the heparin-binding sites, whereas the neuropilin-1 (NRP1)-binding site is encoded by translation of the 8a exon; this is the reason that it cannot bind antiangiogenic VEGFA isoforms

Fig. 4

VEGFA signals through two tyrosine kinase receptors. Signaling of angiogenic isoforms of VEGFA is augmented by a membrane-bound co-receptor, NRP1, which either stabilizes ligand-receptor binding or presents the ligand to the kinase insert domain receptor (a, KDR, left). NRP1 also associates with VEGFA homodimers that bind to FMS-like tyrosine kinase 1 (a, FLT1, right). Antiangiogenic isoforms of VEGFA, however, are unable to bind to NRP1 but still bind either KDR (b, left) or FLT1 (b, right)

Fig. 5

Model of SSC homeostasis with VEGFA isoforms and NRP1 added. We propose that angiogenic isoforms such as VEGFA164 and NRP1 promote the renewal of SSCs or undifferentiated spermatogonia, whereas antiangiogenic isoforms such as VEGFA165b reduce SSC number through cell death or by promoting differentiation prematurely

Fig. 6

SSC niche with VEGFA isoforms added. We presume that angiogenic isoforms of VEGFA promote self-renewal of SSCs, whereas antiangiogenic isoforms of VEGFA promote their differentiation (or death), with these isoforms being predominantly secreted by the Sertoli cell. Furthermore, KDR and NRP1 are located on the germ cells themselves

Fig. 7

VEGFA165b increases TUNEL-positive staining in male germ cells. Micrographs are representative 100× images of testes from a phosphate-buffered saline (PBS, control)-treated mouse (a) and a VEGFA165b (1 µg)-treated mouse (b). Apoptotic spermatogonia fluoresced green (white arrows). The number of TUNEL-positive germ cells per tubule was counted for each treatment (c). Data were considered significant when P<0.05 according to Dunnett’s test in JMP statistical discovery software from SAS