Abstract
Glial cell line-derived neurotrophic factor (GDNF) is essential for the self-renewal and proliferation of spermatogonial stem cells (SSCs) in mice, rats, and rabbits. Although the key extrinsic factors essential for spermatogonial proliferation in other mammals have not been determined, GDNF is one of the potential candidates. In this study, we isolated porcine GDNF (pGDNF) cDNAs from neonatal testis and generated recombinant pGDNF to investigate its biological activity on gonocytes/undifferentiated spermatogonia, including SSCs. In porcine testis, long and short forms of GDNF transcripts, the counterparts of pre-(α)pro and pre-(β)pro GDNF identified in humans and rodents, were expressed. The two transcripts encode identical mature proteins. Recombinant pGDNF supported proliferation of murine SSCs in culture, and their stem cell activity was confirmed by a transplantation assay. Subsequently, porcine gonocytes/undifferentiated spermatogonia were cultured with pGDNF; however, pGDNF did not affect their proliferation. Furthermore, GDNF expression was localised to the vascular smooth muscle cells, and its cognate receptor GFRA1 expression was negligible during spermatogonial proliferation in the testes. These results indicate that although pGDNF retains structural similarity with those of other mammals and conserves the biological activity on the self-renewal of murine SSCs, porcine SSCs likely require extrinsic factors other than GDNF for their proliferation.
Highlights
Glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGFß superfamily, was originally discovered as a survival factor for dopaminergic neurons[1]
Pre-(α)pro GDNF is 26 amino acids longer than pre-(β)pro GDNF, the two transcripts produce an identical mature GDNF after post-translational processing. This is because the pre-(β)pro porcine GDNF (pGDNF) is generated by alternative splicing of the exon encoding the pro-domain and thereby shorten 26 amino acids of pro-domain of the pre-(α)pro pGDNF
We examined whether pGDNF has the ability to expand spermatogonial stem cells (SSCs) in vitro and evaluated its role in spermatogonial proliferation in porcine testes
Summary
Glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGFß superfamily, was originally discovered as a survival factor for dopaminergic neurons[1]. On the other hand, when testis cells from non-rodents such as domestic animals and primates were introduced into recipient mouse testes, colonization and proliferation of spermatogonia were observed, no donor-derived spermatogenesis was reconstituted[15,16,17,18] These results suggested that exogenous factors for spermatogonial proliferation are conserved between mouse and non-rodent mammalian species, but differentiation factors are species-specific. Following transplantation into immunocompromised mouse testes, the cultured rabbit undifferentiated spermatogonia were shown to colonize in the recipient seminiferous tubules and developed clusters of spermatogonia, which retained the original phenotype and maintained at least for six months[19] These results indicate that self-renewing proliferation of rabbit undifferentiated spermatogonia requires GDNF and suggest that they likely contained SSCs because of their long-lived nature. Expression of pGDNF in postnatal testes was investigated to evaluate its role in spermatogonial proliferation in the testes
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