FGF2 Has Distinct Molecular Functions from GDNF in the Mouse Germline Niche

摘要

class="head no_bottom_margin" id="sec1title">IntroductionSpermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia and the origin of spermatogenesis. They reside in a specialized microenvironment called the germline niche located at the periphery of seminiferous tubules in mammalian testes. The germline niche permits SSCs to produce both stem cells and committed progenitors called differentiating spermatogonia by repeated self-renewal and differentiation. Differentiating spermatogonia further amplify their population by several mitotic divisions and enter meiotic division to produce spermatozoa ().It is well established that glial cell line-derived neurotrophic factor (GDNF) ensures the survival and self-renewal of SSCs. demonstrated that GDNF is indispensable for SSC self-renewal by analyzing Gdnf transgenic/deficient mice. took advantage of GDNF functions to establish cultured SSCs termed germline stem (GS) cells from mouse pup testes. This technique enables identification of cytokines and chemicals that affect the behavior of SSCs.Our recent study revealed that fibroblast growth factor 2 (FGF2) is another bona fide self-renewal factor for SSCs (). FGF2-cultured spermatogonia (F-SPG) cultured for more than 4 months under GDNF-free conditions colonized infertile mouse testes and restored fertility by spermatogonial transplantation. Compared with GDNF-cultured spermatogonia (G-SPG), F-SPG exhibit more differentiated characteristics such as lower SSC activity and higher KIT expression in addition to a difference in MAP2K1/2 dependency. These data suggest that FGF2 plays a distinct role in regulating undifferentiated spermatogonia in vivo.It is also known that retinoic acid (RA) is required for spermatogonial differentiation (). Of three RA receptor isotypes, retinoic acid receptor γ (RARG) has been reported to specifically contribute to this process (). Recently, demonstrated that RARG expression is sufficient for induction of RA susceptibility in undifferentiated spermatogonia expressing GFRA1 that triggers GDNF signaling in combination with transmembrane receptor tyrosine kinase RET (). For further understanding, it should be elucidated how RARG⁺ spermatogonia are derived from GFRA1⁺ undifferentiated spermatogonia and how RA signals are regulated within the germline niche.Although FGF2 is considered to be a possible candidate that induces the RARG⁺ subset of undifferentiated spermatogonia, the absence of an animal model has hampered the analysis. Fgf2-transgenic mice are fertile and not reported to have abnormal testes (). This is because transgenic mice might not show extremely strong transgene expression depending on the gene. Furthermore, FGF2-knockout mice show no obvious defects in testicular functions (), which may be because of functional complementation by other FGF molecules.To overcome such circumstances, we used the biodegradable gelatin microsphere (BGM) system to provide strong FGF2 stimuli only in the testis. This system was established and reported primarily by . BGMs were prepared by glutaraldehyde-mediated crosslinking of acidic gelatin (microsphere diameter: 30–100 μm). FGF2 can be adsorbed onto BGMs and released depending on the biodegradation of BGMs (). BGMs were first applied to deliver FGF2 into the skin to induce neovascularization () and later applied to deliver bone morphogenetic protein 2 and transforming growth factor β1 for heart and bone regeneration (, , href="#bib21" rid="bib21" class=" bibr popnode">Marui et al., 2007). href="#bib34" rid="bib34" class=" bibr popnode">Uchida et al. (2016) also employed a similar system using different material to induce hyperproliferation of undifferentiated spermatogonia by GDNF treatment, as observed in the previous report of href="#bib22" rid="bib22" class=" bibr popnode">Meng et al. (2000). These reports encouraged employment of the BGM system.In this study, we analyzed the molecular functions of FGF2 in the mouse germline niche using BGMs for direct FGF2 stimulation in vivo. Although we found that FGF2 expanded GFRA1⁺ spermatogonia, these cells exhibited a phenotype distinct from that of GDNF-expanded GFRA1⁺ spermatogonia. Moreover, FGF2 modified germline niche functions to be more appropriate for spermatogonial differentiation.