8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024