Mammalian testes utilize multiple modalities to moderate spermatogonial responsiveness to retinoic acid

Abstract

What is knownThe foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia that proliferate before responding to retinoic acid (RA). RA converts undifferentiated into differentiating spermatogonia committed to entering meiosis. This RA‐regulated fate transition is essential for maintaining normal ratios of spermatogonia during steady‐state spermatogenesis in adult testes. Imbalances in spermatogonial populations often result in infertility – enhanced differentiation leads to eventual germline depletion, while impaired differentiation increases the likelihood of developing testicular cancer.Study ObjectiveDefine the extent cell autonomous and non‐autonomous mechanisms in adult mammalian spermatogonia regulate their response to RA.HypothesisOnly spermatogonia primed to differentiate will respond to RA and precociously progress through spermatogenesis.MethodologyAdult mice were treated in vivo with vehicle (DMSO), RA, or talarozole (RA catabolism inhibitor) and euthanized 12‐48 hours later. Testes were removed and various assays used to assess populations for responsiveness to RA and capacity to precociously differentiate and enter meiosis. Concurrently, a model of synchronized spermatogenesis was used to define the potential of sequential subsets of differentiating spermatogonia to respond to RA and precociously enter meiosis. In complementary ex vivo experiments, mouse testes and patient‐derived testicular biopsies were cultured with DMSO‐alone, RA, and/or talarozole for 6‐24 hours to evaluate RA responsiveness and changes in spermatogonia fate using immunostaining and qRT‐PCR.Main resultsExogenous RA elicited a rapid (12 hour) and significant increase in expression of the RA‐responsive gene ‘stimulated by retinoic acid 8’ (STRA8) in undifferentiated (ZBTB16+) spermatogonia. STRA8 expression decreased over the next 36 hours, such that numbers of STRA8+/ZBTB16+ spermatogonia were comparable in testes from vehicle‐ and RA‐treated mice. This transient increase in STRA8 affected spermatogonia at all stages of spermatogenesis. Exogenous RA also elicited STRA8 expression in differentiating (KIT+) spermatogonia in adults and synchronized mice. Despite this capacity for responding to exogenous RA, obvious precocious progression was not observed within the testis. Talarozole treatment elicited STRA8 expression in spermatogonia akin to RA treatments. Finally, qRT‐PCR showed adult mouse and human testes regulate responsiveness to RA similarly, based on mRNA expression profiles.ConclusionIn response to exogenous RA, undifferentiated spermatogonia transiently upregulate expression of RA‐responsive genes, but this did not result in precocious progression into meiosis.ImplicationsThe adult mammalian testis is a well‐buffered system in which exogenous RA has little effect on cell fate, thus preserving the normal timing and progression of spermatogenesis.