Regulation of the cytoskeleton during spermatogenesis

Abstract

Spermatogenesis is a complex process that takes place in the seminiferous tubules of the mammalian testes to produce spermatozoa. The epithelium of the seminiferous tubules is comprised of germ cells (GC), at different stages of development, and Sertoli cells (SC). The SC has the task of creating the blood-testis barrier (BTB), providing nutritional support to GCs, and supporting GC transport across the BTB and epithelium. As the SC is involved in such critical and dynamic processes, it is conceivable that SCs are tightly regulated in order to ensure the cellular events of spermatogenesis are carried out flawlessly. A dynamic cytoskeleton enables the SC to do this. Of the three major classes of filamentous proteins in the SC cytoskeleton: actin filaments, intermediate filaments, and microtubules (MT), actin filaments are the best-studied component in SCs. However, the cytoskeleton cannot be fully understood by looking at its individual components, because they all work together to form the cytoskeleton. Researchers across different fields have found that the relationship between actin filaments and MTs is greater than originally thought. The current understanding of SC MTs is that they are essential for structural support of the cell and providing tracks for GC transport across the epithelium, but, beyond this function, little is known about their regulation and interaction with other cytoskeleton components. Herein it is proposed that SC MTs are involved in regulating the ectoplasmic specialization (ES) and BTB, which are actin-rich cell junction sites within the seminiferous epithelium, as well as transport during spermatogenesis. The studies presented here aim to address the regulation of SC MTs during spermatogenesis.

The functions of MT regulatory proteins: MAP/MT affinity-regulating kinases 2 and 4 (MARK2, MARK4) and end-binding protein 1 (EB1), were investigated. MARK2 and MARK4 were shown to be involved in maintenance of the ES and BTB by regulating MTs. Knockdown of MARK2 versus MARK4 in vitro by RNAi in cultured rat SCs revealed that the isoforms differentially regulated the SC ES and BTB. Loss of MARK2 function led to perturbation of TJ-permeability barrier via disruption of TJ protein distribution. Similarly, MARK4 knockdown perturbed the TJ-permeability barrier, however effects were more pronounced than in MARK2 RNAi cells, as its knockdown led to mislocalization of basal ES and TJ proteins. Knockdown of EB1 also perturbed the TJ-permeability barrier by causing mislocalization of junction proteins. Reduction of either MARK2, MARK4, or EB1 disrupted MT distribution throughout SCs, leading to mislocalization of junction proteins. Additionally, it was discovered that alterations in MT arrangement in SCs affected the actin cytoskeleton, which directly supports BTB function. Following these studies, the adjudin model was used to relate these findings to in vivo conditions. Adjudin is a potential male contraceptive and disrupts actin-based adhesion to cause premature GC/spermatid loss. Despite actin adhesion disruption, some spermatids failed to be transported out of the epithelium by the MT tracks, which were also disrupted. Findings from these studies provide evidence that MT and actin cytoskeletons work in coordination to regulate the ES, BTB, and transport during spermatogenesis.