Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

Abstract

Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr(407), known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons.

Transport of germ cells across the seminiferous epithelium during spermatogenesis—the involvement of both actin- and microtubule-based cytoskeletons

“…[55][56][57] It was noted that while spermatids near the tubule lumen at stage VI to early VIII were depleting from the epithelium, analogous to the release of sperm at spermiation, many step 19 spermatids remained entrapped deep inside the epithelium. 8 These step 19 spermatids were consistently found in the epithelium of stages IX, X, XI and XII tubules, until they were eventually engulfed and degraded by Sertoli cells. 8 More striking is the observation that the actin-based apical ES that anchored these spermatids deep inside the epithelium was found to be degenerated wherein the apical ES adhesion proteins (e.g., ß1-integrin, nectin-3) and the underlying F-actin were either downregulated or grossly disorganized or mis-localized.…”

“…8 These step 19 spermatids were consistently found in the epithelium of stages IX, X, XI and XII tubules, until they were eventually engulfed and degraded by Sertoli cells. 8 More striking is the observation that the actin-based apical ES that anchored these spermatids deep inside the epithelium was found to be degenerated wherein the apical ES adhesion proteins (e.g., ß1-integrin, nectin-3) and the underlying F-actin were either downregulated or grossly disorganized or mis-localized. 8 These findings suggest that even though apical ES and the underlying F-actin cytoskeletal network that supported step 19 spermatid adhesion had been compromised by adjudin treatment, these spermatids located deep inside the epithelium failed to be transported to the tubule lumen for their release, unlike those spermatids that resided near the tubule lumen which were emptied into the tubule lumen earlier.…”

“…(C) A simplified model on the support of cargo transport by myosin VIIa in the testis using the F-actin filaments as tracks. (D) Localization of F-actin (green fluorescence, visualized by FITC-phalloidin as earlier described 8 ) in the cross-section of an adult rat testis of a stage VIII tubule, when step 19 spermatids have lined up at the tubule lumen to prepare for their release at spermiation when F-actin expression at the Sertoli cell-step 19 spermatid interface begins to diminish. F-actin is notably detected at the BTB (annotated by yellow arrowheads) near the basement membrane.…”

“…A recent report, 8 using an animal model based on treatment of rats with adjudin (at 50 mg/kg b.w., by oral gavage) known to induce exfoliation of germ cells (beginning with elongating/elongated spermatids within »6 to 9 hr following drug administration, to be followed by round spermatids (in »3 days) and spermatocytes (in »6.5 days)), 53 due to initial disruption of the actin-rich testis-specific apical ES to be followed by desmosome and/or gap junction by this drug, 54 has shed light on the role of these cytoskeletons to support spermatid transport. This animal model, similar to other drug models such as treatment of rats with environmental toxicant cadmium or other toxicants (e.g., carbendazim, 2,5-hexanedione, colchicine), is known to induce germ cell exfoliation from the testis.…”

“…Studies have shown that germ cell transport during the epithelial cycle is supported by the intimate but also intriguing coordination between the microtubule (MT)-and F-actin-based cytoskeletons in Sertoli cells but also at the Sertoli-germ cell interface. [8][9][10] This likely involves MT-and F-actin-specific cytoskeletal regulatory proteins (e.g., nucleation proteins, severing, crosslinking, bundling proteins), motor proteins, and signaling proteins so that the organization of MTs and actin microfilaments can be rapidly altered, modifying from a linear and bundled network, to a branched and unbundled network. These changes, in turn, confer plasticity to the cytoskeletons, making them flexible enough to support the timely and efficient transport of germ cells across the BTB and/or the adluminal compartment during the epithelial cycle.…”