Testis-specific actin-like 7A (ACTL7A) is an indispensable protein for subacrosomal-associated F-actin formation, acrosomal anchoring, and male fertility

Abstract

Formation of the acrosome during spermiogenesis is an essential process for creating fertilization competent sperm. Of the numerous aspects required for acrosome biogenesis, adherence of the acrosomal outer membrane to the nuclear surface is mediated by the subacrosomal perinuclear theca. However, the cellular dynamics and congruent functions pertaining to these acrosomal anchoring factors are not well understood despite many of them being implicated as potential causes for human male infertility. Actin-like protein 7A (ACTL7A) is one such factor for which deleterious polymorphisms have recently been shown to cause human male infertility. It is thought that acrosomal attachment is coordinated by cytoskeletal associations between the acrosome and nucleus via the acroplaxome. To further illuminate the mechanistic underpinnings of ACTL7A for essential acrosome associations, in this study we investigated its dynamic localization in the developing germline, molecular associations with other cytoskeletal components, and the cellular consequences of ablation. Our intracellular localization data shows ACTL7A to be dynamically present within the nucleus and subacrosomal space and later associated with postacrosomal regions of developing spermatids. Through the generation of an Actl7a knock-out mouse model, we consistently observed disruption of acrosomal biogenesis with abnormal migration of the acrosomal granule, and peeling acrosomes during spermatid elongation. Significantly, we found a complete loss of subacrosomal filamentous actin (F-actin) structures in knock-out spermatids suggesting a regulatory role for subacrosomal F-actin. Considering our reported data together with existing literature, we propose a mechanistic model explaining the essential role for ACTL7A for acroplaxome associated F-actin, acrosomal attachment integrity and male fertility.

Novel Nuclear Roles for Testis-Specific ACTL7A and ACTL7B Supported byIn VivoCharacterizations and AI FacilitatedIn SilicoMechanistic Modeling with Implications for Epigenetic Regulation in Spermiogenesis

“…As a first step in establishing in situ evidence for the potential of ACTL7A and ACTL7B to contribute to nuclear regulation, we also provide here evidence of nuclear localization and trafficking regulation, transcriptional consequences of targeted ablation in mouse models, and ACTL7A and ACTL7B dependent nuclear association of regulatory HDACs. This builds on our previous work demonstrating that these two ARPs are both separately causative of male infertility in KO mouse models exhibiting ultrastructural defects to the actin-associated acrosomal cytoskeleton (Ferrer et al, 2023) and the flagellum . Given the diverse and at times unexpected roles that many ARPs have been observed to have, we hypothesized that ACTL7A and ACTL7B may functionally play roles beyond observed structural and cytoplasmic roles in spermiogenesis.…”

“…3A), where this nuclear associated localization appeared mottled rather than equally diffuse. Of note, this mottled nuclear expression pattern for ACTL7B expressing HEK cells was consistently observed and did not exhibit the three specific differential nuclear associated localization patterns previously observed in ACTL7A transfected HEK cells (Ferrer et al, 2023). We next assessed localization of the actin and N-terminal subdomains.…”

“…It is possible that ACTL7A is still present in the sperm of 7B KO sperm at low levels insufficient to be detected in standard western blotting practices. Alternatively, it may be that in the absence of ACTL7B the ACTL7A remaining in 7B KO spermatids is not properly incorporated or stabilized into the postacrosomal sheath where ACTL7A has been shown to reside in sperm (Ferrer et al, 2023). Additionally, any ACTL7A still present in 7B KO sperm could be lost during epididymal transit or during cell isolation procedures given the extreme morphological deformities and general fragility that 7B KO sperm portray (Clement et al, 2023).…”

“…Single cell suspensions were prepared as previously described (Ferrer et al 2023). Briefly, adult murine testes with their tunica albuginea removed were placed in 10 ml of Krebs-Ringer bicarbonate buffer (EKRB) (Bellve et al in 1977) and subjected to enzymatic digestion with 2.5 mg of collagenase IV (#SCR103, Sigma-Aldrich, Saint Louis, MO, USA) for 15 min within a shaking incubator at 37°C, 5% CO2, and 150 rpm.…”

“…Both ACTL7A and ACTL7B are testis-specific members of the larger ARP family and are required for male fertility. Our previous findings in knock out mouse models demonstrated that ACTL7A is required for subacrosomal F-actin formation and acrosome stability, and that ACTL7A is present in the intranuclear space of both spermatocytes and round spermatids (Ferrer et al, 2023). Furthermore, our characterization of ACTL7B as a key influencer of sperm formation and flagellar structural integrity included preliminary observations of nuclear localization (Clement et al, 2023; Ferrer et al, submitted).…”