Investigating the roles of cytoskeletal regulators Cylicin 1, Cylicin 2 and actin-related protein M1 in sperm development and function

Abstract

Mammalian germ cells express a vast set of genes specific to the testis, required for the development of sperm cells with their unique structure and function. Sperm-specific cytoskeletal regulators play essential roles in male fertility. Although emerging studies have been investigating these genes, the roles of many of them remain to be elucidated. <br/> Cylicins are testis-specific genes, exclusively expressed during spermiogenesis in mammals. Two Cylicins are identified in mouse and human testis: Cylicin 1 and Cylicin 2. Cylicins are cytoskeletal proteins with lysine-rich repeats, and they are localized in the perinuclear theca (PT) of spermatids and mature sperm. To investigate their roles in male fertility, <em>Cylc1-</em> and <em>Cylc2</em>-deficient mouse lines were established using CRISPR/Cas9 gene editing. <em>Cylc1</em>-deficiency caused male subfertility, while <em>Cylc2</em>^-/-, <em>Cylc1</em>^-/y <em>Cylc2</em>^+/- and <em>Cylc1</em>^-/y <em>Cylc2</em>^-/- male mice were infertile. The most prominent phenotypical observation upon loss of Cylicins in the impaired assembly of posterior region of the PT – calyx. Furthermore, reduced counts of epididymal sperm and severe morphological abnormalities of the sperm head and tail resulted in reduced sperm motility. Proteomics analysis revealed that loss of Cylicins leads to drastic changes in abundance of various proteins within the sperm cytoskeletal compartment, suggesting that Cylicins have a role in maintenance of the sperm head structure and protein composition. Of note, an infertile patient carrying with a hemizygous variant in <em>CYLC1</em> and a heterozygous variant in <em>CYLC2</em>, and with morphological anomalies of the sperm was identified, highlighting the relevance of Cylicins in human sperm. <br/> Next, a mouse model with a null allele for <em>Arpm1</em> was analyzed. Actin-related protein M1 (ARPM1) is a testis-specific member of Actin-related protein family which can be found in the PT of round to elongating spermatids in mouse. <em>Arpm1</em>-deficient male mice were subfertile, displaying morphological anomalies of the acrosome in roughly 50% of the sperm cells. Loss of ARPM1 leads to deregulation of <em>cis</em>- and <em>trans</em>-Golgi trafficking which are required for proper acrosome development during spermiogenesis. Co-immunoprecipitation revealed that ARPM1 interacts with other proteins that localize in the PT (ACTRT1, ACTRT2, ACTL7A) and the sperm surface protein ZPBP, additionally to its already known interaction with PFN3. Based on these results, the role of ARPM1 as a structural component of the PT was proposed to contribute to the cytoskeletal scaffold that connects acrosome and sperm nucleus. In addition, ARPM1 stabilizes the localization of ZPBP to facilitate fertilization, and it tethers PFN3 which regulates Golgi-trafficking during acrosome biogenesis. <br/> Taken together the results of this research project represent a significant step forward in elucidating the role of PT proteins in sperm function and male fertility.