Sperm capacitation is associated with phosphorylation of the testis-specific radial spoke protein Rsph6a†

Abstract

Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remain largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, Western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.

Study of expression of genes potentially responsible for reduced fitness in patients with myotonic dystrophy type 1 and identification of new biomarkers of testicular function

“…Currently, there are no human studies in the literature. This protein is important for normal ciliary and flagellar function (Abbasi et al, ; Eriksson, Ansved, Anvret, & Carey, ; Paudel et al, ). In humans, the expression of RSPH6A is testis‐specific, and the protein localizes in particular in the sperm flagella.…”

“…Previous studies demonstrated that RSPH6A is specifically expressed in human testes and that the knockout (KO) male mice are infertile due to short, immotile SPZ. However, currently little is known about the role played by RSPH6A protein in the mammalian flagellar axoneme and its implication in male fertility (Abbasi et al, ; Paudel et al, ).…”

CRISPR/CAS9-mediated amino acid substitution reveals phosphorylation residues of RSPH6A are not essential for male fertility in mice†

“…Super-resolution imaging with stochastic optical reconstruction microscopy suggests that the catalytic PKA subunit can be localized in the sperm axoneme [ 7 ]. RSPH6A localized in the axoneme was identified as a PKA substrate candidate using phospho-proteomic analysis [ 2 ]; however, this study reveals that S17 and S20 of RSPH6A are not essential for male fertility. As is the case for RSPH6A [ 4 ], disruption of axonemal proteins often leads to impaired flagellar formation [ 8 ], which makes it challenging to analyze their function in mature spermatozoa using a straight KO approach.…”

“…Although it is well known that phosphorylation events that start with protein kinase A (PKA) are essential for capacitation [ 1 ], downstream phosphorylated molecules and their functions are not well understood. Using phospho-proteomic and subsequent biochemical analyses, we previously identified phosphorylation residues (S17 and S20) of radial spoke head 6 homolog A (RSPH6A) that were associated with capacitation [ 2 ]. RSPH6A is an evolutionarily conserved and testis-enriched protein, and thought to be a component of the radial spokes in the flagellar axoneme [ 2–4 ].…”

“…Using phospho-proteomic and subsequent biochemical analyses, we previously identified phosphorylation residues (S17 and S20) of radial spoke head 6 homolog A (RSPH6A) that were associated with capacitation [ 2 ]. RSPH6A is an evolutionarily conserved and testis-enriched protein, and thought to be a component of the radial spokes in the flagellar axoneme [ 2–4 ]. In a previous study, we generated Rsph6a knock-out (KO) mice to analyze its function; however, because Rsph6a KO spermatozoa exhibited abnormal flagellar formation [ 4 ], this hampered the analysis of RSPH6A in sperm capacitation.…”