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. 2019 Feb 1;100(2):440-454.
doi: 10.1093/biolre/ioy202.

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

Bidur Paudel  1 , María Gracia Gervasi  1 , James Porambo  2 , Diego A Caraballo  3 , Darya A Tourzani  1 , Jesse Mager  1 , Mark D Platt  2 , Ana María Salicioni  1 , Pablo E Visconti  1
Affiliations

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

Bidur Paudel et al. Biol Reprod. .

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 remains 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.

Keywords: axoneme; capacitation; phosphorylation; radial spoke; sperm.

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Figures

Figure 1.
Figure 1.
Enrichment of Rsph6a phosphopeptide in capacitated mouse sperm. Single ion chromatograms for the differentially labeled phosphopeptide RApSQGpSeR from (A) the d0 capacitated/d3 noncapacitated analysis and (B) the d3 capacitated/d0 noncapacitated analysis. Predicted monoisotopic masses for the ions of type b and y are shown above and below the sequences, respectively. Ions observed in the associated MS/MS spectrum are underlined and those that lose phosphoric acid are presented in bold type. The label “Δ” denotes loss of phosphoric acid from the corresponding ion of type b or y. “R-OMe” represents the Fisher esterified C-terminal arginine, while “e” represents the Fisher esterified glutamic acid. Masses for b and y ions, which are shifted by +3 amu in the deuterated spectra, are presented in blue, while those ions shifted by +6 amu are presented in red. The Cap/NCap ratio for each analysis (11.44 in experiment A and 7.94 in experiment B) was used to calculate an overall odds ratio of 9.53, in order to remove potential errors associated with the comparison of the deuterated and nondeuterated peptide species [8]. Spectra shown are representative of at least three separate biological replicates, run under two conditions (d0 and d3) as explained above.
Figure 2.
Figure 2.
(A) Rsph6a isoform 1 and isoform 2 amino acid sequences. Antibodies epitopes are underlined in each isoform (E2 or E3). The sequence corresponding to the phosphopeptide detected by MS/MS is shown in red, boldface, and larger font. (B) Chordates phylogeny showing the origin of mammalian RSPH6A and RSPH4A. The chordates common ancestor had one RSPH gene (indicated as ancestral RSP), which was duplicated along the Therian (marsupials and placental mammals) branch. Each copy acquired an extra N-terminus. Within placentals, one copy retained the canonical function (RSPH4A) while the other is tissue-specific (RSPH6A) as a result of a neofunctionalization process. In marsupials, RSPH6A is no longer a functional gene as a consequence of a pseudogenization process.
Figure 3.
Figure 3.
Rsph6a is expressed in spermatocytes. (A) Schematic diagrams showing two mouse Rsph6a isoforms (I1 and I2). Isoform I2 is formed by splicing of a 765-bp exon in isoform I1. Numbers inside exons indicate exon order (bottom). Same color arrows indicate exon-specific forward and reverse primers designed along the length of the +Rsph6a gene (top). Numbers above each exon represent exon size. (B) Rsph6a gene expression analysis of several mouse tissues by RT-PCR using exon-specific primers as shown in (A). Primers used for Rsph4a are listed in the Methods section. (C) Rsph6a gene expression analysis of mouse testes at various developmental ages by RT-PCR using exon-specific primers as shown in (A). For both RT-PCR reactions, β-actin was used as a loading control. (D) Quantitative PCR analysis on mouse testes using F4/R4 primers. Shown are pooled results from three biological replicates. For each biological replicate, three reactions were run per assay, and the same experiment was repeated on separate dates at least two times. For normalization, β-actin was run in parallel along with other tissues and the values obtained for 4.0-week-old mouse testis were used as the “calibrator”; the 2formula image-ΔΔCT method (fold change) was applied to report mRNA expression. Error bars represent the standard error of the means (±). Statistical differences in expression are indicated by asterisks (P < 0.05) when compared to Rsph6a expression found in 1- to 2-week-old mouse testes. (E) In situ hybridization showing expression of Rsph6a (left panel a and b) and Tssk6 mRNA (right panel c and d) in mouse testis sections. Black rectangular boxes represent enlarged portion (insets); black arrows show Rsph6a-positive secondary spermatocytes, and Tssk6-positive postmeiotic spermatids, respectively. Scale bars, 50 μm.
Figure 4.
Figure 4.
Validation of a custom-made anti-Rsph6a antibody (E3) in recombinant Rsph6a isoforms and in mouse sperm. (A) Representative western blot showing detection of recombinant Rsph6a isoforms (I1 and I2) when incubated with E3 antibody alone (A, left panel) or in the in the presence of 200-molar excess of blocking peptide (P1) (A, right panel). (B) Protein lysates extracted from mouse sperm treated under capacitating (C) or noncapacitating (N) conditions were subjected to western analysis and detection of Rsph6a was done with mAb E3 antibody. In B (left panel), three distinct proteins are shown upon detection with mAb E3. Antibody competition with the E3 antigenic peptide (P1) is shown in B (right panel). PVDF membranes were stripped and sequentially probed with anti-tubulin antibody as loading control. (C) Immunofluorescence detection of Rsph6a in mouse sperm when probed with mAb E3 alone or in the presence of blocking peptide: (a, e) DIC images; (b) immunofluorescence with E3 alone or (f) when incubated with blocking peptide (E3-P1); (c) merged a-b; (d) inset showing magnified image from (c). Scale bars, 20 μm. Western blotting and Immunofluorescence images shown are representative of at least three independent experiments.
Figure 5.
Figure 5.
Identification of phosphorylated Rsph6a by two-dimensional gel electrophoresis. (A) 2D-PAGE of mouse sperm lysates under noncapacitating (NCAP) or (B) capacitating (CAP) conditions probed with mAb E3 antibody. (C) 2D-PAGE analysis of mouse sperm lysates incubated under capacitating conditions in the presence of 50 μM H-89 inhibitor (CAP + H-89) or (D) 50 μM of LRE1 inhibitor (CAP + LRE1) probed with mAb E3 antibody. Numbers indicate the Rsph6a spots at different isoelectric points, and changes in phosphorylated Rsph6a are observed upon each treatment. Images are representative results of at least three independent experiments.
Figure 6.
Figure 6.
Model. (A) Schematic representation of the mouse sperm. The head and flagellar regions mid-piece, principal piece, and end piece are shown. A dotted line indicates the cross-section schematized in B. (B) Schematic representation of the principal piece cross-section. Fibrous sheath, outer dense fibers, and axonemal components are indicated. Numbers 1 to 9 indicate the microtubule doublet pair. C1 and C2 indicate the central pair of microtubules. Protein kinase A (PKA) is shown in the radial spoke stalk, and potential sites of phosphorylation of Rsph6a by PKA during sperm capacitation are indicated by a (P).
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