MFP1/MSD-1 and MFP2/NSPH-2 co-localize with MSP during C. elegans spermatogenesis

Abstract

Until recently, the only verified component of Fibrous Bodies (FBs) within Caenorhabditis elegans spermatocytes was the Major Sperm Protein (MSP), a nematode-specific cytoskeletal element. Earlier studies in the pig parasite Ascaris suum had identified accessory proteins that facilitate MSP polymerization and depolymerization within the pseudopod of crawling spermatozoa. In this study, we show that C. elegans homologs of the two Ascaris accessory proteins MFP1 and MFP2 co-localize with MSP in both the pseudopods of C. elegans sperm and the FBs of C. elegans spermatocytes.

Figure 1

(A) Overview of spermatogenesis and fibrous body–membranous organelle (FB-MO) development. (Top) Spermatocytes develop on a syncytial rachis then detach to undergo the meiotic divisions. Following anaphase II, components which are no longer needed partition to a central residual body (RB). (Bottom) Within meiotic prophase spermatocytes, fibrous bodies (FBs) develop on the cytosolic face of the Golgi-derived membranous organelle (MO). During the budding division, FB-MO complexes partition to the spermatids. MOs then dock with the plasma membrane as the FBs disassemble and release MSP (Major Sperm Protein) dimers into the cytosol. During sperm activation, MSP localizes to the pseudopod, and MOs fuse with the plasma membrane. (B) Alignment of C. elegans ZK265.3/NSPH-2 (Nematode Specific Peptide family, group H) and C04G2.9/NSPH-3.2 with Ascaris MFP2 (MSP Fiber Protein) performed using the EMBL-EBI Clustal Omega Multiple Sequence Alignment tool (Madeira et al., 2019). Peptide for the anti-NSPH-2 antibody is underlined. (C-D) DAPI and anti-NSPH-2 labelling in hermaphrodites with genetic knockouts of nsph-3.2 and nsph-2 respectively. (C) Left to right developmental sequence of developing spermatocytes and spermatids. (D) Hermaphrodite spermatozoa within spermatheca. No primary antibody controls were performed on nsph-3.2 knockout animals. (E-G) Sperm spreads and individually staged cells with (E) anti-MSP labelling (n=63, 9 experiments), (F) anti-NSPH-2 labelling (n=50, 7 experiments), and (G) labelling against the MFP1 homolog, MSD-1 (Major Sperm protein Domain containing) (n=46, 3 experiments). (H-I) Co-labelling of MSP with either (H) anti-NSPH-2 or (I) anti-MSD-1. In spermatozoa (enlarged), the patterns were either fully (top) or partially overlapping (bottom). For NSPH-2/MSP n=16; for MSD-1/MSP n=14. Scale bars = 10 mm for C, D and E-G left; 5 mm for H, I and E-G right. Abbreviations: Karyosome spermatocytes (K), Metaphase spermatocytes (M), Budding Figures (B), Spermatids (S), and Spermatozoa (Z).