Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity

Abstract

The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme. Its primary components are A-kinase anchoring proteins (AKAPs) 3 and 4, which suggests that the FS affects flagellar beating via the scaffolding of signaling pathways necessary for motility. Sperm proteins ROPN1 and ROPN1L bind AKAP3. To determine the role of ROPN1 and ROPN1L in sperm function, we created mice deficient in ROPN1 (RKO), mice deficient in ROPN1L (RLKO), and double knockout mice (DKO). All three strains of mice had normal testicular morphology and spermatogenesis. Only the DKOs had obvious defects in sperm morphology (thinning and shredding of the principal piece), which was accompanied by a reduction in AKAP3 levels. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. RKO mice had moderately impaired motility and increased levels of ROPN1L. DKO sperm were immotile. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. Together these data indicate that ROPN1L and ROPN1 compensate for each other in the absence of the opposing protein, possibly to maintain AKAP3 incorporation in the FS. Sperm from mice lacking ROPN1L exhibited reductions in both cAMP-dependent protein kinase (PKA) phosphorylation of a 270-kDa protein (perhaps FSCB), and in capacitation-induced tyrosine phosphorylation. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. These data demonstrate that mutations in ROPN1 and ROPN1L can cause defects in FS integrity, sperm motility, and PKA-dependent signaling processes, leading to male infertility.

FIG. 1

The principal piece of mature double-knockout (DKO) spermatozoa exhibits structural defects, including loss of FS protein AKAP3. A) Sperm from WT, RLKO, RKO, and DKO mice were fixed and stained with rabbit polyclonal AKAP3 antibody and examined at original magnification ×100. In WT panels: mp, midpiece; pp, principal piece. Arrows in DKO phase point to principal piece defects. Insets are enlargements of dashed boxes to show detail. B) AKAP3, AKAP4, ODF2, and tubulin Western blots of caudal epididymal sperm. C) Diagram of the principal piece. D) AKAP3, AKAP4, ODF2, and tubulin Western blots of soluble (lysates) and insoluble (pellets) testis proteins from WT and DKO mice. These are representative of at least three independent experiments, each using unique sets of animals. Bars = 10 μm.

FIG. 2

ROPN1L and ROPN1 compensate for each other during spermatogenesis in opposing gene trap animals through additional localization and increased expression. Immunohistochemical staining of WT, RLKO, and RKO testes (A), and caput epididymal sections (B) using rabbit polyclonal antibodies to ROPN1L and ROPN1 shows diaminobenzidine-positive (brown) staining. Blue hematoxylin counterstain stains nucleic acid-rich regions. Arrows in A point to areas of common (black arrows) or differential (green arrows) staining between WT and knockout tissues. In C, these antibodies were also used to demonstrate a large increase in ROPN1L levels in RKO caudal epididymal sperm (upper panel, compare lanes 1 and 3). These pictures are representative of three independent experiments using three unique sets of animals. Bars = 60 μm.

FIG. 3

CASA of motility parameters from WT, RLKO, and RKO mice. Motility parameters include percentage of sperm that are motile (% motile), percentage of sperm that exhibit progressive motility (% prog.), VAP, VSL, VCL, amplitude of lateral head displacement (ALH), BCF, and ratios of velocity for linearity VSL:VCL (LIN) and straightness VSL:VAP (STR). Error bars are ± SEM. Statistical significance is indicated by *P < 0.05. These pictures are representative of three independent experiments using three unique sets of animals.

FIG. 4

PKA and tyrosine phosphorylation of caudal epididymal sperm proteins is altered in the absence of ROPN1L and ROPN1. Caudal epididymal spermatozoa were isolated from WT, RLKO, and RKO mice in noncapacitating M2 media (capacitating media [−], lanes 1–3) or capacitating M16 media (capacitating media [+], lanes 4–6) and were incubated for 60 min at 37°C/5% CO₂. Sperm were then collected, counted, and lysed in boiling SDS sample buffer prior to SDS-PAGE, transfer, and Western blot analyses. In A, rabbit polyclonal antibody that detects substrates phosphorylated by PKA was used to look for alterations in PKA signaling, and differences were quantitated by densitometric analyses with ImageJ. Error bars are ± SEM. Statistical significance is indicated by *P < 0.05. In B, Western blot analysis using goat polyclonal antibody to FSCB, and in C using mouse monoclonal antibody 4G10, which detects proteins phosphorylated on tyrosine residues. Tubulin Western blot analyses were performed as loading controls. These pictures are representative of at least three independent experiments, each using unique sets of animals.