Statistical significance is indicated by * P < 0.05. 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. Sperm were then collected, counted, and lysed in boiling SDS sample buffer prior to SDS-PAGE, transfer, and Western blot analyses. 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☌/5% CO 2. PKA and tyrosine phosphorylation of caudal epididymal sperm proteins is altered in the absence of ROPN1L and ROPN1. 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. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. 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. 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. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. RKO mice had moderately impaired motility and increased levels of ROPN1L. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. 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. All three strains of mice had normal testicular morphology and spermatogenesis. 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). Sperm proteins ROPN1 and ROPN1L bind AKAP3. 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. The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme.
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