Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1

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.

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa

“…CABYR is one of four proteins known to have this domain. Knockout studies analysing two more of these proteins, Ropporin 1 (ROPN1), as well as a similar protein, Ropporin1-like (ROPN1L), indicated defects in the integrity of the fibrous sheath (double KO) exhibiting thinning of the principal piece and a loss of AKAP3 (Fiedler et al, 2013). Unfortunately, there was no further investigation of the ultrastructure of these spermatozoa so it is unclear whether the integrity of the fibrous sheath remained intact.…”

“…Unfortunately, there was no further investigation of the ultrastructure of these spermatozoa so it is unclear whether the integrity of the fibrous sheath remained intact. The Ropn1 Ropn1l double KO mouse line exhibits a complete failure in fertilization (Fiedler et al, 2013). The fourth R2D2-domain-containing protein, SPA17 has not yet been knocked out, although it too can bind to AKAP3 (Lea et al, 2004) and is localized to the fibrous sheath (Chiriva-Internati et al, 2009), indicating that knockout might potentially result in a phenotype similar to that upon loss of CABYR.…”

“…The fibrous sheath is believed to regulate the degree of flexibility, flagellar motion and shape of the flagellar beat during the lifespan of a sperm cell (Eddy et al, 2003). The main structural components of the fibrous sheath come from two proteins of the A-kinase anchoring family (AKAPs), which includes AKAP3 and AKAP4 (Fiedler et al, 2013). As such, the fibrous sheath acts as a scaffold for several signalling and/or metabolic pathways (Li et al, 2010).…”

Functional anatomy of the mammalian sperm flagellum

“…This modification, combined with the freedom of ODF motion in the midpiece region, not only substantially increases the available bending torque, it also makes the proximal portion of the 9 1 2 central axoneme largely insensitive to the effects of the large t-forces developed during vigorous motility. While it is now relatively well established that the 9 1 2 axoneme proper is the source of power for the motility of mammalian sperm, it is also well documented that the periaxonemal structures are essential for normal motility and fertilizing capacity (Haidl et al, 1991;David et al, 1993;Pilder et al, 1997;Serres et al, 1986;Fiedler et al, 2013).…”

“…This modification, combined with the freedom of ODF motion in the midpiece region, not only substantially increases the available bending torque, it also makes the proximal portion of the 9 1 2 central axoneme largely insensitive to the effects of the large t-forces developed during vigorous motility. While it is now relatively well established that the 9 1 2 axoneme proper is the source of power for the motility of mammalian sperm, it is also well documented that the periaxonemal structures are essential for normal motility and fertilizing capacity (Haidl et al, 1991;David et al, 1993;Pilder et al, 1997;Serres et al, 1986;Fiedler et al, 2013).The periaxonemal structures appear to be especially critical to generate the form of motility that occurs in mammalian sperm after incubation in the female tract and is referred to as hyperactivated motility. This form of motility was first described by (Yanagimachi, 1970), and shown to occur in the oviduct (Katz and Yanagimachi, 1980).…”