Testis-expressed profilins 3 and 4 show distinct functional characteristics and localize in the acroplaxome-manchette complex in spermatids

Background Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the somatic profilins, profilin-1 (PFN1) and profilin-2 (PFN2). Results Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. β-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. Conclusion The testis-specific isoform PFN3 showed major hallmarks of the well characterized somatic profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans

Overexpression of Full-Length Centrobin Rescues Limb Malformation but Not Male Fertility of the Hypodactylous (hd) Rats

Rat hypodactyly (hd) mutation is characterized by abnormal spermatogenesis and sperm decapitation, limb malformation (missing digits II and III) and growth retardation. We have previously reported centrobin (centrosome BRCA2-interacting protein) truncation at the C-terminus in the hd mutant. Here, we report data from a transgenic rescue experiment carried out to determine a role of centrobin in pathogenesis of hd. The transgenic construct, consisting of full-length-coding cDNA linked to a ubiquitous strong promoter/enhancer combination, was inserted to chromosome 16 into a LINE repeat. No known gene is present in the vicinity of the insertion site. Transgenic centrobin was expressed in all tissues tested, including testis. Transgenic animals show normal body weight and limb morphology as well as average weight of testis and epididymis. Yet, abnormal spermatogenesis and sperm decapitation persisted in the transgenic animals. Western blotting showed the coexistence of full-length and truncated or partially degraded centrobin in sperm of the rescued transgenic animals. Immunocytochemistry showed a buildup of centrobin and ODF2 (outer dense fiber 2) at the sperm decapitation site in the hd mutant and rescued transgenic rats. Additional findings included bulge-like formations and thread-like focal dissociations along the sperm flagellum and the organization of multiple whorls of truncated sperm flagella in the epididymal lumen. We conclude that centrobin is essential for normal patterning of the limb autopod. Centrobin may be required for stabilizing the attachment of the sperm head to flagellum and for maintaining the structural integrity of the sperm flagellum. We postulate that the presence of truncated centrobin, coexisting with full-length centrobin, together with incorrect timing of transgenic centrobin expression may hamper the rescue of fertility in hd male rats

Histology and Cell Biology

xviii,619 hal,;ill,;28 c

Histology and cell biology : An Introduction to pathology

XV+619hlm.;27c

Cytoskeletal track selection during cargo transport in spermatids is relevant to male fertility

Spermatids generate diverse and unusual actin and microtubule populations during spermiogenesis to fulfill mechanical and cargo transport functions assisted by motor and non-motor proteins. Disruption of cargo transport may lead to teratozoospermia and consequent male infertility. How motor and non-motor proteins utilize the cytoskeleton to transport cargos during sperm development is not clear. Filamentous actin (F-actin) and the associated motor protein myosin Va participate in the transport of Golgi-derived proacrosomal vesicles to the acrosome and along the manchette. The acrosome is stabilized by the acroplaxome, a cytoskeletal plate anchored to the nuclear envelope. The acroplaxome plate harbors F-actin and actin-like proteins as well as several other proteins, including keratin 5/Sak57, Ran GTPase, Hook1, dynactin p150Glued, cenexin-derived ODF2, testis-expressed profilin-3 and profilin-4, testis-expressed Fer tyrosine kinase (FerT), members of the ubiquitin-proteasome system and cortactin. Spermatids express transcripts encoding the non-spliced form of cortactin, a F-actin-regulatory protein. Tyrosine phosphorylated cortactin and FerT coexist in the acrosome-acroplaxome complex. Hook1 and p150Glued, known to participate in vesicle cargo transport, are sequentially seen from the acroplaxome to the manchette to the head-tail coupling apparatus (HTCA). The golgin Golgi-microtubule associated protein GMAP210 resides in the cis-Golgi whereas the intraflagellar protein IFT88 localizes in the trans-Golgi network. Like Hook1 and p150Glued, GMAP210 and IFT88 colocalize at the cytosolic side of proacrosomal vesicles and, following vesicle fusion, become part of the outer and inner acrosomal membranes before relocating to the acroplaxome, manchette and HTCA. A hallmark of the manchette and axoneme is microtubule heterogeneity, determined by the abundance of acetylated, tysosinated and glutamylated tubulin isoforms produced by post-translational modifications. We postulate that the construction of the male gamete requires microtubule and F-actin tracks and specific molecular motors and associated non-motor proteins for the directional positioning of vesicular and non-vesicular cargos at specific intracellular sites