Terminal differentiation of male germ cells into functional spermatozoa requires shaping and condensation of the nucleus as well as the formation of sperm-specific structures. A transient microtubular structure, the manchette, is mandatory for sperm head shaping and the development of the connecting piece and the sperm tail. The connecting piece or head-to-tail coupling apparatus (HTCA) mediates the tight linkage of sperm head and tail causing decapitation and infertility when faulty. Using mice as the experimental model, several proteins have already been identified affecting the linkage complex, manchette or tail formation when missing. However, our current knowledge is far too rudimentary to even draft an interacting protein network. Depletion of the major outer dense fiber protein 1 (ODF1) mainly caused decapitation and male infertility but validated binding partners collaborating in the formation of sperm-specific structures are largely unknown. Amongst all candidate proteins affecting the HTCA when missing, the structural protein CCDC42 attracted our attention. The coiled-coil domain containing 42 (CCDC42) is important for HTCA and sperm tail formation but is otherwise largely uncharacterized. We show here that CCDC42 is expressed in spermatids and localizes to the manchette, the connecting piece and the tail. Beyond that, we show that CCDC42 is not restricted to male germ cells but is also expressed in somatic cells in which it localizes to the centrosome. Although centrosomal and sperm tail location seems to be irrespective of ODF1 we asked whether both proteins may form an interacting network in the male germ cell. We additionally considered ODF2, a prevalent protein involved in the formation of spermatid-specific cytoskeletal structures, as a putative binding partner. Our data depict for the first time the subcellular location of CCDC42 in spermatids and deepen our knowledge about the composition of the spermatid/sperm-specific structures. The presence of CCDC42 in the centrosome of somatic cells together with the obvious restricted male-specific phenotype when missing strongly argues for a compensatory function by other still unknown proteins most likely of the same family.
The centrosome, as the main microtubule-organizing center, safeguards chromosome segregation by supporting the bipolar spindle. Centrosome aberrations are causally related to chromosome segregation disorders, both characterizing cancer cells. Thus, a restriction to only having one centrosome per cell and cell cycledependent duplication of the centrosome is mandatory. Duplicated centrosomes remain physically connected, in order to function as a single entity, until onset of mitosis when centrosome disjunction is licensed by disassembly of linker proteins and accumulation of β-catenin. The crucial role β-catenin plays in centrosome disjunction inevitably demands for restricting its premature accumulation. ODF2 (also known as cenexin) is an essential centrosomal component, but its relevance for the interphase centrosome has not been elucidated. We show here that ODF2 plays a central role in centrosome cohesion. Depletion of ODF2 induces premature centrosome splitting and formation of tripolar spindles that are likely caused by the observed accumulation of centrosomal β-catenin. Our data collectively indicate that ODF2 restricts β-catenin accumulation at the centrosome, thus preventing premature centrosome disjunction.
The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell’s environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders.
Development of spermatozoa requires remodelling and formation of particular structures. In elongating spermatids, the transient microtubular manchette contributes to the formation of the head-tail coupling apparatus (HTCA) and the sperm tail. The HTCA derives from the centrosome in that the proximal centriole inserts into the nuclear indentation and the distal centriole gives rise to the sperm flagellum. Although impairments in the formation of HTCA and sperm tail cause male infertility their molecular constituents are only partially known. The WD40-protein CFAP52 is implicated in motile cilia, but its relevance for male germ cell differentiation is not known. Here we show that CFAP52 is widespread expressed and localizes to a subset of microtubular structures. In male germ cells, CFAP52 is a component of the transient manchette and the sperm tail. However, expression of Cfap52 is not restricted to motile cilia-bearing cells. In NIH3T3 cells, CFAP52 localizes to the centrosome, the basal body, and the mitotic spindle poles, but not to the primary cilium. Our results demonstrate that CFAP52 is not restricted to motile cilia but instead most likely functions in constituting the centrosome/basal body matrix and the sperm tail. The cilia and flagella associated protein 52, CFAP52 (also named WDR16, WD repeat domain 16, WDRPUH, FLJ37528) is a member of the large WD40-repeat protein family and is widely expressed in eukaryotes. WD40repeats, which are also known as WD or beta-transducin repeats, are short motifs of 40-60 amino acids often terminating in the dipeptide WD. The WD40-domain often comprises several of the WD40-repeats each forming a beta-pleated sheet in a propeller blade 1,2. WD40 proteins are crucially involved in several physiological functions as diverse as signal transduction, RNA processing, remodeling the cytoskeleton, regulation of vesicular traffic, and cell cycle control 3-5. Their immediate purpose most likely is to serve as platforms for the assembly of protein complexes and to mediate protein-protein interactions 6. The WD40 protein family member WDR16 is highly conserved having homologs in invertebrates and vertebrates including flies and humans 7. In rat, expression of WDR16 was mainly observed in testis and in ependymal cells of the brain, and with reduced expression in lungs. Its expression profile, especially during ependymal development, indicated a correlation to the presence of motile cilia whereas in cells harboring primary cilia WDR16 could not be detected 7. By immunofluorescence, WDR16 was found in the cytosol of rat testicular cells, especially in early spermatocytes and pachytene spermatocytes showing a uniform and unspecific localization. Sperm flagella stained negative for WDR16 but in Western blots the tail fraction of bull sperm revealed WDR16 7. Overall, these results indicated that WDR16 is a marker for motile cilia-bearing cells. WDR16 depletion in zebrafish caused hydrocephalus, however, ciliary movement remained intact. Similarly, the ependymal layer did not display visibl...
The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 (Atat1) as being causative. Our observations demonstrate that cells with reduced expression of Atat1 and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of Atat1 and tubulin acetylation are therefore selective forces in cell competition.
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