Outer Dense Fibers Research Articles

A missense mutation in the Capza3 gene and disruption of F-actin organization in spermatids of repro32 infertile male mice

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific “capping protein (actin filament) muscle Z-line, alpha 3” gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse.

Identificación de diferencias proteómicas en muestras oligozoospérmicas

ObjectivesOligozoospermia is one of the most common findings present in subfertile males, but its aetiology remains unknown in most cases. In this work we have used current proteomic tools to identify the different proteins present in oligozoospermic semen samples and therefore potentially involved in infertility. MethodsWe compared the expression of 75 sperm protein spots in 3 pools of 5 oligozoospermic samples to that of 5 semen donor controls using two-dimensional proteomic analysis. ResultsA total of14 protein spots have been identified with a > 2 fold different amount in the oligozoospermic samples as compared to controls. Proteins detected as increased are, creatine kinase B (CKB), histidine triad nucleotide-binding protein 1 (HINT1), succinyl CoA 3 ketoacid coenzyme A transferase1 precursor (OXCT1), transgelin-2 (TAGLN2), 2 spots corresponding to cytoskeletal actin (ACTB), glutathione S-transferase Mu 3 (GSTM3), annexin A5 (ANXA5), cyclin dependent kinase 5 (CDK5), and proteins detected as decreased are ATP synthase beta chain, mitochondrial precursor (ATP5B), outer dense fibre protein 2 (ODF2), tubulin beta subunit (TUBB2), capping protein (actin filament) muscle Z-line, beta (CAPZB), triosephosphate isomerase 1 (PTI1). DiscussionWe have identified several proteins present in different amounts in oligozoospermic sperm samples as compared to control samples. These proteins could be now be further considered as candidates for the development of diagnostic markers and open up the opportunity to gain further insight into the pathogenic mechanisms involved in oligozoospermia.

Bend propagation in the flagella of migrating human sperm, and its modulation by viscosity

A pre-requisite for sexual reproduction is successful unification of the male and female gametes; in externally-fertilising echinoderms the male gamete is brought into close proximity to the female gamete through chemotaxis, the associated signalling and flagellar beat changes being elegantly characterised in several species. In the human, sperm traverse a relatively high-viscosity mucus coating the tract surfaces, there being a tantalising possible role for chemotaxis. To understand human sperm migration and guidance, studies must therefore employ similar viscous in vitro environments. High frame rate digital imaging is used for the first time to characterise the flagellar movement of migrating sperm in low and high viscosities. While qualitative features have been reported previously, we show in precise spatial and temporal detail waveform evolution along the flagellum. In low viscosity the flagellum continuously moves out of the focal plane, compromising the measurement of true curvature, nonetheless the presence of torsion can be inferred. In high viscosities curvature can be accurately determined and we show how waves propagate at approximately constant speed. Progressing waves increase in curvature approximately linearly except for a sharper increase over a distance approximately 20-27 microm from the head/midpiece junction. Curvature modulation, likely influenced by the outer dense fibres, creates the characteristic waveforms of high viscosity swimming, with remarkably effective cell progression against greatly increased resistance, even in high viscosity liquids. Assessment of motility in physiological viscosities will be essential in future basic research, studies of chemotaxis and novel diagnostics.

Dietary selenium deficiency as well as excess supplementation induces multiple defects in mouse epididymal spermatozoa: understanding the role of selenium in male fertility

Selenium (Se) is essential for male fertility. The present study was carried out to observe the defects associated with Se deficiency as well as excess Se supplementation by analyzing the sperm ultrastructure and chromatin organization. Different Se status mice were generated viz. Se deficient (group I), Se adequate (group II) and Se excess (group III) by feeding the respective diets for a period of 4 (group Ia, IIa and IIIa) and 8 weeks (group Ib, IIb and IIIb). Reduction in sperm concentration, motility and percentage fertility was observed in Se deficient and Se excess groups. Electron microscopy revealed mitochondrial swelling and gaps between adjacent mitochondria in mice fed Se-deficient diet for 4 weeks. At 8 weeks, several abnormalities such as loose contact of the mitochondrial helix with the plasma membrane, loss of mitochondria, retention of cytoplasmic droplet, fracturing of outer dense fibres and presence of both the midpiece and the principal piece cross-sections in a common plasma membrane were observed. In Se excess group, the predominant defect was the frequent presence of equidistant, cross-sectioned midpieces of the tail embedded in a common cytoplasm. These defects are indicative of loss of sperm motility. Spermatozoa from Se-deficient mice had incompletely condensed chromatin and indicated an increase in occurrence of DNA strand breaks. The animals fed Se excess diet also indicated increase in DNA breaks but this was significantly less than the deficient diet fed groups. Our study reveals the defects associated with Se deficiency that result in loss of reproductive ability and also reflects its possible harmful effects on spermatozoa after prolonged consumption at supranutritional level.

Meichroacidin Containing the Membrane Occupation and Recognition Nexus Motif Is Essential for Spermatozoa Morphogenesis

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.

Overexpression of RPGR Leads to Male Infertility in Mice Due to Defects in Flagellar Assembly1

Male infertility is one possible consequence of a group of disorders arising from dysfunction of cilia. Ciliopathies include primary ciliary dyskinesia, polycystic kidney disease, Usher syndrome, nephronophthisis, Bardet-Biedl syndrome, Alstrom syndrome, and Meckel-Gruber syndrome as well as some forms of retinal degenerations. Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are best known for leading to retinal degeneration but have also been associated with ciliary dysfunctions affecting other tissues. To further study the involvement of RPGR in ciliopathies, transgenic mouse lines overexpressing RPGR were generated. Animals carrying the transgene in varying copy numbers were investigated. We found that infertility due to aberrant spermatozoa correlated with increased copy numbers. In animals with moderately increased gene copies of Rpgr, structural disorganization in the flagellar midpiece, outer dense fibers, and fibrous sheath was apparent. In contrast, in animals with high copy numbers, condensed sperm heads were present, but the flagellum was absent in the vast majority of spermatozoa, although early steps of flagellar biogenesis were observed. This complexity of defects in flagellar assembly suggests a role of RPGR in intraflagellar transport processes.

Hydrostatic pressure pre-treatment affects the protein profile of boar sperm before and after freezing–thawing

A sublethal environmental stress, high-hydrostatic pressure (HHP) was reported to significantly improve the motility, viability and fertility parameters of frozen bull and boar semen. However, the mechanism of how HHP treatment improves survival rates at sperm cryopreservation remains unclear. The purpose of this study was to evaluate the effect of HHP treatment of fresh boar semen on the protein profile of boar sperm before and after freezing. Fresh, extended semen of eight boars was split, one part was treated with 200, 300 or 400 bar for 90 min using a custom made pressuring device before the start of the semen freezing procedure, and the other part was prepared without HHP treatment. After thawing, samples were checked for motility. The effect of HHP treatment on the post-thaw motility of frozen semen was significant ( P = 0.02). Post-thaw motility of each treatment groups increased compared to control (46% vs. 52%, 56% and 56%; control vs. 200 bar, 300 bar and 400 bar treatments). Samples for protein analysis were collected from the 300 bar treatment group before HHP treatment at room temperature (25 ± 3 °C), at 5 °C of the cooling process and after thawing with or without HHP treatment. The sperm were lysed using a urea-pyranoside-dithiothreitol buffer to extract their proteins for protein analysis. Approximately 800 μg total proteins were assayed by two-dimensional gel electrophoresis and stained with colloidal Coomassie blue. The levels of 125 protein spots were quantified. The results revealed that the levels of 7 protein spots differed significantly among treatments. The identities of various protein constituents were identified by mass spectrometry and database searching. Ubiquinol-cytochrome c reductase complex core protein 1, perilipin, and carbohydrate-binding protein AWN precursor were identified as HHP response proteins being significantly higher in HHP-treated samples. Testis-specific glyceraldehyde 3-phosphate dehydrogenase, outer dense fiber of sperm tails 2 isoform 10, cytosolic 5′-nucleotidase 1B, and quinone oxidoreductase represented the cooling and freezing related proteins. The differing levels of these identified proteins could be valuable for further exploring the protective mechanism of the HHP treatment in frozen–thawed porcine sperm.

Genomic origin, processing and developmental expression of testicular outer dense fiber 2 (ODF2) transcripts and a novel nucleolar localization of ODF2 protein

Outer dense fibers are a major constituent of the sperm tail and outer dense fiber 2 (ODF2) protein is one of their major components. ODF2 shares partial homology with cenexin 1 and cenexin 2, regarded as centriolar proteins. We show that ODF2 and cenexin 2 transcripts are the product of differential splicing of a single gene, designated Cenexin/ODF2 and that cenexin 1 is an incomplete clone of ODF2. ODF2 terminates in exon 20b whereas in cenexin 2 this exon is spliced out and translation terminates in exon 24. We demonstrate a transcriptional switch during rat testicular development, from somatic-type to testis-type ODF2 and cenexin transcripts during the onset of meiosis. The switch is completed when spermiogenesis is established. ODF2 immunoreactive sites were visualized in the acroplaxome, along the sperm tail and the centrosome-derived sperm head-to-tail coupling apparatus. An unexpected finding was the presence of ODF2 antigenic sites, but not cenexin antigenic sites, in the dense fibrillar component of the nucleolus of Sertoli cells, spermatogonia and primary spermatocytes. The characterization of the genomic origin, processing and developmental expression of ODF2 transcript isoforms and their protein products can help reconcile differences in the literature on the role of ODF2 and cenexin in the centrosome. Furthermore, the finding of ODF2 in the dense fibrillar component of the nucleolus suggests that this protein, in addition to its presence in sperm outer dense fibers and centrosome, highlights and adds to the nucleolar function during spermatogenesis and early embryogenesis.

Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase.

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.

Spermatotoxic effect of aflatoxin B1 in rat: extrusion of outer dense fibres and associated axonemal microtubule doublets of sperm flagellum

Male Wistar rats were treated with aflatoxin B1 (AFB1). Live as well as methanol-fixed cauda epididymal spermatozoa were stained with acridine orange (AO) and ethidium bromide (EB) and observed under a fluorescence microscope. Giemsa-stained smears were observed in a bright field microscope. Unstained smears were observed with phase contrast illumination. The axoneme of more than 10% of the spermatozoa of treated rats had the outer dense fibres (ODFs), in varying numbers, and the associated axonemal microtubule doublets of the flagellum extruded either at midpiece-principal piece junction or connecting piece. This could be perceived in all light microscopic preparations, but AO-EB staining offered an advantage of the assessment of the viability as well. TEM observation of sections of the testis and cauda epididymidis also revealed ODF extrusion, as seen in the transverse sections of sperm flagella missing one or more ODFs and the associated axonemal microtubule doublets. In a few such sections, the extruded elements were seen in the cytoplasm, outside the mitochondrial sheath or peripheral sheath. Marginal to severe mitochondrial pathologies were observed in the spermatozoa and elongated spermatids, suggesting a link between AFB1-induced sperm mitochondrial pathology and extrusion of ODFs. However, the possibility that AFB1 treatment would disrupt the cytoskeletal proteins of the flagellum, resulting in the extrusion of ODFs, cannot be excluded. This sperm abnormality is reported for the first time as produced by a dietary toxin. Dietary aflatoxins, therefore, could also be contributory factors for the deterioration of the reproductive health of men.

Abnormal elongation of midpiece, absence of axoneme and outer dense fibers at principal piece level, supernumerary microtubules: a sperm defect of possible genetic origin?

To characterize a flagellar defect involving 95% of the sperm population from an infertile man. Case report. Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy. A 42-year-old infertile man with severe asthenozoospermia. Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission electron microscopy (TEM) and scanning electron microscopy (SEM), tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization for chromosomes 18, X, and Y. Ultrastructural abnormalities of the flagellum detected by methods listed. Ultrastructural analysis revealed, in 95% of sperm cells, the total absence of the axoneme and outer dense fibers at the principal piece level, whereas the midpiece appeared abnormally long. Tubulin localization showed a total disorganization of the axoneme with a network of microtubular structures emerging randomly at any level of the flagellum. Fluorescence in situ hybridization analysis was normal. We report a rare sperm tail defect, characterized by abnormal elongation of the midpiece and absence of the axoneme and the outer dense fibers at the principal piece level in 95% of flagella. This defect occurs in the vast majority of the sperm population from a sterile man, and therefore a genetic origin could be hypothesized.

Molecular dissection of ODF2/Cenexin revealed a short stretch of amino acids necessary for targeting to the centrosome and the primary cilium

The outer dense fiber protein ODF2 is the major component of the sperm tail cytoskeleton and a critical component of the mature centriole of the centrosome. Centriole maturation involves the formation of appendages and the recruitment of ODF2/Cenexin. ODF2 and Cenexin are alternative splice variants that differ in a short stretch of amino acids at their N-terminal regions encoded by exon 3b. Whereas Cenexin is ubiquitously expressed, Odf2 is the predominant transcript of testes [Hüber, D., Hoyer-Fender, S., 2007. Alternative splicing of exon 3b gives rise to ODF2 and Cenexin. Cytogenet. Genome Res. 119, doi:10.1159/000109621]. Here, we show that testicular expression of Odf2 correlates with spermiogenesis and ongoing sperm tail formation thus implicating functional differences between ODF2 and Cenexin. By generation of a series of ODF2/Cenexin deletion constructs fused to GFP and inspection of their subcellular localization in transfected NIH3T3 cells we found that a peptide of 42 amino acids specific for Cenexin is necessary for targeting ODF2/Cenexin to the centrosome and the primary cilium. Additionally, this region is also necessary for the formation of ODF2/Cenexin fibers that are associated with acetylated microtubules. Centrosomal targeting of ODF2/Cenexin does not depend on dynein-mediated transport further supporting an alternative targeting mechanism. However, part of the C-terminal coiled-coil region of ODF2 is also important in centrosomal/ciliary targeting and fiber formation presumably by supporting self-association and the formation of higher-order structures.

Germ cell-less like-2 protein is a new component of outer dense fibers in rat sperm flagella

We have analyzed the expression profiles of ten genes in terms of testis development and organ specificity in rat, which were selected from 215 round spermatid-specific transcripts listed in a database. Out of the ten genes, we directed our attention to one gene, a germ cell-less like-2 gene (gcl-2), a homolog of Drosophila gcl gene (gcl), which is a component of the germ plasma and required for primordial germ cell formation. Rat genome contains duplicate rat gcl-2 (rgcl-2) genes, rgcl-2A and rgcl-2B, both of which are located at Xq13. RT-PCR analysis showed that the expression of the two genes was up-regulated during testis development and that they were predominantly expressed in the testis. Both rgcl-2A and rgcl-2B encode a protein of 498 amino acid residues, showing 90.56% identity at the amino acid level. Confocal laser scanning microscopy revealed that rgcl-2 protein was synthesized in the cytoplasm of elongating spermatids and at least a part of it was integrated into the middle piece of spermatozoa during spermiogenesis. Immunogold electron microscopy uncovered that rgcl-2 was localized at the abaxial (convex) surface of outer dense fibers (ODF) of rat sperm flagella. Therefore, we concluded that rgcl-2 is a new component of ODF in sperm flagella.

Alternative splicing of exon 3b gives rise to ODF2 and Cenexin

ODF2 was first identified as the major component of the sperm tail outer dense fibers. Additionally, ODF2 is a critical component of the mature centriole of the animal centrosome where it locates to the distal appendages. Moreover, generation of primary cilia strictly depends on ODF2. The mature centriole is characterized further by recruitment of Cenexin. Albeit highly similar in sequence the relationship between ODF2 and Cenexin has not been investigated. We demonstrate here that ODF2 and Cenexin are alternative splice products by identifying a novel exon 3b encoding Cenexin specific amino acids. Even though ODF2 is the main isoform in testicular tissue RT-PCR analyses revealed that isoforms are not restricted to specific tissues.

Localisation and function of voltage-dependent anion channels (VDAC) in bovine spermatozoa

Sperm motility, regulation of cell volume, sperm capacitation, acrosome reaction and tight binding of spermatozoa to the zona pellucida are crucial events in the process of fertilisation. Voltage-dependent anion channels (VDAC) are highly conserved pore-forming proteins implicated in apoptosis, metabolite transport between mitochondria and cytosol, energy metabolism, and cell volume regulation in somatic cells. Several studies have demonstrated the presence of VDAC in cell compartments other than mitochondria. In previous studies using immunofluorescence, we were able to localise VDAC2 and VDAC3 in outer dense fibres of the bovine sperm flagellum. Furthermore, we described the presence of VDAC2 in the head of bovine sperm. In the present study, we confirm the localisation of VDAC2 in the acrosomal region of bovine spermatozoa using immunoelectron microscopy. After incubation with anti-VDAC antibodies raised against each VDAC isoform, bovine spermatozoa showed an increased loss of the acrosomal cap, noticeable changes in the surface of the head, coiled tails and an increased cell volume. The incubation of bovine spermatozoa with anti-VDAC antibodies might lead to alteration of the intracellular ion concentration that causes changes in the cell volume, followed by destabilization of the cytoskeleton and, finally, to loss of the acrosomal cap.

The Expression of GLUT8, GLUT9a, and GLUT9b in the Mouse Testis and Sperm

The objective of this study is to confirm the precise glucose transporter (GLUT) 8 localization and determine the expression of GLUT9a and GLUT9b by Western blot and confocal and immunoelectron microscopy in the mouse testis and sperm. GLUT8, GLUT9a, and GLUT9b proteins are expressed in the most intraseminiferous tubula cells and Leydig cells. GLUT8 localizes in the midpiece and principal piece as well as in the acrosomal region of the sperm. Immunoelectron microscopic analysis shows that GLUT8 is strongly detectable at the acrosome and neck region of the sperm. In the midpiece, GLUT8 localizes at the outer dense fibers (odf) as well as at the circumference of the spiral mitochondria. In the principal piece, GLUT8 localizes at the odf. GLUT9a strictly localizes in the midpiece, but GLUT9b localizes in the acrosome, midpiece, and principal piece of the sperm. These results suggest that glucose uptake via GLUT8, GLUT9a, and GLUT9b likely affects normal spermatogenesis, steroidogenesis, and sperm function in the mouse.

Three dimensional rendering of the mitochondrial sheath morphogenesis during mouse spermiogenesis

In the middle piece of mouse sperm tail, the idea of the mitochondria wrapping in a sinistral (left-handed) double helical structure was generally accepted. In the existing model, mitochondria aligned in four longitudinal rows (stage 1) and twisted dextrally (right-handed) (stage 2) and began to stagger, where opposing rows of mitochondria contacted each other to form a sinistral double helix (stage 3), finally, the end-on touching mitochondria further elongated to their mature length (stage 4). However, in this model, mitochondria need to shift a long distance and reposition themselves. Since no gaps have been found in mitochondrial sheath, repositioning of most mitochondria along the middle piece is unlikely to happen. Therefore, we reapproached the questions through three-dimensional rendering to provide a new model for mitochondrial sheath formation. In our proposed model, four dextrally arranged spherical mitochondrial arrays were considered stage 1 (resembles stage 2 of the old model). In stage 2 (resembles stage 3 of the old model), a critical difference was found that pairs of mitochondria from the opposing arrays formed ring-like structures instead of a sinistral double helix. In stage 3, which was not observed in the old model, mitochondria staggered in a specific pattern to form the sinistral double helix. In stage 4, mitochondria elongated from crescent-shaped to rod-shaped structures. The new model proposed here would allow each mitochondrion to stay at where they attached first and elongate laterally from two directions to reach their final double helical structure without unreasonable great distance shift along the outer dense fibers.

Analysis of Ppp1cc-Null Mice Suggests a Role for PP1gamma2 in Sperm Morphogenesis1

Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.

Spatial Distribution of Actin and Tubulin in Human Sperm Nuclear Matrix‐intermediate Filament Whole Mounts—A New Paradigm

Sperm is a highly differentiated cell streamlined for fertilization. The function is thus heavily dependent on the cytoskeletal organization. Conventional methods limit the appreciation and correlation of this intricate cytoskeletal filament network in the context of an entire sperm. Our recent successful localization of nonmuscle myosin IIA on sperm nuclear matrix-intermediate filament (NM-IF) preparations from fertile men by embedment-free electron microscopy (EF-EM), prompted us to investigate the antigenic distribution of two major cytoskeletal proteins-actin and tubulin. The NM-IF preparations were subjected to a cocktail of buffered paraformaldehyde (2%) with a low concentration of glutaraldehyde (0.05%). These proteins were localized by indirect immunogold technique using EF-EM on sperm NM-IF whole mounts. Ultrastructure analysis revealed well preserved centrioles, outer dense fibers, axonemal filaments, and submitochondrial reticulum in the sperm NM-IF. Immunoreactive actin was localized along the length of the sperm whereas beta-tubulin was present in the axoneme alone. The spatial distribution of actin and tubulin in normal human sperm NM-IF reported here together with that of myosin on whole mount offers a powerful technique to understand sperm cytoskeletal supramolecular structure.

Identification of BCAP, a new protein associated with basal bodies and centrioles

Cilia exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Defects in this organelle can lead to lethal pathologies in humans, including primary ciliary dyskinesia. An understanding of the cilia formation process would lead to better characterization of defects involved in such pathologies. In the present study, we identified a gene encoding a novel human protein, BCAP for Basal body Centriole-Associated Protein, which shares homologies with a previously described protein, Outer Dense Fiber 2 (ODF2). ODF2, a major component of the sperm tail cytoskeleton, is required for the formation of mother centriole distal/subdistal appendages and the generation of primary cilia. Here, we show that the bcap gene contains 18 alternatively spliced exons and encodes five different isoforms, three long and two short ones. BCAP is preferentially expressed in cilia/flagella containing tissues. Moreover, its expression is correlated with cilia formation during mucociliary differentiation of human nasal epithelial cells. Using immunofluorescence analyses, BCAP was localized within basal bodies of ciliated cells and within centrioles of proliferating cells. In light of the several spliced isoforms of BCAP and the particular localization of the protein, BCAP isoforms could play distinct roles in cilia and in centrosomes.