Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin

The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (K(d) = approximately 5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

Novel actin-related proteins Arp-T1 and Arp-T2 as components of the cytoskeletal calyx of the mammalian sperm head

The calyx is a large cytoskeletal component of the perinuclear theca of the mammalian sperm head, displaying remarkable morphological interspecies differences, which is biochemically characterized by resistance to high ionic strength and detergents and by a special protein composition, including the basic proteins calicin, cylicin I and II, and two major actin-capping proteins. In our calyx preparations from bull spermatozoa we have noted two major acidic components which upon partial amino acid sequencing have been identified as novel members of the subfamily of actin-related proteins (Arps). Antibodies raised against the corresponding human proteins, termed Arp-T1 and Arp-T2, have been used to detect the proteins by immunoblotting and immunofluorescence microscopy, demonstrating their specific synthesis in the testis, late in spermatid differentiation, and their localization in the calyx. The discovery of two novel Arps as major components in a cytoskeletal, nonmotile structure of mammalian spermatozoa suggests that certain members of this family of proteins may serve functions other than nucleation of actin filaments, and possible biological roles of such Arps in spermatozoa are discussed.     

Widespread occurrence of calicin, a basic cytoskeletal protein of sperm cells, in diverse mammalian species

A novel cytoskeletal element consisting of dense webs of thin (3-14 nm) filaments surrounding the nucleus of the sperm head has recently been isolated and shown to be associated with certain major basic proteins. Using antibodies specific for calicin, a prominent Mr-60,000 cytoskeletal protein of the posterior calyx of bull sperm heads detected in immunoblotting on gel electrophoretically separated polypeptides as well as in immunofluorescence and immunoelectron microscopy, we show that the same--or an immunologically related--polypeptide occurs in sperm heads of other species with greatly different morphology, including human, boar, guinea pig, hamster, rat and mouse. The calicin localization in the various species is described and discussed in relation to the specific sperm morphology.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis.

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking. Copyright 2001 Academic Press.     

Distribution and localization of calmodulin-binding proteins in bull spermatozoa

Previous studies from our laboratory have shown that a decrease in the calmodulin binding properties of a few sperm proteins occurs during the capacitation process, an effect associated with a decrease in intracellular calmodulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay assay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, whereas p30 is found in the fraction enriched with sperm heads. This latter calmodulin binding protein, p30, appears to be associated with the perinuclear theca. None of these binding proteins was solubilized by nonionic detergents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 was extracted only in conditions reported to isolate the perinuclear theca. Cellular localization of calmodulin binding proteins was also achieved by incubating spermatozoa fixed on slides with biotinylated calmodulin and revealed in a further step by fluorescein-conjugated streptavidin. Using this procedure, it was found that calmodulin binds to the sub- and postacrosomal areas of the sperm head along with the midpiece in the presence of Ca(2+). Only a sharp band of fluorescence at the subacrosomal area was observed when this procedure was performed in the absence of Ca(2+) in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when spermatozoa were incubated under capacitating conditions, in the presence of heparin, in agreement with the published effect of capacitation on calmodulin binding proteins.     

The composition, protein genesis and significance of the inner acrosomal membrane of eutherian sperm

As a consequence of the acrosomal reaction during fertilization, the inner acrosomal membrane (IAM) becomes exposed and forms the leading edge of the sperm for adhesive binding to and subsequent penetration of the zona-pellucida (ZP) of the metaphase-II-arrested oocyte. A premise of this review is that the IAM of spermatozoa anchors receptors and enzymes (on its extracellular side) that are required for sperm attachment to and penetration of the ZP. We propose a sperm cell fractionation strategy that allows for direct access to proteins bound to the extracellular side of the IAM. We review the types of integral and peripheral IAM proteins that have been found by this approach and that have been implicated in ZP recognition and lysis. We also propose a scheme for the origin and assembly of these proteins within the developing acrosome during spermiogenesis. During development, the extravesicular side of the membrane of the acrosomic vesicle is coated by peripheral proteins that transport and bind this secretory vesicle to the spermatid nucleus. The part of the membrane that binds to the nucleus becomes the IAM, while its extravesicular protein coat, which is retained between the IAM and the nuclear envelope of spermatozoa becomes the subacrosomal layer of the perinuclear theca (SAL-PT). Another premise of this review is that the IAM of spermatozoa is bound with proteins (on its intracellular side), namely the SAL-PT proteins, which hold the clue to the mechanism of acrosomal-nuclear docking. We propose a sperm cell fractionation strategy that allows for direct access to SAL-PT proteins. We then review the types of SAL-PT proteins that have been found by this approach and that have been implicated in transporting and binding the acrosome to the sperm nucleus.     

Involvement of classical bipartite/karyopherin nuclear import pathway components in acrosomal trafficking and assembly during bovine and murid spermiogenesis

This study arose from our finding that SubH2Bv, a histone H2B variant residing in the subacrosomal compartment of mammalian spermatozoa, contains a bipartite nuclear localization signal (bNLS) but in spite of this did not enter the spermatid nucleus. Instead, it associated with proacrosomic and acrosomic vesicles, which were targeted to the nuclear surface to form the acrosome. On this basis we proposed that SubH2Bv targets proacrosomic/acrosomic vesicles from the Golgi apparatus to the nuclear envelope by utilizing the classical bipartite/karyopherin alpha (KPNA) nuclear import pathway. To test the protein's nuclear targeting ability, SubH2Bv, with and without targeted mutations of the basic residues of bNLS, as well as bNLS alone, were transfected into mammalian cells as GFP-fusion proteins. Only the intact bNLS conferred nuclear entry. Subsequently, we showed that a KPNA, most likely KPNA6, occupies the same sperm head compartment and follows the same pattern of acrosomal association during spermiogenesis as SubH2Bv. Sperm head fractionation combined with Western blotting located this KPNA to the subacrosomal layer of the perinuclear theca, while immunocytochemistry of testicular sections showed that it associates with the surface of proacrosomic/acrosomic vesicles during acrosomal biogenesis. The identical sperm-localization and testicular-expression patterns between KPNA and SubH2Bv suggested a potential binding interaction between these proteins. This was supported by recombinant SubH2Bv affinity pull-down assays on germ cell extracts. The results of this study provide a compelling argument that these two nuclear homing proteins work in concert to direct the acrosomic vesicle to the nucleus. Their final residence in the subacrosomal layer of the perinuclear theca of spermatozoa indicates a role for SubH2Bv and KPNA in acrosomal-nuclear docking.     

Comparative distribution of short dystrophin superfamily products in various guinea pig spermatozoa domains

In this study, the presence and cellular distribution of dystrophin family products (i.e. Dp71d, Dp71f-like protein and dystrobrevin) was examined by indirect immunofluorescence and Western blotting in guinea pig spermatozoa. Two dystrophin-associated proteins, beta-dystroglycan and alpha-syntrophin, and nNOS a protein frequently associated with alpha-syntrophin, were determined. In spermatozoa lacking plasma membrane and acrosome, Dp71f-like protein was found in the postacrosomal perinuclear theca and also in the middle piece of the flagellum. In the flagellum, Dp71f-like protein is localized together with alpha-syntrophin and nNOS. Dp71d was present in the plasma membrane of the middle piece with beta-dystroglycan, alpha-syntrophin and nNOS. Dp71d was also present in plasma membrane of the post acrosomal region, but only with nNOS. Finally, dystrobrevin was located all along skeletal flagellum structures and in the subacrosomal hemisphere of the perinuclear theca. This distinct and complementary distribution in various domains of spermatozoa may reveal a specific function for each short dystrophin family product, in the stabilization of the domains where they are located.     

An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development

We have been interested in determining the minimally required elements in the sperm head that are necessary in order for the paternal genome to participate in embryogenesis. We used an ionic detergent, mixed alkyltrimethylammonium bromide (ATAB), plus dithiothreitol (DTT) to remove the acrosome and almost all of the perinuclear theca, leaving only the sperm nucleus morphologically intact. We also tested the stability of the sperm nuclear matrix by the ability to form nuclear halos. Sperm nuclei washed in freshly prepared 0.5% ATAB + 2 mM DTT completely decondensed when extracted with salt, but nuclei washed in the same buffer that was 1 wk old, and then extracted with salt, produced nuclear halos, indicating stable nuclear matrices. When we treated sperm heads with freshly prepared ATAB+DTT and injected them into oocytes, none of the oocytes developed into live offspring. In contrast, sperm heads treated in the same way but with 1-wk-old ATAB+DTT solution could support development of about 30% of the oocytes to live offspring. Electron microscopy demonstrated that most of the perinuclear theca had been removed in both cases. These data suggest that at least in the mouse, the only component of the spermatozoa that is crucial for participation in embryologic development is the sperm nucleus with a stable nuclear matrix.     

A perinuclear theca substructure is formed during epididymal guinea pig sperm maturation and disappears in acrosome reacted cells

The perinuclear theca (PT) is a unique cytoskeletal mammalian sperm structure that surrounds the nucleus. Using negatively stained whole-mount preparations, we detected a PT substructure on the apical region of the postacrosomal theca layer of guinea pig spermatozoa. The PT substructure consists of projections resembling eyelashes, circling the sperm head. The PT substructure was absent in caput but appeared in corpus epidydimal spermatozoa. The same finding was observed in sheep and rabbit spermatozoa. The PT substructure persisted in capacitating spermatozoa, but was absent in acrosome reacted gametes. No labeling of the PT substructure was observed by the immunogold technique using antibodies against calmodulin, spectrin, myosin, and vimentin. A 34-kDa band appeared as a possible PT substructure protein. The PT was positive to the antibodies and the presence of the above-mentioned proteins was confirmed by Western blot. F-actin gold label was observed in mature spermatozoa on the PT substructure base zone. Results using cytochalasin D and phalloidin point to a role of F-actin in the PT substructure formation/disassembly processes. Ca(2+), bicarbonate, and proteases might be involved in the mechanism of the substructure disassembly. Novel PT morphological changes occurring during sperm epidydimal maturation and at acrosome reaction, respectively, are discussed in relation to the PT stability and function.     

Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human spermatozoa

Acrosomeless round-headed spermatozoa from three men were studied under electron microscopy and indirect immunofluorescene microscopy using the anti-calicin antibody that recognizes a basic protein of the sperm perinuclear theca (Longo et al., 1987). Electron microscopy revealed the existence of anomalies of the nuclear envelope, the nuclear matrix underlying the nuclear envelope, and the perinuclear layer. The absence of sperm labeling with the anti-calicin antibody confirmed that the formation of the perinuclear theca was impaired. Data obtained from both mature spermatozoa and ejaculated spermatids suggest that i) round-headed sperm head anomalies result from a failure of differentiation of the sperm-specific skeletal complex related to the nucleus, and ii) the acrosome spreading over the nucleus, the nuclear elongation and the post-acrosomal sheath formation are dependent on such nuclear-perinuclear differentiations. In contrast, chromatin condensation, cytokinesis and some events of the acrosomal shaping appear not to depend on those nuclear-related differentiations. The possible processes allowing the maintenance of the sperm head structures and their subsequent morphogenesis are discussed.     

Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit

Using a monoclonal antibody as a highly specific probe and a seminal particle-free fraction of rabbit ejaculated spermatozoa, actin has been localized in the postacrosomal region of mature rabbit spermatozoa. The sperm actin has been extracted and identified on two-dimensional PAGE immunoblots as a single spot of pI = 5.45 and Mr = 43,000. Rabbit sperm actin is present in a nonfilamentous form and is not removed by removing the plasma membrane. Unlike mature spermatozoa, however, filamentous actin is present in spermatogenic cells, as determined by rhodamine phalloidin staining. Starting as diffusely distributed in spermatocytes, actin accumulates in the subacrosomal space and appears as a band in conjunction with the developing acrosome. This band lengthens throughout the spermatid stage and becomes continuous with the postacrosomal region staining in testicular spermatozoa. Actin may therefore function during spermatogenesis to both shape the acrosome to the nucleus and to anchor inner acrosomal membrane proteins.     

Somatic histones are components of the perinuclear theca in bovine spermatozoa

The perinuclear theca is a non-ionic detergent-resistant, electron-dense layer surrounding the condensed nucleus of mammalian sperm. The known proteins originating from the perinuclear theca have implicated the structure in a variety of important cellular processes during spermiogenesis and fertilization. Nonetheless, the composition of the perinuclear theca remains largely unexplored. We have isolated a group of low molecular mass (14-19 kDa) perinuclear theca-derived proteins from acrosome-depleted bovine sperm heads by salt (1 M KCl) extraction and have identified them as core somatic histones. N-terminal sequencing and immunoblotting with anti-histone antibodies confirmed the presence of both intact and proteolytically cleaved somatic histones H3, H2B, H2A, and H4. Identical proteins were isolated using 2% SDS or 1 N HCl extractions. Subsequent acid and SDS extractions of intact bovine sperm revealed the presence of all four intact histone subtypes, with minimal proteolysis. Two-dimensional acid/urea/Triton-SDS-PAGE, coupled with immunoblotting analysis, confirmed the somatic nature of these perinuclear theca-derived histones. Estimates of the abundance of perinuclear theca-derived histones showed that up to 0.2 pg per sperm of each histone subtype was present. Immunogold labeling at the ultrastructural level localized all four core somatic histones to the post-acrosomal sheath region of bovine epididymal sperm, when probed with affinity-purified anti-histone antibodies. Little immunoreactivity was detected in residual perinuclear theca structures following the extractions. Taken together, these findings indicate the unprecedented and stable localization of non-nuclear somatic histones in bovine sperm perinuclear theca.     

Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.     

Identification and cellular location of glutamine synthetase in human sperm

Glutamine synthetase (GS) catalyzes the de novo synthesis of glutamine, an amino acid that has been shown to influence sperm motility in mammals. To date, no information is available about GS content in human sperm. In this study, we have characterized the presence and cellular location of GS in fresh human normozoospermic samples. We have detected a single band corresponding to GS by Western blot. Confocal analysis has revealed GS immunoreactivity in the post-acrosomal head region. Moreover, double-labeling experiments with either F-actin or calicin have demonstrated GS confinement in the post-acrosomal region of the perinuclear theca. These data have been validated by a post-embedding ultra-structural study. The presence of GS in the post-acrosomal region of the perinuclear theca suggests that human sperm can carry out in glutamine synthesis.     

Molecular characterization of mammalian cylicin, a basic protein of the sperm head cytoskeleton

The cytoskeletal calyx structure surrounding part of the nucleus of the mammalian sperm head contains two major kinds of basic proteins, i.e., the approximately 60-kD calicin and a group of very basic (IEP > pH 10) polypeptides ranging in size from approximately 58 to approximately 100 kD ("multiple band proteins," MBPs). We have produced MBP-specific mAbs and have isolated a bovine and a human cDNA clone encoding one of these proteins, termed "cylicin" (from the Greek word c eta kv lambda l zeta for cup or beaker). Bovine cylicin I of a calculated molecular weight of 74,788 contains a high proportion (29%) of positively charged amino acids, resulting in an IEP of 10.55, numerous KKD tripeptides, and is characterized by an organization of the central part of the molecule in nine repeating units of maximally 41 amino acids each of which according to prediction analysis should tend to form an alpha helix. The identity of the polypeptide has been proven by direct amino acid sequencing of > 14 different fragments and by experiments using antibodies raised against a partial cDNA-derived protein segment produced in E. coli. By Northern blot analysis we have identified the 2.4-kb cylicin I mRNA only in testis. The unusual cytoskeletal protein cylicin is compared with other proteins and its possible architectural role during spermiogenesis is discussed.