Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

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.     

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.     

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.     

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.     

Protein composition of the ventral processes on the sperm head of Australian hydromyine rodents

The sperm head of the plains rat, an Australian hydromyine rodent, is highly complex in structure and contains, in addition to an apical hook, two large ventral processes (VPs) that extend from its upper concave surface and that are largely composed of a huge extension of the sperm head cytoskeleton surrounded by postacrosomal dense lamina. In this study we have attempted to determine their protein composition. For this, the VPs were isolated, the proteins within them separated by SDS-PAGE, and the resultant polypeptide bands Western blotted and probed with antibodies against laboratory rat perforatorial and bull perinuclear theca sperm proteins. Antibodies were also used to determine the perforatorial and perinuclear theca proteins by immunogold labeling of transmission electron microscopic sections. The results indicate that the material within the VPs is largely composed of perforatorial cross-reacting proteins together with F-actin with the dominant protein being PERF 15. The perinuclear theca proteins are, by contrast, restricted to a narrow region adjacent to the acrosomal and nuclear membranes. In conclusion, this study has shown that the VPs of the spermatozoa of Australian rodents are perforatorial-like appendages that contain similar proteins to the perforatorium of the apical hook together with F-actin; their functional significance remains unknown.     

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.     

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.     

Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus

Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.     

Post-testicular development of a novel membrane substructure within the equatorial segment of ram,bull, boar,and goat spermatozoa as viewed by atomic force microscopy

Atomic force microscopy has been used to investigate changes in the plasma membrane overlying the head region of mammalian spermatozoa (bull, boar, ram, goat, stallion, mouse, and monkey) during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle. On ejaculated ram, bull, boar, and goat spermatozoa the postacrosomal plasma membrane has a more irregular surface than that covering the acrosome. The equatorial segment, by contrast, is relatively smooth except for an unusual semicircular substructure within it that has a coarse uneven appearance. This substructure (referred to as the equatorial subsegment) is situated adjacent to the boundary between the postacrosomal region and the equatorial segment itself and seems to be confined to the order Artiodactyla as it has not been observed on stallion, mouse, or monkey spermatozoa. The equatorial subsegment develops during epididymal maturation, and following induction of the acrosome reaction with Ca(2+) ionophore A23187, its topography changes from a finely ridged appearance to that resembling truncated papillae. A monoclonal antibody to the equatorial subsegment binds only to permeabilized spermatozoa, suggesting that the subsegment is related to the underlying perinuclear theca that surrounds the sperm nucleus. A role for the equatorial subsegment in mediating fusion with the oolemma at fertilization is discussed.     

Ultrastructural study of spermatogenesis in the free-living marine nematode <Emphasis Type="Italic">Paracyatholaimus pugettensis</Emphasis> Weiser et Hopper, 1967 (Chromadorida: Cyatholaimidae)

Spermatogenesis and the morphology of mature sperm in the free-living chromadorid Paracyatholaimus pugettensis from the Sea of Japan were studied using transmission electron microscopy. In spermatocytes fibrous bodies (FBs) appear; in spermatids, the synthetic apparatus is located in the residual body, whereas the main cell body (MCB) houses the nucleus, mitochondria, and FBs. The nucleus of the spermatid consists of a loose fibrous chromatin that is not surrounded by a nuclear envelope; centrioles lie in the perinuclear cytoplasm. The plasma membrane of the spermatid MCB forms numerous filopodia. Immature spermatozoa from the proximal part of the testis are polygonal cells with a central nucleus. The latter is surrounded by mitochondria and FBs with poorly defined boundaries. The immature spermatozoa bear lamellipodia all along their surface. Mature spermatozoa are polarized cells with an anterior pseudopodium, which is filled with filaments that make up the cytoskeleton; the MCB houses a nucleus that is surrounded by mitochondria and osmiphilic bodies. In many ultrastructural characteristics, the spermatozoa of P. Pugettensis are similar to those of most nematode species studied so far (i.e., they are ameboid, have no acrosome, axoneme, or nuclear envelope). On the other hand, as in other chromadorids, no aberrant membrane organelles were observed during spermatogenesis of P. Pugettensis.Original Russian Text Copyright © 2004 by Biologiya Morya, Zograf, Yushin.     

The Fluorescent Dye 3, 3' Dihexyloxacarbocyanine Iodide Selectively Stains the Midpiece and Apical Region of the Heads of Murid Rodent Spermatozoa

Fluorescence microscopy of caudal epididymal spermatozoa stained with 3, 3' dihexyloxacarbocyanine iodide (DiOC₆(3)) showed intense fluorescence along the concave surface of the apical hook of spermatozoa of Rattus species and along the upper concave margin of the sperm head in Mus musculus In the spermatozoa of Hydromys chrysogaster, Melomys cervinipes, and Pseudomys australis, the two ventral processes also fluoresced brightly. In P. australis, fluorescence in the apical hook of sperm heads was largely localized to its upper and lower surfaces. The sperm of N. alexis did not show consistent positive fluorescence. The localization of fluorescence in these spermatozoa after staining with DiOC₆(3) was mainly restricted to regions where a large accumulation of perinuclear theca material lies beneath the plasmalemma. The reason for this remains to be determined, but DiOC₆(3) may be useful for quickly demonstrating areas of abundant perinuclear thecal material in sperm heads of eutherian mammals by light microscopy.     

The disruption in actin-perinuclear theca interactions are related with changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen

The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen–thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen–thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P < 0.001), and significant changes in sperm chromatin nuclear decondensation (P < 0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P < 0.05). Evaluation with rodamine-labeled actin (0.2 μM) showed two different patterns with differences in percentages before and after cryopreservation (P < 0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing–thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.     

The perinuclear matrix as a structural element of the mouse sperm nucleus.

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.     

The Fluorescent Dye 3, 3′ Dihexyloxacarbocyanine Iodide Selectively Stains the Midpiece and Apical Region of the Heads of Murid Rodent Spermatozoa

Fluorescence microscopy of caudal epididymal spermatozoa stained with 3, 3′ dihexyloxacarbocyanine iodide (DiOC₆(3)) showed intense fluorescence along the concave surface of the apical hook of spermatozoa of Rattus species and along the upper concave margin of the sperm head in Mus musculus In the spermatozoa of Hydromys chrysogaster, Melomys cervinipes, and Pseudomys australis, the two ventral processes also fluoresced brightly. In P. australis, fluorescence in the apical hook of sperm heads was largely localized to its upper and lower surfaces. The sperm of N. alexis did not show consistent positive fluorescence. The localization of fluorescence in these spermatozoa after staining with DiOC₆(3) was mainly restricted to regions where a large accumulation of perinuclear theca material lies beneath the plasmalemma. The reason for this remains to be determined, but DiOC₆(3) may be useful for quickly demonstrating areas of abundant perinuclear thecal material in sperm heads of eutherian mammals by light microscopy.     

Identification of a sperm nuclear annulus: a sperm DNA anchor

We report the isolation and characterization of a new nuclear structure from spermatozoa of the golden Syrian hamster which we term the nuclear annulus. The nuclear annulus was located at the implantation fossa, the point at which the tail is joined to the sperm head, inside the nucleus adjacent to the inner nuclear envelope. Extraction of sperm nuclei with 2 M NaCl and 10 mM dithiothreitol caused the solubilization of the protamines and DNA decondensation. This released the DNA from its structural constraints except that the DNA remained anchored to the nuclear annulus, which survived the extraction procedure. Nuclear annuli were isolated and examined by scanning electron microscopy. The nuclear annulus was a double-crescent, ring-shaped structure, 2.10 +/- 0.16 micron long and 1.36 +/- 0.13 micron wide. We believe that the nuclear annulus may play an important role in the organization of sperm DNA.     

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.     

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.     

Sperm head shaping in ratites: New insights,yet more questions

Head shaping in mammalian sperm is regulated by a number of factors including acrosome formation, nuclear condensation and the action of the microtubular manchette. A role has also been suggested for the attendant Sertoli cells and the perinuclear theca (PT). In comparison, relatively little information is available on this topic in birds and the presence of a PT per se has not been described in this vertebrate order. This study revealed that a similar combination of factors contributed to head shaping in the ostrich, emu and rhea, although the Sertoli cells seem to play a limited role in ratites. A fibro-granular structure analogous to the mammalian PT was identified, consisting of sub- and post-acrosomal components. The latter was characterized by stage-specific finger-like projections that appeared to emanate from the cytoplasmic face of the nuclear envelope. They were particularly obvious beneath the base of the acrosome, and closely aligned, but not connected to, the manchette microtubules. During the final stages of chromatin condensation and elongation of the sperm head the projections abruptly disappeared. They appear to play a role in stabilizing the shape of the sperm head during the caudal translocation of the spermatid cytoplasm.     

An ultrastructural analysis of the mature spermatozoon of Anguispira alternata (say) (pulmonata,stylommatophora)

Mature spermatozoa from the hermaphroditic duct of adult snails were examined using various techniques of light microscopy as well as scanning and transmission electron microscopy. The sperm are approximately 557 μm in length including a dextrally spiral head approximately 13 μm long. The head consists of an electron-dense nucleus sculptured into a double-ridged spiral and an acrosome projecting approximately 0.45 μm beyond the apex of the nucleus. The acrosome consists of a membrane-bound vesicle approximately 0.1 μm in diameter and a column of homogeneous material which extends along one side of the terminal spiral of the nucleus. This material is separated from the nucleus by the nuclear envelope. The neck region, though similar to that found in other pulmonates, possesses a unique coiled structure surrounding the central doublet of the axoneme. The midpiece axoneme possesses a 9+9+2 configuration anteriorly grading into a 9+2 pattern for the majority of its length. There are three mitochondrial helices – one primary and two secondary – in the midpiece. Only the primary helix persists throughout the midpiece.