Subacrosomal Space Research Articles

Ultrastructure of the mature spermatozoon of the bivalveEstellarca olivacea(Mollusca: Bivalvia: Arcidae) and its phylogenetic implications

Ultrastructure of mature spermatozoa of Estellarca olivacea was studied by transmission electron microscopy and its phylogenetic implications are discussed for the first time in this paper. The mature spermatozoon is composed of a head which contains a cone-shaped acrosome, a round nucleus and a tail region. The subacrosomal space is less electron dense which contains a homogeneous material. No axial rod and a basal plate were observed in subacrosomal space. No anterior invagination exists in the nucleus, but an inverted shallow V-shaped posterior invagination is visible. Nuclear lacunae could be seen clearly although the nucleus is highly condensed. Within the mid-piece of the spermatozoon there exist five spherical mitochondria while the long whip-like end portion is composed of an axoneme with the typical 9+2 structure. The spermatozoon of Estellarca olivacea is a product of the evolution of the reproductive system of the family Arcidae. Whether the particular acrosome, subacrosomal space, or the highly condensed nucleus might be adaptations of high fertilization rate in the particular environment of this species is discussed.

Spermatozoa and spermatogenesis in the northern quahaug Mercenaria mercenaria (Mollusca, Bivalvia)

We studied the ultrastructure of spermatogenesis and spermatozoa in the northern quahaug, the clam Mercenaria mercenaria. Spermatogenetic cells gradually elongate. Mitochondria gradually fuse and increase in size and electron density. During spermatid differentiation, proacrosomal vesicles migrate towards the presumptive anterior pole of the nucleus and eventually form the acrosome. The spermatozoon of M. mercenaria is of a primitive type. It is composed of head, mid-piece, and tail. The acrosome shows a subacrosomal space with a short conical contour. The slightly curved nucleus of the spermatozoon contains fine-grained dense chromatin. The middle piece consists of a centriolar complex which is surrounded by four mitochondria. The flagellum has a standard “9 + 2” microtubular structure. The ultrastructure of spermatozoa and spermatogenesis of M. mercenaria shares a number of features with other species of the family Veneridae. M. mercenaria may be a suitable model species for further investigations into the mechanisms of spermatogenesis in the Bivalvia.

Ultrastructure of the mature spermatozoon of the bivalve Scapharca broughtoni (Mollusca: Bivalvia: Arcidae)

The ultrastructure of mature spermatozoa of the giant clam bivalve Scapharca broughtoni was investigated by transmission electron microscopy for the first time. The mature spermatozoon consists of a head which is composed of a cone-shaped acrosome, a round nucleus, and a tail region. A subacrosomal space contains an axial rod and a basal plate, the latter lying between the acrosome and the nucleus. Although the nucleus lacks an anterior invagination, an inverted shallow V-shaped posterior invagination is present within the nucleus. Within the middle portion of the spermatozoon lie five spherical mitochondria while the long whip-like end portion is composed of an axoneme with the typical 9 + 2 structure. Our conclusion is that the spermatozoon of S. broughtoni is of the type I anacrosomal “aquasperm”, and the morphology of acrosome and nucleus are an adaptation to external fertilization.

Sperm morphology in five species of cicadettine cicadas (Hemiptera: Cicadomorpha: Cicadidae)

Mature spermatozoa from five species of cicadas of the subfamily Cicadettinae ( Quintilia wealei, Melampsalta leucoptera, Stagira simplex, Xosopsaltria thunbergi and Monomatapa matoposa) were examined by light and electron microscopy. In each species sperm are elongate, aggregated into organized bundles with their heads embedded in a homogenous matrix to form spermatodesmata, and exhibit polymegaly. The head of the sperm consist of an anteriorly positioned conical acrosome that has a tubular substructure and a deep, posterior invagination that forms the subacrosomal space (eccentrically positioned anteriorly). The acrosome is flattened anteriorly; posteriorly it extends along either side of the nucleus as two tubular processes that gradually decrease in diameter. The filiform nucleus tapers anteriorly and intrudes into the subscrosomal space. Posteriorly the nucleus has a lateral invagination that houses material of the so-called centriolar adjunct. Posterior to the centriolar adjuct and the nucleus are two crystalline mitochondrial derivatives and a centriole, respectively, the latter giving rise to the axoneme, which has a 9 + 9 + 2 arrangement of microtubules. In these respects the sperm are similar to those of platypleurine cicadas. However, some features seem unique to cicadettines, including the structural organization of an enlarged centriolar adjunct and the dimensions of the tails. The enlarged centriolar adjunct has a lamella-like substructure and can be considered a synapomorphic character in the Cicadettinae. It is, therefore, potentially useful in the separation of this subfamily from the Cicadinae. In addition, the great length of the sperm nucleus of long-headed sperm in M. matoposa could be a synapomorphy of this genus and related taphurine and cicadettine species.

Effects of lithium carbonate on rat seminiferous tubules: an ultrastructural study

Lithium salts are commonly used for treatment of bipolar disorder but prolonged treatment with therapeutic doses induces substantial toxic effects. In the present study we examined the effects of lithium carbonate on the ultrastructure of rat seminiferous tubules. Rats were exposed to lithium carbonate at doses of 35 mg/kg/day for 21 days. After lithium treatment, the tunica propria widened and folded together with convolutions of the basement membrane, myoid cells and lymphatic endothelium. In the seminiferous epithelium loss of germ cell attachment and appearance of expanded intercellular spaces between spermatogenic cells were observed. Early stages of spermatogenic cells showed nuclear protrusions or swellings because of an extensive enlargement of the outer nuclear membrane. Round spermatids exhibited abnormally shaped acrosomes and dilation of the subacrosomal space. Many abnormal, degenerated late spermatids with random orientation were seen towards the basal and adluminal compartments of the seminiferous epithelium. In addition, spermatids exhibited alteration in F-actin bundle ectoplasmic specialization and contained many mitochondria-associated granular bodies.

Morphology of the spermatozoon in two sympatric species of Fissurella Bruguière, 1789 (Mollusca: Vetigastropoda) from Southern Chile

Summary Light and electron microscopy were used to analyze the morphology and ultrastructure of the spermatozoon of Fissurella nigra and Fissurella picta, two sympatric species of keyhole limpets from the southern Chilean coast. The spermatozoa of both species are of the aquasperm type, typical of species with external fertilization. Each species had its own distinctive spermatic morphology, particularly in relation to size. Proceeding from anterior to posterior, the spermatozoa of both species each consists of an elongated head with a conical acrosome having a deep subacrosomal space and truncated conical nucleus, followed by a midpiece containing five mitochondria associated in a compact ring around the proximal and distal centrioles, and, at the end, a flagellum. The spermatozoon head of F. nigra is almost twice as long as that of F. picta. This difference constitutes the first morphological evidence that the size of the spermatozoon could represent a candidate for the maintenance of reproductive isolation between these two sympatric species.

Spermiogenesis in Psilochorus simoni (Berland, 1911) (Pholcidae, Araneae): Evidence for considerable within-family variation in sperm structure and development

A large number of characters and considerable variation among taxa make animal sperm cells promising objects for phylogenetic studies. However, our knowledge about sperm structure and development in spiders is still rudimentary. In pholcids, previous studies of two species representing different subfamily level taxa have revealed conspicuous differences. Here, we report on a representative of a third subfamily level taxon, confirming substantial variation in sperm structure and development within the family. The male genital system in Psilochorus simoni (Berland, 1911) consists of paired testes and deferent ducts which lead into a common ejaculatory duct. The somatic cells of the testes show a high secretory activity, and produce at least two different kinds of secretion. The spermatozoa show features already known from other Pholcidae as well as unique characters. The acrosomal vacuole is tube-like with a narrow subacrosomal space. The axoneme migrates deep into the nucleus and is finally located near the acrosomal vacuole. Thus, the postcentriolar elongation of the nucleus is very long. A centriolar adjunct is not present and after the coiling process the implantation fossa is completely filled with glycogen which is also found in larger amounts within the cytoplasm of the sperm cell. After the coiling process, a vesicular area is present that becomes most prominent in the periphery of the sperm cell and surrounds the axoneme and parts of the nucleus. The secretion sheath surrounding the mature spermatozoon is already formed in the lumen of the testis, possibly by a secretion present in the testis but absent in the deferent duct. Sperm are transferred as cleistospermia. Results are compared with previous studies on pholcid spermiogenesis and sperm structure.

Spermatogenesis and Spermatozoa in Stygocapitella subterranea (Annelida, Parergodrilidae), an Enigmatic Supralittoral Polychaete

The intertidal polychaete species Stygocapitella subterranea (Parergodrilidae) is characterised by extraordinary biology and morphology, resembling those of clitellates and Hrabeiella periglandulata, a terrestrial species of Annelida. An ultrastructural study of the spermatogenesis and spermatozoa was undertaken to elucidate whether these similarities might exhibit adaptive characters typical of annelids with highly derived reproductive modes. A second goal was to find out whether there are some common apomorphic features between S. subterranea and its sister taxon, Parergodrilus heideri, instead of the differences observed on the light-microscopic level, as well as to look for potential synapomorphies to support a suggested relationship to Orbiniidae and Questidae. Spermatogenesis conforms to the general pattern typical of Annelida. Spermatids develop on large cytophores comprising at least 128 cells. The spermatozoa are extremely thread-like and, with a length of about 320 μm, are among the longest spermatozoa known for annelids. The acrosome is elongated and consists only of an acrosomal vesicle with a large subacrosomal space. A conspicuous feature is the incomplete chromatin condensation, resembling late spermatids. In the long midpiece, there is a single ring-shaped mitochondrial derivative, which develops by fusion out of a multiple array of eight mitochondria surrounding the axoneme. There is a distinct annulus between midpiece and tail. The proximal part of the tail is immobile; the axoneme is surrounded by a thick layer of cytoplasm and bears a velum-like extension. In addition to characters apomorphic for S. subterranea, these latter three features exhibit certain similarities to P. heideri that are likely to be synapomorphic. Unfortunately, a relationship of Parergodrilidae to an orbiniid/questid clade does not receive additional support from spermatozoal characters. Similarities with either Clitellata or H. periglandulata are likely to be primarily related to corresponding features of their reproductive biology rather than to phylogenetic relationship.

Fine structure of the spermatozoon of Diopatra neapolitana (Polychaeta, Onuphidae)

The identification of Diopatra species lacks of clear diagnostic features of taxonomic importance and the knowledge of their reproductive characters is scant. The spermatozoa of Diopatra neapolitana were ultrastructurally investigated by electron microscopy in order to correlate the mode of reproduction with sperm cells morphology. The mature male gamete has a depressed subspherical nucleus, a cone-like acrosome, and a long flagellum. The acrosome is conical in shape and radially symmetrical, with a base diameter twice the height. Within the acrosome vesicle, the basal region includes a very electron-dense thickened ring composed of paracrystalline substances. The subacrosomal space is filled with a poorly electron-dense material, with straight filaments axially arranged to form a perforatorium. The nucleus contains the complete axial canal, holding the hind perforatorium region. The middle piece consists of five mitochondria with well-distinct membranes and tubulo-vesicular cristae. Two centrioles are located perpendicularly to each other. The proximal one lies in the central fossa and the distal one, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a 9+2 arrangement of microtubules. In general, the spermatozoon of D. neapolitana conforms exteriorly to the typical ect-aquasperm; the acrosome complex ultrastructure, however, shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning—free-swimming larvae) of D. neapolitana belonging to Santa Gilla population, and may be helpful to solve the taxonomic problems of the D. neapolitana complex as well.

Ultrastructure of the spermatozoon of Talpa romana (Mammalia, Lipotyphla)

In this study, we used SEM and TEM to investigate the ultrastructure of spermatozoa from the cauda epididymis of Talpa romana. For comparison, we also analysed spermatozoa from the cauda epididymis of T. europaea captured in the same area. The male gamete of T. romana has a flattened head with an elliptic profile, consisting of a large acrosome and a nuclear region separated by a thin subacrosomal space. At the tip of the nucleus, the subacrosomal space ends in a finger-shaped projection. The tail includes a connecting piece, middle piece, principal piece and end piece. The male gametes of T. romana are substantially similar to those of T. europaea. A comparison with other species of insectivores permits extension of the similarity of sperm features to Scalopus aquaticus and Condylura cristata. Many spermatozoa from the cauda epididymis of T. romana and T. europaea have the tail bent at the annulus, and this is always associated with remnants of cytoplasmic droplets. This morphology is considered to be a common phenomenon.

Testicular expression and distribution of the rat bcl2 modifying factor in response to reduced intratesticular testosterone.

The Bcl2 modifying factor (Bmf) is a pro-apoptotic member of the Bcl2 family of apoptosis-related proteins that has been shown to initiate apoptosis in response to the loss of attachment of cells from their basal lamina (anoikis). Experimental reduction in intratesticular testosterone concentration brings about the death of spermatids as a consequence of their sloughing from Sertoli cells. Given the role of Bmf in anoikis in other systems, we hypothesized that Bmf would be expressed in germ cells and that its expression and normal distribution might be altered under conditions that induce widespread germ cell loss. To test these hypotheses, we demonstrated that Bmf indeed is expressed in the testis and cloned the full-length rat Bmf cDNA. Immunohistochemistry revealed that Bmf is present in the subacrosomal space of postmeiotic spermatids from step 4 to 16 of spermiogenesis. To test the hypothesis that Bmf expression and distribution are altered by conditions that elicit anoikis, intratesticular testosterone was reduced by implanting Silastic capsules containing testosterone and estradiol into adult rats for 8 weeks. As hypothesized, this resulted in a significant change in Bmf distribution relative to untreated animals. In particular, Bmf exhibited a loss of its normal subacrosomal distribution, becoming redistributed throughout the cytoplasm and nucleus, and appeared in cells in which it is not normally expressed (e.g., pachytene spermatocytes). Additionally, Bmf mRNA expression increased in response to lowered testosterone. These results suggest that Bmf may well be involved in germ cell apoptosis and/or anoikis in response to decreased intratesticular testosterone concentration.

Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head.

Nuclear shaping is a critical event during sperm development as demonstrated by the incidence of male infertility associated with abnormal sperm ad shaping. Herein, we demonstrate that mouse and rat spermatids assemble in the subacrosomal space a cytoskeletal scaffold containing F-actin and Sak57, a keratin ortholog. The cytoskeletal plate, designated acroplaxome, anchors the developing acrosome to the nuclear envelope. The acroplaxome consists of a marginal ring containing keratin 5 10-nm-thick filaments and F-actin. The ring is closely associated with the leading edge of the acrosome and to the nuclear envelope during the elongation of the spermatid head. Anchorage of the acroplaxome to the gradually shaping nucleus is not disrupted by hypotonic treatment and brief Triton X-100 extraction. By examining spermiogenesis in the azh mutant mouse, characterized by abnormal spermatid/sperm head shaping, we have determined that a deformity of the spermatid nucleus is restricted to the acroplaxome region. These findings lead to the suggestion that the acroplaxome nucleates an F-actin-keratin-containing assembly with the purpose of stabilizing and anchoring the developing acrosome during spermatid nuclear elongation. The acroplaxome may also provide a mechanical planar scaffold modulating external clutching forces generated by a stack of Sertoli cell F-actin-containing hoops encircling the elongating spermatid nucleus.

Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs Glycymeris sp. (Pteriomorphia) and Eurhomalea rufa (Heterodonta)

Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30–40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae.

Vesicles in the subacrosomal space and partial diaphragms in the subacrosomal nuclear envelope of round spermatids of a rat injected intravenously with gold labeled-testosterone-bovine serum albumin conjugate: vesicular trafficking from acrosome to nucleus.

Colloidal gold labeled-testosterone-bovine serum albumin conjugate (testosterone-BSA-gold) injected into the vascular system of rats is taken up by endocytosis into round spermatids. Based on observation of silver deposits indicating testosterone-BSA-gold with silver enhancement, we have suggested that testosterone-BSA-gold enters the nuclei through not only the postacrosomal nuclear envelope but also the subacrosomal nuclear envelope (SNE) via the acrosome (Nishimura and Nakano, 1997). However, it was unclear how testosterone-BSA-gold in the acrosome entered the nucleoplasm. Spermatids showing silver deposits on the subacrosomal space were observed under electron microscope without silver enhancement, to clarify the courses of translocation. In the spermatids, vesicles with the gold particles were seen in the subacrosomal space. Some of the vesicles were in contact with the SNE. A part of the outer nuclear membrane projected into the space. Furthermore, local single-bilayer nuclear membranes, which seemed to partially lack nuclear lamina, were present in the SNE. These results indicate the possibility that the vesicles mediate the transport of testosterone-BSA-gold from acrosome to nucleus, and that the vesicle membrane fuses with not only the outer nuclear membrane but also a shared bilayer in the SNE.

Ultrastructure of spermatogenesis in Cerastoderma glaucum (Cardiacea) and Spisula subtruncata (Mactracea)

Summary In Cerastoderma glaucum, Sertoli cells are rich in lipids, glycogen and lysosomes, and premeiotic cells exhibited nuage, a prominent Golgi complex and endoplasmic reticulum cisternae encircling the nucleus. The Golgi complex gives rise to proacrosomal vesicles during mid-spermiogenesis, and the round acrosomal vesicle, with a dense fibrillar core, migrates laterally while linked to the plasma membrane as it develops the subacrosomal material. In its final position, the vesicle becomes cap-shaped (0.6 μm) and differentiates into apical light and basal dense regions. The elongated and helicoidal nucleus (8–9.9 μm) has a thin tip (0.3 μm) that invades the subacrosomal space, and in the midpiece (0.8 μm) two of the four mitochondria extend laterally to the nucleus (1.5–2.1 μm). In Spisula subtruncata, Sertoli cells are rich in lipids, glycogen and phagocytosed sperm. Premeiotic cells exhibit nuage, a prominent Golgi complex that gives rise to proacrosomal vesicles from the leptotene stage and a flagellimi that is extruded at zygotene. The acrosomal vesicle forms during the round spermatid stage and differentiates into a large and dense basal region and an apical light region. It then migrates while linked to the plasma membrane by its apical pole. Development of the subacrosomal perforatorium is associated with nuage materials and endoplasmic reticulum vesicles. The mature cap-shaped (0.6 μm) acrosomal vesicle exhibits a large apical and irregular region with floccular contents and a basal dense region. The round nucleus becomes barrel-shaped (1.5 μm) and the midpiece (0.8 μm), with four mitochondria, contains a few glycogen particles.

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.

Estrutura fina do espermatozóide de Mellita quinquiesperforata Leske (Echinodermata) do litoral norte do Brasil

The fine structure of the spermatozoon in the Mellita quinquiesperforata Leske, 1778 was studied. Collected from the Amazon region, were described by light and transmission electron microscopy. The spermatozoon consisted of a acrosome complex, nucleus, four to six mitochondria, two centrioles and flagellum. The acrosome was composed of a acrosome complex, the acrosomal vesicle and the subacrosomal space. The nucleus are composed by dense material. The middle piece contains 4-6 mitochondria which are arranged around the centrioles, the 9p+0, whose axoneme has the classic 9p+2 microtubular construction.

Ultraestrutura do espermatozóide de Ucides cordatus Linnaeus (Crustacea, Ocypodidae) do litoral norte do Brasil

Ultrastructure of spermatozoa of the crab Ucides cordatus Linnaeus, 1763 is described and illustrated. Three parts can be distinguished: 1. the elongate-ovoid acrosome located anteriorly; 2. the cytoplasmic region; and 3. the nuclear region. The acrosome is composed of a thick electron-dense wall and a less electron-dense subacrosomal space. The cytoplasm region connects the acrosome with the nucleus, and three arms filled with microtubules arise from this region. The nucleus is polymorphous. The spermatozoon of Ucides cordatus follows the general pattern described for other Decapods.

Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa.

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.

SPERMOGENESIS AND SPERM STRUCTURE IN THREE SPECIES OF PATELLOIDA AND ONE SPECIES OF NIPPONACMAEA (PATELLOGASTROPODA: ACMAEOIDEA)

The spermatozoa of Patelloida profunda albonotata, P. saccharina, P. pygmaea and Nipponacmaea schrenkii (Lottiidae) are described by transmission electron microscopy. All have ect-aquasperm, typical of invertebrates using external fertilization. The sperm of all four species have a cylindrical nucleus (length: breadth > 4: 1 in P. p. albonotata and P. saccharina; <4: 1 in P. pygmaea and N. schrenkii) which tapers towards the rounded anterior end. All have an acrosome with a posterior acrosomal lobe which extends into the centre of the subacrosomal space. In P. p. albonotata and P. saccharina the acrosomal contents are undifferentiated and the posterior lobe extends to the nucleus, being separated from it by flocculent material. In P. pygmaea the acrosomal contents are differentiated, the lobe is relatively short and the subacrosomal space is filled with material with a fibrous appearance. The acrosome of N. schrenkii is undifferentiated and the posterior lobe is no more than a bulge. The sperm of P. p. albonotata and N. sacchrina have a small (0.25 μm long) cytoplasmic collar which surrounds the axoneme anteriorly whereas in P. pygmaea and N. schrenkii the cytoplasmic collar is longer (1 μm) and is swollen by an electron-dense vesicle. The composition and function of this vesicle is unknown. The spermatozoa of Patelloida and Nipponacmaea have structural features which are similar to sperm of the Lottiidae providing some support for the placement of these genera in the Lottiidae as proposed by Lindberg & Hedegaard (1996) and Sasaki & Okutani (1993) respectively. The similarities of the sperm of P. pygmaea to N.schrenkii raise some doubts about the tax-onomic status of the former species. Spermiogenesis in all four species is similar to that described for other Acmaeoidea and Patelloidea. In P. pygmaea and N. schrenkii, however, in addition to the acrosomal vesicle, the Golgi body produces a number of electron-dense vesicles which fuse and eventually form a single vesicle in the collar of the mid-piece.