Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

A study of the chorion and the follicle cells in relation to the sperm-egg interaction in the ascidian,Ciona intestinalis

We present ultrastructural observations on the processes underlying interaction between the spermatozoon and the egg envelopes in Ciona intestinalis. The morphological and cytochemical basis of sperm binding to the chorion and the subsequent steps such as the acrosome reaction and penetration of the spermatozoon through the chorion are described. Some speculations are also presented on the role of the follicle cells in fertilization.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

The sperm ultrastructure of Merluccius merluccius (Teleostei,Gadiformes): phylogenetic considerations

The anacrosomal aquasperm of the gadiform Merluccius merluccius is ultrastructurally similar to the advanced type II spermatozoa (perciform‐type sperm) typically found in most Perciformes. The perciform‐type spermatozoon is characterized by the lateral insertion of the flagellum and the location of the centrioles outside the nuclear fossa. Apart from these characteristics, the spermatozoon of M. merluccius is remarkable because of the mutually parallel arrangement of the centrioles, a rare feature among fishes, which is considered an apomorphic condition for animal sperm cells. Within the superorder Paracanthopterygii, which contains a large diversity of sperm patterns resulting from a high number of apomorphies, a perciform‐type sperm is present only in the order Gadiformes. The significance of the presence of perciform‐type spermatozoa in the three investigated gadiform families is discussed in a phylogenetic context.     

Binucleate and biflagellate spermatozoa in Tricholepidion gertschi Wygodzynsky (Insecta, Zygentoma)

The phenomenon of sperm pairing is known from some species of the apterygotan insect order Zygentoma, and has been described as the close apposition of two sperm cells. When released from the testes, they are single cells; pairing taking place in the deferent ducts. In a study of the relic species Tricholepidion gertschi, Zygentoma, sperm pairing was found to be due to a true fusion of two partners along their entire sperm head regions. The spermatozoon thus formed has two acrosomes, two nuclei and two separate sperm tails. The biflagellate spermatozoon swims with coordinated movements of its two flagella only when the two flagella lie close together but is totally uncoordinated when separate. The spermatozoon is about 50 microm long, thus much shorter than those of related apterygotan species. The mechanism of sperm cell fusion is unclear, although it appears that a 55-nm wide layer of electron dense substance, here termed the peripheral lamina, may play a role in delimiting the extent of sperm fusion.     

Spermiogenesis and sperm ultrastructure of Cyathocephalus truncatus (Pallas, 1781) Kessler, 1868 (Cestoda: Spathebothriidea)

Spermiogenesis and sperm ultrastructure of adult Cyathocephalus truncatus, a member of presumably basal group of "true" cestodes (Eucestoda), have been examined for the first time by using transmission electron microscopy. The process of sperm formation corresponds in basic pattern to that of the Pseudophyllidea. In addition, the 2 pairs of electron-dense attachment zones are present in median cytoplasmic process of C. truncatus. However, mature spermatozoa of C. truncatus differ significantly from those of the pseudophyllideans, especially in the morphology of the proximal and distal spermatozoon extremities. The proximal extremity of the mature spermatozoon lacks a crested body, which is present in more derived cestodes and some pseudophyllideans. The distal end of the mature spermatozoa exhibits different morphology in the gametes from testes and those from receptaculum seminis. New for the Eucestoda is a finding that a lateral cytoplasmic extension creates the distal end of the spermatozoa from testes, resembling sperm of some Monogenea and Digenea. In contrast, the distal extremity of the spermatozoa from receptaculum seminis contains only a nucleus. Despite the above-mentioned peculiarities, the ultrastructural data on sperm/spermiogenesis suggest close relationships of the Spathebothriidea and Pseudophyllidea.     

The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis

Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.     

THE SPERMATOZOON OF ARTHROPODA: ZOOTERMOPSIS NEVADENSIS AND ISOPTERAN SPERM PHYLOGENY

In this paper, the ultrastructure of the spermatozoon of Zootermopsis nevadensis (Isoptera, Hodotermitidae) and of some Rhinotermitidae and Termitidae is described. Zootermopsis sperm is rod like, aflagellate, immotile, and without an acrosome; it is composed of a filiform nucleus encircled by a monolayered microtubular manchette, and a few mitochondria. This spermatozoon was previously thought to be flagellate, and therefore the most primitive in Isoptera: our present study suggests a new phylogenetical position for Hodotermitidae. All the species of Rhinotermitidae and Termitidae studied by us show a similar spheroidal sperm model, devoid of acrosome, flagellum and manchette at spermatid stage, and are made up of only a round nucleus, two mitochondria and a centriole. This widely distributed model seems to be the more evolved in the order. The nature of sperm evolution in the Isoptera is considered.     

High concentration of inositol 1,4,5-trisphosphate in sea urchin sperm

We measured inositol 1,4,5-trisphosphate (InsP3) content of sea urchin gametes by using a specific protein binding assay, and found that a spermatozoon contains 4 x 10(-19) to 1 x 10(-18) moles of InsP3 before the acrosome reaction. Since the acrosome reaction has previously been shown to increase the InsP3 content of sperm severalfold, our measurement indicates that a spermatozoon contains at least 2 x 10(-18) moles of InsP3 at fertilization, corresponding to a concentration in the spermatozoon of about 1 mM. The threshold for activation of eggs by injection of InsP3 dissolved in a much larger volume of solution has been found to be about 3 x 10(-18) moles, corresponding to a concentration in the injectate of 1 microM. This suggests that sea urchin sperm may contain enough InsP3 to activate eggs. With an electroporation method, we also showed that sperm extract acts on eggs only from inside, consistent with a primary messenger role for InsP3.     

Ultrastructural Observations on Fertilization in Dionchus remorae (Platyhelminthes,Monogenea, Dionchidae)

Ultrastructural observations are presented for some of the stages occurring during fertilization in Dionchus remorae (a gill parasite of Echeneis naucrates) and are believed to be the first published concerning a monogenean. Fertilized female germ cells were found in the ovary. Several loops of the spermatozoon were present within the oocyte cytoplasm; the sperm nucleus became electron lucent and the parallel peripheral doublets of the axonemes became increasingly divergent. The cortical granules in the oocyte were not released immediately after penetration by the spermatozoon. The homogeneity apparently found in the oocyte ultrastructure and process of fertilization in the monogeneans and digeneans contrasts with the variety that exists in their sperm ultrastructure.     

The Spermatozoon of Siboglinum (Pogonophora)

The spermatozoon and some spermatid stages of Siboglinum (Pogonophora) have been examined by light and electron microscopy. In the spermatozoon a helical acrosome, a helical nucleus and a “body” with axonema follow each other in normal sequence. Head and tail are joined by a very short neck region containing two modified centrioles. The posterior portion of the nucleus is surrounded by a mitochondrial sheath consisting of three tightly wound mitochondrial helices. In the main portion of the tail the 9+2 unit is sorrounded by a granular sheath of dense material. In the neck region a centriole adjunct develops into a dense substance containing about nine rods. At an early stage, when the centriolar apparatus and flagellum become associated with the nucleus, three large mitochondria with fairly regular cristae are seen at the base of the nucleus. A well developed Golgi apparatus is present in early stages. Rows of microtubules are observed encircling the spermatid nucleus. Compared with the primitive type of spermatozoon the pogonophore sperm shows elongated and specialized nucleus, acrosome and mitochondria. It is concluded that the ancestral form must have had a fairly primitive spermatozoon and that evolution has proceeded towards a modified sperm with complicated spiral structure in connection with the evolution of a modified biology of fertilization, viz. specialized spermatophores. It is not known how the spermatophore discharges the spermatozoa nor how the spermatozoa find their way to the eggs. Two kinds of sperms are produced in the gonads of Siboglinum. The atypical sperm is smaller than the typical one.     

Ultrastructure of the biflagellate spermatozoon of tomopteris helgolandica greef, 1879 (annelida,polychaeta)

The spermatozoon of the polychaete Tomopteris helgolandica is of an aberrant type with two flagella, each measuring about 40μm. The nucleus is roughly conical and weakly bent. At the anterior end it is rounded and covered only by the nuclear and plasma membranes. Membraneous, electron-dense structures are applied laterally to the nucleus. These structures may have a helical arrangement. The middle piece contains about ten mitochondria, two centrioles, and two centriolar satellite complexes. The centriolar regions are connected with the posterior part of the nucleus. The axonemes of the two tail flagella lack the usual central complex with central tubules, radial spokes, or related structures. No arms seem to be present on the A tubules of the doublets. In the middle piece the tail flagella are surrounded by invaginations of the plasma membrane forming flagellar canals. The sperm has a bilateral symmetry whereas the primitive sperm has a radial symmetry. The occurrence of two tail flagella in this spermatozoon has no phylogenetical connection with biflagellate spermatozoa in other animal groups. A series of mutations has resulted in the development of two flagella emerging from the two centrioles, the lack of a central complex in the axoneme, and the lack of a typical acrosome. In the Polychaeta, sperm structure is generally more related to function that to phylogenetics. During swimming the spermatozoon of Tomopteris rotates around its longitudinal axis.     

Immotile,aflagellate spermatozoa in Ptiliidae coleopterans

The first aflagellate and immotile Coleopteran spermatozoon is described in a group of species belonging to the family Ptiliidae. The mature spermatozoon is devoid of flagellum, centrioles and mitochondria, includes a three-layered acrosomal complex (extraacrosomal layer, acrosome and perforatorium) and a long nucleus made up of two regions of different densities. A compact submembranary capsule and a thick glycocalyx are also present. Motility organelles are absent during the whole spermiogenesis, which is not regressive. The new structures peculiar for this type of sperma are designed for protection. No other sperm models with these characteristics have been described so far.     

Trypsin-like enzymes during fertilization in the shrimp Rhynchocinetes typus

The spermatozoon of Rhynchocinetes typus is atypical because it is nonmotile and lacks head and tail. The body has a rigid spike. Neither an acrosome-like structure nor changes during gamete interaction which could be interpreted as an acrosome reaction have been observed in this species. Nevertheless, the spermatozoon exerts a lytic effect on the extracellular envelope of the egg, and in this way it penetrates through egg-coats, forming a channel. In this research we found that crude spermatozoa extracts analyzed by gelatin SDS-PAGE developed one band of protease activity that was completely inhibited by SBTI (soybean trypsin inhibitor) and pAB (p-aminobenzamidine). In sperm extracts an enzymatic activity was determined, using BAEE (N-benzoil-L-arginine ethyl ester), but not ATEE, as substrate. This activity was inhibited by SBTI and pAB. We observed that in vitro fertilization was inhibited by spermatozoon incubation with the trypsin inhibitors SBTI, PMSF (phenyl-methanesulphonyl fluoride), and pAB. Additionally, we observed that when whole isolated egg-coats were incubated with sperm extract and then analyzed by SDS-PAGE, one band of the egg-coats disappeared. These results have been interpreted as sperm trypsin-like enzyme participation in R. typus sperm passage through the egg-coats. Mol. Reprod. Dev. 46:581–586, 1997. © 1997 Wiley-Liss, Inc.     

暗褐蝈螽与优雅蝈螽精子超微结构的比较研究(直翅目,螽斯科)

研究了暗褐蝈螽Gampsocleis sedakovii(Fischer von Waldheim)和优雅蝈螽G.gratiosa Brunner von Wattenwyl精子的超微结构。这两种蝈螽精子头部的顶体复合体由顶体外层、顶体本体和顶体组成,顶体复合体位于细胞核前端,并包裹部分细胞核;颈部具5纵层细胞器;尾部鞭毛轴丝为典型的9+9+2型,线粒体衍生体部分晶状化。暗褐蝈螽精子较短,顶体复合体夹角较大,精子鞭毛横切面直径稍大;优雅蝈螽精子稍长,顶体复合体夹角较小,精子鞭毛横切面直径较小,两种精子超微结构差异不显著,其生殖隔离机制有待进一步研究。     

Mammalian sperm acrosome: formation, contents, and function

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.     

Structure of the Testes,Spermatozoa and Spermatozeugmata of Mimagoniates barberiRegan, 1907 (Teleostei: Characidae), an Internally Fertilizing,Oviparous Fish

Abstract The testis of Mimagoniates barberiis bipartite. Spermatogenic tissue is restricted to the anterior part. The posterior part of the testis is devoid of spermatogenic tissue and contains numerous efferent ducts filled with mature sperm. Cells in germinal cysts develop synchronously, sperm nuclei and flagella become oriented parallel in the late stages of spermiogenesis. In the caudal portion of the aspermatogenic part all sperms are arranged into unencapsulated sperm bundles — spermatozeugmata. Two types of spermatozeugmata are found both in the caudal portion of the testis and in milt. In the larger, spindle–shaped type, sperm flagella form the spindle tips. In the smaller ones, which have approximately a length of spermatozoon, the sperm are parallel and approximately in register. In both types sperm heads are arranged parallel. A mature spermatozoon is flail–shaped. The sperm head is highly elongated and situated alongside the flagellum, the tip of the head is directed backwards. Large mitochondria are situated on one side of the elongated nucleus only and form the tip of the head. Live spermatozoa move with the centriolar part ahead. Both testis and spermatozoon structure as well as formation of spermatozeugmata in M. barberiare highly derived features which perhaps evolved as adaptations to internal fertilization.     

The spermatozoon of Eurasian murine rodents: Its morphological diversity and evolution

The murine rodents are the most speciose subfamily of mammals. Here the morphology of the spermatozoon, as determined by scanning and transmission electron microscopy of representative species from four Eurasian clades, is described. Much interspecific variability in all components of the spermatozoon was found to occur, although most species have a bilaterally flattened sperm head with a single apical hook of variable length and orientation. Ultrastructural observations indicate that this apical hook invariably contains a nuclear projection as well as a large extension of the subacrosomal cytoskeleton, as a perforatorium rostrally, and a complex asymmetrical acrosomal extension. These spermatozoa also have relatively long tails that are attached to the lower concave surface of the sperm head. Uniquely, in species in the Apodemus clade, the apical hook is orientated caudally. In a few species a highly derived sperm head morphotype that does not contain an apical hook is present. These sperm heads vary in morphology from being globular in two species of Bandicota, to bilaterally flattened and paddle-shaped in Tokudaia and Micromys. In spermatozoa of the latter two genera the subacrosomal cytoskeleton, which is less extensive than in species with a hooked sperm head, forms an apical extension, but that is not the case in Bandicota. In all species where the sperm head lacks an apical hook the acrosome is more symmetrical. The sperm tail is much shorter in these species, with attachment to the head occurring on the ventral surface in Tokudaia and basal in Micromys and the two species of Bandicota. As the sperm head morphotype with a complex apical hook is present in all the major clades of murine rodents, it is likely to be a plesiomorphic character within each of these clades, with the nonhooked sperm heads, which vary greatly in structure between species of the different lineages, probably being independently derived. The ultrastructural organization of the sperm head of Bandicota, but not those of Micromys or Tokudaia, suggest divergence in some of the morphological events associated with sperm-egg interaction at the time of fertilization.     

The morphology of the squirrel spermatozoon: A highly complex male gamete with a massive acrosome

This study describes the morphology of the spermatozoon from the cauda epididymidis of representative members of two squirrel subfamilies, the Sciurinae and Callosciurinae, as determined by fluorescent, scanning, and transmission electron microscopy. All species examined possess a massive apical segment of the sperm acrosome. It varied markedly in the extent of its caudal flexion but was always much larger, and more complex, than that of the spermatozoon of most other rodents so far documented, although somewhat similar to that of some hystricomorph species. Because this sperm form appears to be present within at least two of the three major living clades of Rodentia, it is possible that it is the ancestral condition within this mammalian order. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.