Twin-beam laser velocimeter for the investigation of spermatozoon motility

Previous laser light-scattering studies of spermatozoon motility have been hampered by the large, asymmetric shape of spermatozoa, which causes difficulties in the interpretation of intensity fluctuations in the light scattered from a single laser beam. This paper describes an experimental arrangement for measuring the distribution of transit times for swimming spermatozoa using two slightly separated, focused laser beams. The theory of operation of the instrument is developed to enable the analysis of the experimentally obtained cross-correlation functions. The effects of the pronounced spermatozoon asymmetry and associated intensity modulation in the scattered light are also investigated and shown to be negligible for the twin beam experimental arrangement, provided that the swimming speed distribution has a coefficient of variation (sigma/upsilon greater than 0.1. Results obtained using this apparatus are presented for the velocity distribution of spermatozoa from a variety of bulls.     

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.     

Activity of exoglycosidases in ejaculated spermatozoa of boar and bull

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of alpha-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.     

Ultrastructure of spermatozoa of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae), in the seminal receptacle

The seminal receptacle of Paragonimus ohirai contains not only mature spermatozoa, but also atypical and degenerate ones, suggesting that abnormal spermatozoa are retained in this organ. The spermatozoon is of a parallel biflagellar type with cortical microtubules, consisting of the anterior region, first mitochondrial region, intermediate (amitochondrial) region, second mitochondrial region, posterior nuclear region (PNR) and tail region (TR). The first third of the spermatozoon exhibits typical undulatory movement, while the middle part shows vibratory movement. At the area between head and midsections (H-M area) the peripheral doublets of axonemes are interrupted, and the external ornamentation is distributed widely around this portion. Throughout the immotile PNR and TR, the axonemes lack the dynein arms of their peripheral doublets. H-M, PNR, and TR ultrastructural characteristics are specific in P. ohirai spermatozoon and seem to be closely related to its pattern of movement.     

Structural features of the spermatozoon of a passeridan bird,the Carib grackle,Quiscalus lugubris

The spermatozoon of the Carib grackle, Quiscalus lugubris, a member of the family Icteridae, is generally similar in organization to the passerine-type of spermatozoon, in being highly elongated and displaying a helical structure of the acrosome, nucleus and principal piece of the tail. There are subtle variations in acrosomal structural features between this organelle in the grackle and that in some of the very few passerine species of birds in which the spermatozoon has been studied. The proximal centriole is present, and, thus, the Carib grackle is the third passeridan bird in which this organelle, hitherto regarded as absent in passerine birds, has been described in the spermatozoon. The spermatozoon of this bird also possesses a granular helix, which feature has been found variably even in the scanty available reports on passerine spermatozoa. It is advocated that the spermatozoon be studied in many more species of this large clade of birds. This report provides a basis for the study of spermiogenesis in the Carib grackle, with the aim of exposing, inter alia, a number of developmental features and processes of certain organelles that have received attention, recently, in the spermatozoa of passerine birds.     

Teratospermia and migration of spermatozoa in vitro and in vivo]

The comparative study of morphological anomalies of spermatozoon before and after penetration shows that in vivo or in vitro migration causes a selection of the spermatozoon. This selection is not complete. Anomalies of the midpiece or of the flagella inhibe the spermatozoon progression but morphological anomalies of the head are not a handicap for the migration. this observation seems to indicate that spermatozoa with chromosomic failures can paticipate to the fertilization.     

Identification of an ATPase activity associated with the high molecular weight proteins of the human spermatozoon axonemes

The high salt extract obtained from demembranated human spermatozoa contains high molecular weight proteins. These proteins are associated with an ATPase activity inhibited by sodium orthovanadate. In association with lower molecular weight proteins, they constitute a 20 S particle and are probably localized in the dynein arms (and in the radial spokes) of the human spermatozoon axonemes. Evidence is shown for a biochemical analogy between the dynein ATPases extracted from the invertebrate axonemes and the human dynein-like ATPase described in this study.     

Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa

Septins are polymerizing GTP binding proteins required for cortical organization during cytokinesis and other cellular processes. A mammalian septin gene Sept4 is expressed mainly in postmitotic neural cells and postmeiotic male germ cells. In mouse and human spermatozoa, SEPT4 and other septins are found in the annulus, a cortical ring which separates the middle and principal pieces. Sept4-/- male mice are sterile due to defective morphology and motility of the sperm flagellum. In Sept4 null spermatozoa, the annulus is replaced by a fragile segment lacking cortical material, beneath which kinesin-mediated intraflagellar transport stalls. The sterility is rescued by injection of sperm into oocytes, demonstrating that each Sept4 null spermatozoon carries an intact haploid genome. The annulus/septin ring is also disorganized in spermatozoa from a subset of human patients with asthenospermia syndrome. Thus, cortical organization based on circular assembly of the septin cytoskeleton is essential for the structural and mechanical integrity of mammalian spermatozoa.     

Ultrastructure of apyrene and eupyrene spermatozoa from the seminal vesicle of Euptoieta hegesia (Lepidoptera: Nymphalidae)

The ultrastructure of the seminal vesicle's spermatozoa of the butterfly Euptoieta hegesia was analyzed. The apyrene spermatozoa measure about 300 microm in length and swim freely in a secretion. The anterior end consists in a cap with a cylindrical extension and a globular structure. The flagellum has a 9+9+2 axoneme, two mitochondrial derivatives with paracrystalline matrices and an external coat formed by concentric layers. The eupyrene spermatozoa measure about 550 microm in length and are grouped into bundles. The anterior end consists in an amorphous globule. Posterior to this globule, a coat with a dense material covers the spermatozoon where an acrosome and a nucleus appear. The flagellum has a 9+9+2 axoneme and two mitochondrial derivatives. External to the coat and attached to the dense material, there is a reticular appendage, which has a paracrystalline core and extends to the distal tip of the spermatozoon.     

Spermatozoa morphology and ultrastructure in Nile perch,Lates niloticus (Linnaeus, 1758)

Lates niloticus is a valuable commercial fish species with good potential for aquaculture. However, there is limited information on the type and structure of the Nile perch spermatozoon, which could potentially aid in culture of this species. Here, we describe the spermatozoon ultrastructure in L. niloticus using transmission and scanning electron microscopy. The spermatozoon had a round head-shape, medio-laterally flat, no acrosome, a short midpiece located laterally to the nucleus, uniflagella with one wing. The head of the spermatozoon contained the nucleus, centriolar system, proximal part of the flagellum, and cytoplasmic channel. Centrioles were arranged at an angle of 90° to each other, forming a T-shape, parallel to the nucleus. The midpiece was cylindrical, loaded with cytoplasm, five to seven spherical mitochondria; and the flagellum’s plasma membrane extended to form one lateral wing. The spermatozoa were classified as type II spermatozoa. L. niloticus spermatozoon differed from that of its Australian congener L. calcarifer, especially in the centriole arrangement and nuclear shape, length of the midpiece and the number of mitochondria and lateral wings.     

Ultrastructure of the Spermatozoon of an Opisthobranch, Tornatina sp. (Mollusca, Gastropoda, Retusidae)

The spermatozoon of Tornatina sp. has been studied with phase-contrast light microscopy and transmission electron microscopy. The head of the spermatozoon consists of an elongate acrosome which caps the apex of an unusually complex, helical nucleus. This elaborate nuclear morphology has not been previously reported, but possibly is found in other opisthobranch gastropod spermatozoa. An axoneme is inserted deeply into the base of the nucleus whilst posterior from the nucleus, the axoneme is ensheathed successively by the mitochondrial derivative (midpiece) and 'glycogen' granules (glycogen piece). The midpiece exhibits fine structure similar to that observed in other euthyneuran spermatozoa (paracrystalline and matrix materials) and possesses a single helical compartment filled with what are probably glycogen granules. A dense ring structure occurs at the junction of the midpiece and glycogen piece. The spermatozoon of Tornatina and other gastropods (prosobranch and euthyneuran) are compared.     

Freeze-dried sperm fertilization leads to full-term development in rabbits

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be "dead" in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.     

Ultrastructural study of the spermatozoon of Heterolebes maculosus (Digenea,Opistholebetidae), a parasite of the porcupinefish Diodon hystrix (Pisces,Teleostei)

This paper describes the ultrastructure of the mature spermatozoon of Heterolebes maculosus. It is the first study of this kind concerning the Opistholebetidae (Platyhelminthes, Digenea). The ultrastructural elements observed in the spermatozoon are: two axonemes with 9+“1” pattern of Trepaxonemata and their attachment zones, two mitochondria, a nucleus, cortical microtubules, external ornamentation of the plasma membrane and spine-like bodies. The number and the disposition of cortical microtubules, the organisation of 11 cortical microtubules disposed in semi-circle around the first mitochondrion in the external ornamentation region and the organisation of the posterior part of the spermatozoon are discussed. Three principal types of posterior part of digenean spermatozoa are proposed. The similarity between the spermatozoon of the Opistholebetidae H. maculosus and Opecoelidae enables us to confirm that these two families are closely related.     

Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea,Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei,Carangidae)

The mature spermatozoon of Sclerodistomum italicum is filiform, tapered at both ends and shows the following features: 2 axonemes of the 9 + “1” pattern of the Trepaxonemata, mitochondrion, nucleus and parallel cortical microtubules. The specific features of the spermatozoon of S. italicum include the simultaneous presence of two types of extramembranous ornamentations, the presence of short cortical microtubules in the anterior part of the spermatozoon and the presence of only one bundle of cortical microtubules in the median part of the spermatozoon. Thus far these structures are known only in the Hemiuroidea. The presence of filamentous ornamentation in the anterior extremity of the spermatozoon has not previously been described in the Sclerodistomidae. Similar to spermatozoa of other hemiuroideans, S. italicum lack spine-like bodies described in spermatozoa of many digenean taxa. The posterior extremity of the spermatozoon exhibits the same ultrastructural characteristics typical of the Hemiuroidea.     

Surface of the rooster spermatozoon changes in passing through the Wolffian duct

Fluid secreted by the rooster Wolffian duct contains several proteins separable on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) gels. Antibodies against these fluid components were obtained by immunizing rabbits, and the IgG fraction was then purified. As judged by indirect immunofluorescence, purified IgG against rooster duct fluid did not bind to any testicular spermatozoa. However, it bound distinctly to the whole surface of spermatozoa from the initial (epididymal) region and more intensely to all spermatozoa from the mid- and terminal regions of the Wolffian duct of the rooster, though not at all to mature duck or pigeon spermatozoa. Thus, in the rooster, as in therian mammals, the surface of the spermatozoon clearly acquires specific components secreted by the Wolffian duct. It should not be assumed that such surface change in rooster spermatozoa is entirely comparable, in a functional sense, to that undergone by mammalian spermatozoa, in which this seems directly related to fertilizing ability. Unlike those of mammals, rooster spermatozoa do not seem to require capacitation, and some spermatozoa in the testis already are competent to fertilize. Components acquired in the Wolffian duct by the rooster spermatozoon may bear on other aspects, perhaps sperm transport and/or survival in the female.     

大鳞副泥鳅精子结构研究

用光学显微镜和透射电子显微镜对大鳞副泥鳅精子的结构进行研究。结果表明,大鳞副泥鳅的精子主要分为头部、中段和尾部;头部无顶体,在光学显微镜下近圆形,在透射电子显微镜下纵切亦近圆形,主要由细胞核组成,核内染色质致密,核内有核空泡,核外可见清晰核膜,核外可见细胞质,细胞质很少且紧贴细胞核,细胞质外是质膜,质膜在细胞质外呈波浪状;头部后端有一较浅的植入窝,约占核的1/4,植入窝的长轴几乎与细胞核的长轴平行,植入窝内有中心粒复合体;精子的中片与头部无明显分割,位于头部的后方,由中心粒复合体和袖套组成,袖套两边不对称,中心粒复合体由近端中心粒和远端中心粒组成,近端中心粒与远端中心粒之间呈一钝角;精子尾部无侧鳍,可分为主段和末段,尾部主段具有典型的＂9＋2＂的轴丝结构。精子头长为（1.79±0.28）μm,中片长为（1.86±0.42）μm,尾长为（28.06±2.78）μm,全长为（31.65±2.82）μm。