Penetration of spermatozoon into the ovum and transformation of the sperm nucleus into the male pronucleus in the domestic fowl,Gallus gallus

Summary The apex of the sperm head which has undergone the acrosome reaction comes in contact with the plasma membrane of the ovum. After the entire surface of the inner acrosomal membrane has come into close contact with the plasma membrane of the ovum, the two membranes fuse to form a continuous membrane. All parts of the spermatozoon that are devoid of plasma membrane penetrate into the ooplasm. As the head of the spermatozoon moves deeper into the ooplasm, the chromatin begins to disperse, and the head of spermatozoon is transformed into a large spherical nucleus with low electron density. At a later stage of the transformation, many small vesicles appear around the nucleus and subsequently fuse to form two continuous membranes. These membranes represent the male pronuclear envelope. The condensation of the chromatin occurs in places in the nucleus, so that the male pronucleus is formed. During the course of the formation of the male pronucleus, the subacrosomal rod and tail become detached from the head and disintegrate.The authors are greatly indebted to assoc. Prof. Dr. Osamu Koga for his valuable advices. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

The passage of spermatozoa through the vitelline membrane in the domestic fowl,Gallus gallus

Summary The developing outer layer of the vitelline membrane of the ovum in the posterior part of the infundibulum of the domestic fowl contains many spermatozoa in nearly parallel orientation with its inner layer. When the acrosomal region of a spermatozoon approaches or contacts the inner layer, promptly undergoes the acrosome reaction. The outer acrosomal membrane and overlying plasma membrane fuse together and the apical region of the acrosome opens, so that the acrosomal contents are released. Meanwhile the spermatozoon remains a time in contact with the surface of the inner layer, and the network of the inner layer just under the tip of the sperm head begins to be dissolved. This dissolution extends downward forming a tunnel, approximately 9 m in diameter. The spermatozoon then passes through the inner layer obliquely via the central region of the tunnel and arrives at the perivitelline space.The authors are greatly indebted to assoc. prof. Dr. Osamu Koga for his valuable advices. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Fate of spermatozoa that do not participate in fertilization in the domestic fowl

Summary The fate of spermatozoa that do not participate in fertilization was investigated by electron microscopy. After artificial insemination, we observed several spermatozoa between the fibers of the outer layer of the vitelline membrane of the ovum. One or more spermatozoa were also found in a phagocytic vesicle of macrophages located in the intercellular space of the mucosal epithelium of the infundibulum or in the outer layer of the vitelline membrane.From these observations, we assume that the superfluous spermatozoa in the lumen of the anterior part of the oviduct might be removed by inclusion into the outer layer of the vitelline membrane and by phagocytosis by macrophages.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his invaluable advice. The authors also wish to thank Mr. Takayuki Mri for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Specialised plasma membranes in the preovulatory follicle op the fowl

Summary In the hen's follicle processes from the zona granulosa cells extend into the zona radiata. The terminal plasma membrane of these processes has a total thickness of around 160 Å and consists of five layers. Small granules spaced at regular intervals are attached to the cytoplasmic aspect of the inner layer by short stalks. In the 2 mm and 7 mm follicles the plasma membrane of the ovum facing the specialised terminal membranes has a striated appearance and shows a regular arrangement of granules attached by stalks to both its inner and outer aspects. The terminal and striated membranes are separated by an interval although there are areas of closer contact. In the 15 mm and 35 mm pre-ovulatory follicles the plasma membrane round the whole surface of the ovum is now typical striated membrane with bristles and attached granules. No explanation can be given at present of the function of the terminal membranes of the granulosa processes. They may indicate some change in the permeability permitting the intercellular diffusion of particles. It is suggested that the striated ovum plasma membrane is associated with the adsorption and transport of substances into the ovum for yolk synthesis.     

Behavior of the spermatozoon during sperm-blastomere fusion and its significance for fertilization (Saccoglossus kowalevskii: hemichordata)

Summary Association of spermatozoa with blastomeres removed from the egg envelope was studied in order to investigate sperm membrane behavior under circumstances unlike those attending fertilization of normal envelope-enclosed eggs. Under the altered circumstances all parts of the surface of the sperm cell were afforded the opportunity of meeting blastomere plasma membrane. Nevertheless, in each case the spermatozoon first became activated, an acrosomal tubule formed, and it was this organelle alone which fused and established continuity with the blastomere plasma membrane. This behavior of the spermatozoon with respect to the denuded blastomere parallels the behavior of the sperm cell with respect to the envelope-enclosed egg at fertilization. Since the sperm cell behaves so similarly in two situations which are so different with respect to what the spermatozoon encounters, this behavior is considered to reflect the inherent nature of the spermatozoon, and analysis of this behavior in relation to a blastomere is considered to be valid also in relation to an egg during fertilization. It is concluded that membrane fusion, involving the acrosomal tubule of an activated spermatozoon, is the only means by which gametic union is established and that these are obligatory rather than fortuitous features of sperm-egg association in Saccoglossus. It is suggested that these features are probably obligatory also in other species which exhibit this pattern of sperm-egg association.This investigation was supported by Research Grant HD-00007 from the National Institutes of Health, United States Public Health Service.     

The ultrastructure of the spermatozoon and spermiogenesis in Cryptocotyle lingua (digenea: Heterophyidae)

During spermiogenesis two lateral flagellar processes and a median process arising from the apex of the zone of differentiation, fuse to form the elongated unipartite spermatozoon. Two axial units, therefore, with the ‘9+1’ pattern of microtubules are incorporated into the spermatozoon. The nucleus, in the head region, contains dense lamellar subunits arranged in a spiral in the long axis. These are formed by condensation of the chromatin during spermiogenesis. The single elongated mitochondrion, resulting from early fusion of small mitochondria, extends through the head and middle regions of the spermatozoon. Peripheral microtubules, present originally in the zone of differentiation, are arranged in straight dorsal and ventral rows, along the length. β glycogen particles accumulate in the spermatozoa after they have separated from the residual cytoplasm. Spermatozoa are present in the testes on the second day after infection of the bird host and accumulate in the vesicula seminalis from the third day onwards.     

Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster

Spermiogenesis and posttesticular sperm maturation in the epididymis are distinct developmental processes that result in a polarized spermatozoon possessing a plasma membrane partitioned into segment-specific domains of distinct composition and function. The mechanisms that specify the distribution of intracellular organelles and target proteins to restricted membrane domains are not well understood. In this study we examined the expression pattern and distribution of protein farnesyltransferase (FTase) in hamster spermatids and epididymal spermatozoa to determine if protein lipidation may represent a potential mechanism to regulate protein association with specific organelles or the plasma membrane. Round spermatids exhibited only weak immunostaining with antibody against the β-subunit of FTase, whereas elongating spermatids exhibited a high level of FTase expression that was segregated to the cytoplasmic lobe surrounding the anterior flagellum. Although FTase was released with the residual body, mature spermatids retained FTase within the midpiece and cytoplasmic droplet. In epididymal spermatozoa, FTase remained associated with the cytoplasmic droplet during its migration to the midpiece-principal piece junction; following release of the cytoplasmic droplet, no immunodetectable FTase was noted in the midpiece segment. Immunoblotting demonstrated the presence of both the α and β subunits of FTase in sperm lysates. The temporal expression pattern and restricted distribution of FTase in spermatids and epididymal spermatozoa suggest a potential role in regulating protein association with specific organelles and/or membrane domains of the mature spermatozoon. Mol. Reprod. Dev. 48:71–76, 1997. © 1997 Wiley-Liss, Inc.     

The structure and development of the spermatozoon of Heterakis gallinamm (Nematoda)

The structure and development of the spermatozoon of the nematode Heterakis gallinamm has been described. The spermatozoon is amoeboid. The centrioles have the unusual structure of nine single fibres. The nucleus has no limiting membrane and is surrounded by mitochondria and organelles, here called alpha bodies. These alpha bodies appear to arise from Golgi complexes in association with granular endoplasmic reticulum in the spermatocytes and have a fibrillar component which is released into the peripheral cytoplasm of the spermatid when the spermatozoon is formed. There is no refringent cone as in other ascaroid spermatozoa.     

Spermiogenesis and spermatozoon ultrastructure of Diplodiscus subclavatus (Pallas, 1760) (Paramphistomoidea, Diplodiscidae), an intestinal fluke of the pool frog Rana lessonae (Amphibia, Anura)

Spermiogenesis in Diplodiscus subclavatus begins with the formation of the zone of differentiation presenting two centrioles associated with striated roots and an intercentriolar body. The latter presents seven electron-dense layers with a fine central plate and three plates on both sides. The external pair of these electron-dense layers is formed by a granular row. Each centriole develops into a free flagellum, both of them growing orthogonally in relation to the median cytoplasmic process. After the flagellar rotation and before the proximodistal fusion of both flagella with the median cytoplasmic process four attachment zones were already observed in several cross-sections indicating the area of fusion. Spinelike bodies are also observed in the differentiation zone before the fusion of flagella. Finally, the constriction of the ring of arched membranes gives rise to the young spermatozoon that detaches from the residual cytoplasm. The mature spermatozoon of D. subclavatus shows all the classical characters observed in Digenea spermatozoa such as two axonemes of different length of the 9+"1" trepaxonematan pattern, nucleus, mitochondrion, two bundles of parallel cortical microtubules and granules of glycogen. However, some peculiarities such as a well-developed lateral expansion associated with external ornamentation of the plasma membrane and spinelike bodies combined with their area of appearance distinguish the ultrastructural organization of the sperm cells of D. subclavatus from those of other digeneans.     

Ultrastructural study of the male gamete of Pleurogonius truncatus Prudhoe, 1944 (Platyhelminthes, Digenea, Pronocephalidae) parasite of Eretmochelys imbricata (Linnaeus, 1766)

In Pronocephaloidea, the spermatozoa of only two species have been studied today. Because of this, we present in this work data concerning to a third specie, Pleurogonius truncatus Prudhoe, 1944. The mature spermatozoon of P. truncatus possesses two axonemes with the 9+"1" pattern typical of Trepaxonemata, mitochondrion, nucleus, parallel cortical microtubules, spinelike bodies, cytoplasmic expansion and an external ornamentation of the plasma membrane. A particularity of the spermatozoon of P. truncatus is in the ultrastructure of the anterior spermatozoon extremity with only cortical microtubules and ornamentation of the plasma membrane. This type of anterior extremity has never been described until today in Pronocephaloidea. On the other hand, the ultrastructure of the posterior extremity of the spermatozoon confirms that already described in Pronocephalidae.     

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : II. Incorporation with the Egg

This, the last of a series of three papers, deals with the final events which lead to the incorporation of the spermatozoon with the egg. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed at various times up to five minutes after insemination. The first direct contact of sperm head with egg proper is by means of the acrosomal tubules. These deeply indent the egg plasma membrane, and consequently at the apex of the sperm head the surfaces of the two gametes become interdigitated. But at first the sperm and egg plasma membranes maintain their identity and a cross-section through the region of interdigitation shows these two membranes as a number of sets of two closely concentric rings. The egg plasma membrane rises to form a cone which starts to project into the hole which the spermatozoon earlier had produced in the vitelline membrane by means of lysis. But the cone does not literally engulf the sperm head. Instead, where they come into contact, sperm plasma membrane and egg plasma membrane fuse to form one continuous membranous sheet. At this juncture the two gametes have in effect become mutually incorporated and have formed a single fertilized cell with one continuous bounding membrane. At this time, at least, the membrane is a mosaic of mostly egg plasma membrane and a patch of sperm plasma membrane. The evidence indicates that the fusion of the two membranes results from vesiculation of the sperm and egg plasma membranes in the region at which they come to adjoin. Once this fusion of membranes is accomplished, the egg cytoplasm intrudes between the now common membrane and the internal sperm structures, such as the nucleus, and even extends into the flagellum; finally these sperm structures come to lie in the main body of the egg. The vesiculation suggested above appears possibly to resemble pinocytosis, with the difference that the vesicles are formed from the plasma membranes of two cells. At no time, however, is the sperm as a whole engulfed and brought to the interior of the egg within a large vesicle.     

扩张莫尼茨绦虫(圆叶目:裸头科)精细胞分化、精子形成及精子形态

本项研究应用光学显微镜、扫描和透射电子显微镜,观察了扩张莫尼茨绦虫的精细胞分化、精子形成全过程及精子的精细结构。扩张莫尼茨绦虫的精细胞分化过程为：1)初级精原细胞主要发生于幼节的睾丸滤泡中;2)次级精原细胞发生不完全分裂形成16个细胞一簇的初级精母细胞群,以共同的中央细胞质相连;3)初级精母细胞的特征为细胞核中出现联会复合体结构;4)紧接着的第二次成熟分裂,产生64个由中央细胞质相连的细胞核较小的精细胞。精子形成始于精细胞中分化区的形成,成熟精子缺乏线粒体,具有质膜和冠状体、1—4个领域排布的质膜下皮层微管,细胞质中存在电子致密的颗粒状物质,具一个不规则形态的细胞核,具有“9 1”类型的轴丝构造,缺乏轴丝周围鞘。从精子的纵切面上可将精子区分为5个区段(Ⅰ一Ⅴ区)。在精子形成过程中,中心粒基部出现螺旋形小根结构在寄生虫中为首次报导;成熟精子具有游离鞭毛,在绦虫中为首次发现[动物学报49(3)：370—379,2003]。     

FINE STRUCTURE OF THE SPERMATOZOON OF HYDROIDES HEXAGONUS (ANNELIDA), WITH SPECIAL REFERENCE TO THE ACROSOMAL REGION

This paper describes in some detail the structure of the acrosomal region of the spermatozoon of Hydroides as a basis for subsequent papers which will deal with the structural changes which this region undergoes during fertilization. The material was osmium-fixed and mild centrifugation was used to aggregate the spermatozoa from collection to final embedding. The studies concern also the acrosomal regions of frozen-thawed sperm prepared by a method which previously had yielded extracts with egg membrane lytic activity. The plasma membrane closely envelops four readily recognizable regions of the spermatozoon: acrosomal, nuclear, mitochondrial, and flagellar. The acrosome consists of an acrosomal vesicle which is bounded by a single continuous membrane, and its periphery is distinguishable into inner, intermediate, and outer zones. The inner and intermediate zones form a pocket into which the narrowed apex of the nucleus intrudes. Granular material adjoins the inner surface of the acrosomal membrane, and this material is characteristically different for each zone. Centrally, the acrosomal vesicle is spanned by an acrosomal granule: its base is at the inner zone and its apex at the outer zone. The apex of the acrosomal granule flares out and touches the acrosomal membrane over a limited area. In this limited area the adjoining granular material of the outer zone is lacking. The acrosomal membrane of the inner zone is invaginated into about fifteen short tubules. The acrosomal membrane of the outer zone is closely surrounded by the plasma membrane. At the apex of the acrosomal region a small apical vesicle is sandwiched between the plasma membrane and the acrosomal membrane. Numerous frozen-thawed specimens and occasional specimens not so treated show acrosomal regions at the apex of which there is a well defined opening or orifice. Around the rim or lip of this orifice plasma and acrosomal membranes may even be fused into a continuum. The evidence indicates that the apical vesicle and the parts of the plasma and acrosomal membranes which surround it constitute a lid, and the rim of this lid constitutes a natural "fracture line" or rim of dehiscence. Should fracture occur, the lid would be removed and the acrosomal vesicle would be open to the exterior.     

Antigen changes of monoclonal antibody MSH27 in process of post-testicular maturation (in mice)

An anti-mouse spermatozoon monoclonal antibody, MSH27, as well as its purified antigen, can block sperm-egg membrane fusion. As a candidate protein for sperm-egg membrane fusion, the sperm antigen was investigated in the process of post-testicular maturation (PTM). The molecule was produced in testes and located on the plasma membrane of the postacrosomal area of the spermatozoon. However, the epitope recognized by the MSH27 (MSH27Ep) was not exposed until the occurrence of the acrosome reaction. In the process of fertilization, spermatozoa must complete the acrosome reaction before penetrating across the zona pellucidas (ZPs) to approach the plasma membrane of eggs. The effects of the acrosome reaction and penetration of the ZP on the exposure of the MSH27Ep were also studied. It was shown that the percentage of the spermatozoa with the MSH27Ep exposed increased followed with their mature status in PTM. In fact, it bad a linear correlativity with the rate of the acrosome reaction. After spermatozoa had     

Ultrastructural changes in the median eminence of the rat following deafferentation of the basal hypothalamus

Summary This report concerns a light and electron microscopic investigation of the median eminence and dorsal infundibular stem of the rat following surgical isolation (deafferentation). Using a modification of the Halász technique, the basal hypothalamus, including the arcuate nucleus and median eminence were surgically isolated from surrounding structures. Special attention was directed to the contact (external) zone of the median eminence and rostral infundibulum where tuberohypophyseal axons as well as ependymal cell processes abut upon the abluminal basement membrane of the portal perivascular space. The results of this study to date suggest that 9, 20, and 40 days following surgical isolation, there is a distinct increase in the population of tuberohypophyseal dense core vesicles. It is suggested that deafferentation abolishes inhibitory and excitatory input that serves to modify the cellular dynamics of tuberohypophyseal neurosecretory elements. Comments are also made on the presence of cistern-like structures in the lateral median eminence; the presence of vesicle-like inclusions in terminal ependymal processes is discussed in relationship to the role that ependyma may play in linking the third ventricle with the adenohypophysis.This research was supported by USPHS Grants NB 08171 and AM 10002. The authors are indebted to the excellent technical assistance provided by G. Krobisch Dudley. Further, the authors wish to express their thanks to Dr. Adolph Weindl for his valuable advice and criticism, and to Matilde Holzwarth for her helpful assistance.     

Endoproteolytic cleavage in the extracellular domain of the integral plasma membrane protein CE9 precedes its redistribution from the posterior to the anterior tail of the rat spermatozoon during epididymal maturation [published erratum appears in J Cell Biol 1991 Nov;115(3):following 880]

Originally identified as a basolateral domain-specific integral plasma membrane protein of the rat hepatocyte, CE9 mRNA and protein were also detected at high levels in the testis of the rat by Northern and Western blotting and immunoprecipitation. CE9 proved to be a domain-specific integral plasma membrane protein of the rat spermatozoon: on testicular spermatozoa, it was concentrated within the posterior tail domain of the plasma membrane, whereas on vas deferens spermatozoa, CE9 was concentrated within the anterior tail domain. This change in the localization of CE9 was observed to take place in a offgressive fashion during the passage of the spermatozoa from the caput epididymidis to the cauda epididymidis and was preceded by the specific endoproteolytic cleavage of CE9 in the proximal portion of the caput epididymidis. Amino-terminal amino acid microsequencing of CE9 immunoaffinity purified from epididymis suggested that the cleavage occurred on the carboxy-terminal side of arginine-74 in the primary sequence of CE9, resulting in the loss of approximately 40% of the amino acids in the extra-cellular domain of this transmembrane glycoprotein.     

Disintegration of spermatozoa in the infundibular sperm-host glands of the fowl

Summary The disintegration of spermatozoa in the infundibular sperm-host glands of the fowl was investigated by electron microscopy. After the 15th day following artificial insemination, secretory granules in the epithelial cells of the sperm-host glands increase in number and size, and subsequently the contents of the granules are released into the glandular lumen, so that the electron density of the lumen increases. At this stage, spermatozoa stored in the glands begin to undergo degenerative changes starting from the head. The heads become distended and chromatin of the nucleus begins to disperse as small masses, simultaneously with the destruction of the acrosome. As the dispersion of chromatin progresses, mitochondria of the middle piece become distended and irregular in shape, and then disintegrate. At the last stage, most of the organelles have disappeared, but the fibrous sheath and axial-filament complex are still identified.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his invaluable advice. The authors also wish to thank Mr. Takayuki Möri for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

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.     

Substructure of a cytoskeletal complex associated with the hamster sperm acrosome

Whole mount and thin section preparations of intact and selectively disrupted hamster spermatozoa revealed an organized array of cytoplasmic filaments associated with specific regions of the acrosome. The filaments were localized along the ventral surface of the spermatozoon and extended from its tip, distally to the anterior margin of the equatorial segment. Individual filaments were 11-13 nm in diameter and they were aligned parallel to one another to form a two-dimensional sheet oriented in the long axis of the spermatozoon. The filament complex adhered preferentially to the cytoplasmic surface of the outer acrosomal membrane rather than the plasma membrane. Examination of disrupted spermatozoa revealed that the distribution of this cytoskeletal assembly correlated with the distribution of a specific acrosomal matrix component. The possible role of this complex in the acrosome reaction or in the organization of acrosomal matrix domains is discussed.     

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.