Phagocytosis of spermatozoa by the ovum of the domestic fowl,Gallus gallus at the time of fertilization

Summary Spermatozoa with intact acrosomes, as well as those coming into contact with the ovum at a smaller angle, and morphologically abnormal spermatozoa reach the plasma membrane of the ovum via an extensively dissolved zone of the inner layer of the vitelline membrane. This zone is assumed to be formed by overlapping of two or more tunnels formed by spermatozoa that had previously come into contact with the ovum.When a spermatozoon comes into contact with the plasma membrane of the ovum, many cytoplasmic processes extend outwards and cover it. Thereafter, the plasma membranes of the processes fuse, thereby phagocytizing the spermatozoon. It is assumed that the phagocytized spermatozoa cannot undergo transformation into male pronuclei and that they degenerate soon after phagocytosis.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his valuable advice. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)Previous name: Fukashi Okamura     

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)     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

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.     

THE EFFECTS OF NICOTINE ON FERTILIZATION IN THE SEA URCHIN, ARBACIA PUNCTULATA

The number of sperm incorporated into eggs made polyspermic with varying concentrations of nicotine (0.025–0.25%, v/v) appears to be directly related to the concentrations employed. The cortical response is morphologically equivalent to that observed in control preparations. Shortly after their incorporation all of the spermatozoa undergo structural events normally associated with the development of the male pronucleus in monospermic eggs. During the reorganization of the spermatozoa, sperm asters are formed. The number of male pronuclei that initially migrate to and encounter the female pronucleus is usually one to three. When pronuclei come into proximity to one another the surface of the female pronucleus proximal to the advancing male pronuclei flattens and becomes highly convoluted. Subsequently, the pronuclei contact each other and the outer and inner membranes of the pronuclear envelopes fuse, thereby producing the zygote nucleus. The male pronuclei remaining in the zygote after this initial series of pronuclear fusions continue to differentiate, i.e. they enlarge, form nucleolus-like bodies, and undergo further chromatin dispersion. In approximately 90% of the zygotes, all of the remaining male pronuclei progressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 min postinsemination. Mitosis and cleavage of the polyspermic zygote occurs later than in monospermic eggs.     

THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : II. Zygote Formation by Gamete Membrane Fusion

An earlier paper showed that in Saccoglossus the acrosomal tubule makes contact with the egg plasma membrane. The present paper includes evidence that the sperm and egg plasma membranes fuse to establish the single continuous zygote membrane which, consequently, is a mosaic. Contrary to the general hypothesis of Tyler, pinocytosis or phagocytosis plays no role in zygote formation. Contact between the gametes is actually between two newly exposed surfaces: in the spermatozoon, the surface was formerly the interior of the acrosomal vesicle; in the egg, it was membrane previously covered by the egg envelopes. The concept that all the events of fertilization are mediated by a fertilizin-antifertilizin reaction seems an oversimplification of events actually observed: rather, the evidence indicates that a series of specific biochemical interactions probably would be involved. Gamete membrane fusion permits sperm periacrosomal material to meet the egg cytoplasm; if an activating substance exists in the spermatozoon it probably is periacrosomal rather than acrosomal in origin. The contents of the acrosome are expended in the process of delivering the sperm plasma membrane to the egg plasma membrane. After these membranes coalesce, the sperm nucleus and other internal sperm structures move into the egg cytoplasm.     

Morphological observations on sperm–egg interactions during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

The aim of the present study was to determine the morphological changes that take place in the male and female gametes during in vivo fertilization in the Australian marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata. Plastic sections were cut of sperm and eggs recovered from the oviducts of recently mated individuals, and light microscopy of thick, and transmission EM of thin, sections was carried out. It was found that, before penetration of the zona, the spermatozoon came to lie along the outer surface with its rostral tip forming a depression in the zona substance. During penetration, zona material was packed tightly around the spermatozoon, and no large hole was formed. A spermatozoon within the perivitelline space had made contact with the oolemma by way of its apical tip. In a spermatozoon partly incorporated into the ooplasm, fusion appeared to have taken place between its plasma membrane and that of the oolemma. Mucoid coat material became deposited outside the zona at this time; its existence and/or the release of cortical granule content probably prevented polyspermy. Once inside the egg cytoplasm, the sperm head sometimes travelled a considerable distance before chromatin decondensation occurred. In addition, it appeared to rotate somewhat on its axis at this time. Finally, some membranous structures were found around two condensed sperm heads in the ooplasm, which may have been part of the pronuclear envelope. Thus this study on in vivo fertilization in the dunnart documents, for the first time, some aspects of fertilization in an Australian marsupial as seen with the transmission electron microscope; it indicates a few differences from those previously found for the American opossum.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

The Drosophila maternal gene sésame is required for sperm chromatin remodeling at fertilization

The spermatozoon features an extremely condensed and inactive nucleus. The unique sperm chromatin organization is acquired during the late stages of spermatid differentiation by the replacement of somatic histones with sperm-specific chromosomal proteins. At fertilization, the inactive sperm nucleus must be rapidly transformed into a DNA replication competent male pronucleus before the formation of the zygote. The sequential events of this crucial process are well conserved among animals and are controlled by molecules present in the egg. We have previously identified a Drosophila maternal effect mutation called sésame, which specifically arrests male pronucleus formation at a late stage of chromatin decondensation. In this study, we show that sésame affects maternal histone incorporation in the male pronucleus, a situation that is expected to prevent nucleosomal organization of the paternal chromatin. As an apparent consequence, the male pronucleus is arrested before the first S-phase and does not condense mitotic chromosomes. However, centromeric heterochromatin is present on paternal centromeres, which occasionally interact with microtubules. The abnormal chromatin organization of the male pronucleus does not prevent the formation of a male pronuclear envelope, which breaks down and reassembles in synchrony with maternally derived nuclei present in the same cytoplasm.     

Experimental hybridization among Oryzias species. II. Karyogamy and abnormality of chromosome separation in the cleavage of interspecific hybrids between Oryzias latipes and O. javanicus

In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.     

Analyse de la structure chromatinienne du sperme par la cytométrie en flux à l’acridine orange. — intérêt clinique

During spermatozogenesis, sperm nucleosomal DNA type, linked to histones, is transformed into a special form bound to small basic proteins, the protamines. This process allows sperm nucleus to condense and mature. This maturation process is achieved in the epididymis. When a spermatozoon enters an oocyte, protamines are released and replaced by histones, enabling the sperm nucleus to expand into a male pronucleus. Flow cytometry using acridine orange staining is an objective and quantitative technique well-adapted for the investigation of the chromatin structure at the level of each spermatozoon, as well as the whole ejaculate. This technique allows tracing the young sperm cell nuclei from the diploid stage, through tetraploid until the final haploid stage in mature spermatozoa. It allows also following the sperm maturation process during the epididymal transit. It detects various sperm chromatin condensation defects, hypocondensation, hypercondensation or other aberrations, as well as decondensation defects by using an in vitro assay. These defects may impair sperm fertilizing ability, even after sperm microinjection into the oocyte. Better understanding of sperm chromatin integrity and stability prerequisites might help us improving the quality of various technologies used in assisted medical procreation.     

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

Summary Ultrastructural studies on sperm-egg interaction at the time of fertilization inLaternula limicola were performed. The temporary-acrosome did not change morphologically while the sperm passed through the egg investments. At the onset of sperm entrance into the egg, however, the temporary-acrosome and mitochondria were eliminated from the sperm. Afterwards the sperm was engulfed by the egg surface without membrane fusion of the gametes. After entry the sperm nucleus was surrounded by four membranes: the plasma membranes of the egg and of the sperm, and the membranes of the sperm nuclear envelope. As the sperm nucleus differentiated into the male pronucleus, the plasma membranes of both the sperm and egg were initially vesiculated, then dispersed into the egg cytoplasm. Finally, the sperm nuclear envelope changed into the male pronuclear membrane accompanying sperm chromatin dispersion.     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

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.     

大鲵精子的超微结构研究

本文运用透射电镜和扫描电镜研究了大鲵(Andrias davidianus)精子的超微结构,大鲵精子由头部(head),中片(midpiece)和尾部(tail)三部分组成。头部有棒状细胞核,核内染色质高度浓缩,细胞核前方呈细丝状,但非顶体结构。头部后端凹陷,称为植入窝(implantation fossa),植入窝内有线粒体和中心粒等细胞器结构,此区域为精子的中片。精子尾部细长,主要由轴丝和附属纤维(accessory fiber)组成,轴丝的外面具有波动膜。     

The sperm-egg fusion in the nematode Parascaris equorum

Summary

The process of fertilization and the sperm storage in the female apparatus in Parascaris equorum is described in this paper. The sperm approaches the egg by means of pseudopodia containing bundles of microfilaments. The sperm and egg membranes fuse and the sperm penetrates progressively into the ovum. The egg and sperm plasma membranes and glycocalyces disappear at the point of fusion. At the end of fertilization, they are reformed at the egg's surface, while the egg and sperm chromatin begins to decondense. Spermatozoa are stored in the female apparatus prior to fertilization; here they come into contact with the epithelial cells of the spermatheca, protruding pseudopodia rich in microfilaments into the cellular body.