Molecular basis of fertilization in the mouse

Recent studies of mouse fertilization have identified two complementary gamete receptors that mediate sperm-egg binding. Sperm surface β1,4-galactosyltransferase (GalTase) binds to specific oligosaccharides of the egg coat (zona pellucida) glycoprotein ZP3. Evidence suggests that these same molecules may stimulate the acrosome reaction in sperm. After the acrosome reaction, it is thought that sperm remain adherent to the zona by binding another glycoprotein, ZP2. The acrosome-reacted sperm releases hydrolytic enzymes, including acrosin and N-acetylglucosaminidase, enabling it to penetrate the zona pellucida. After the penetrating sperm binds to the egg membrane and activates development, N-acetylglucosaminidase is exocytosed from egg cortical granules and, as part of the zona block to polyspermy, globally removes the sperm GalTase binding site from ZP3 oligosaccharides.     

The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.     

Interspecies interaction of zona-free ova with spermatozoa in mouse,rat and hamster

Mouse, rat and hamster zona-free eggs were penetrated in vitro by spermatozoa of their own species and by alien spermatozoa of mouse, rat and hamster. The tested combinations showed very distinct differences in penetration ability. Mouse zona-free eggs were penetrated by spermatozoa of their own species only. Rat zona-free eggs were penetrated by their own and mouse spermatozoa. Hamster zona-free eggs were penetrated by their own, mouse and rat spermatozoa. Several proteolytic enzymes used for lysis of zona pellucida, time of sperm preincubation and sperm concentration did not affect sperm-egg interaction. It is concluded that the species specificity of egg plasma membrane in the rodents tested is probably based on some specific surface components.     

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.     

ELECTRON MICROSCOPIC OBSERVATIONS OF GUINEA PIG SPERMATOZOA PENETRATING EGGS IN VITRO*

Guinea pig ovarian oocytes matured in vitro were inseminated in vitro with capacitated, acrosome-reacted spermatozoa and sperm penetration through the zona pellucida and into the egg cytoplasm were examined. Sperm heads passing through the zona pellucida had already lost all their acrosomal elements except for the inner acrosomal membrane and the equatorial segment. It was often observed that the texture of the zona material around the sperm head was distorted, giving the impression that the zona pellucida was parted, at least partially, by a shearing force produced by the sperm head advancing through the zona. When eggs were freed from their zonae pellucidae and inseminated, the acrosome-reacted spermatozoa immediately bound to the egg surfaces and began to fuse with the eggs; whereas the spermatozoa with intact acrosomes failed to do so. Fusion began between the egg plasma membrane and the sperm plasma membrane at the central region of the sperm head. The anterior half of the sperm head was engulfed by the egg in a phagocytic fashion, while its posterior half was incorporated into the egg by a fussion between egg and sperm plasma membranes. Incorporation of the sperm tail into the egg was achieved by fusion between the sperm and egg plasma membranes.     

Ultrastructural observations on in vivo fertilisation in the brushtail possum, Trichosurus vulpecula, following PMSG/LH superovulation and artificial insemination

Information on the dynamics of gamete interaction in marsupials is very limited and not available for any species from the major Australian Order Diprotodontia which includes most of the more familiar animals such as kangaroos, possums and the koala. This study addressed this deficiency by examining the ultrastructure of in vivo fertilised eggs from common brushtail possums (Trichosurus vulpecula). Females were superovulated by treatment with 15 IU PMSG and then 4 mg porcine LH 3 days later, and inseminations were performed 910-13 h after LH) using epididymal spermatozoa. Between 33 and 39 h after LH injection females were killed, reproductive tracts excised and the oviduct ampulla segment flushed for eggs. Three of the six eggs examined were fertilised as judged by the presence of sperm remnants in the cytoplasm. On the basis of these eggs it was found that sperm penetration left a large hole in the zona pellucida (ZP), suggesting that sperm zona penetration occurs primarily by the enzymatic action of acrosomal enzymes. Sperm lying within the perivitelline space were lacking both an outer acrosomal membrane and the associated acrosomal contents, while both these structures were found on sperm embedded within the mucoid layer, which is consistent with induction of the acrosome reaction by binding to the ZP. Once inside the egg cytoplasm, the sperm head travelled only a short distance before chromatin decondensation occurred. Fertilised eggs showed signs of cytoplasmic activation including cytoskeleton association with apparently dividing mitochondria and prominent rough endoplasmic reticulum. Unfertilised eggs appeared to be undergoing degenerative changes and lacked any evidence of activation. This study was demonstrated that superovulation and laparoscopic intravaginal artificial insemination provide a system through which perifertilisation events in the possum and other monovular Australian marsupials can be examined experimentally.     

Cytochalasin-D retards sperm incorporation deep into the egg cytoplasm but not membrane fusion with the egg plasma membrane

The fertilization process is impaired when spermatozoa are previously incubated with Cytochalasin-D (Cyt-D). Although this fact reveals the participation of polymerized actin in fertilization, the specific event obstructed by Cyt-D treatment has not been determined. To identify this event, we capacitated guinea pig spermatozoa in minimal capacitating medium with pyruvate and lactate (MCM-PL) with Cyt-D, to inseminate hamster zona pellucida (ZP)-free eggs. Cyt-D (70 microM) decreased F-actin relative concentration in capacitated spermatozoa to a larger extent than in spermatozoa incubated under control conditions. Cyt-D also cancelled the F-actin increase normally observed in acrosome-reacted cells, and decreased the number of these cells with normal F-actin localization at the equatorial zone. Insemination of eggs with Cyt-D treated spermatozoa did not change early fertilization events such as the egg cortical reaction (CR), membranes fusion, and egg F-actin new localization, but clearly retarded, by 16 hr, spermatozoa incorporation deep into the egg cytoplasm, and decondensation of egg metaphase II chromosomes. These results show that actin polymerization is necessary for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane fusion nor egg activation early steps.     

A model for sperm-egg binding and fusion based on ADAMs and integrins

Once a sperm meets an egg, several events must occur in order for fertilization to proceed. Sperm must bind to the zona pellucida, undergo the acrosome reaction, penetrate the zona pellucida and then bind to and fuse with the egg plasma membrane. Shortly thereafter, the egg must be activated for zygotic development. This review focuses on mammalian sperm-egg plasma membrane binding and fusion, and in particular on the roles of two families of cell-adhesion molecules, ADAMs and integrins, in this important union.     

Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

This light and transmission electron microscopical study shows that the first polar body is given off before ovulation and that part of its cell membrane and that of the surrounding oocyte have long microvilli at the time of its ejection. Several layers of cumulus cells initially surround the secondary oocyte and first polar body, but the ovulated oocytes in the oviducts in the process of being fertilized do not have cumulus cells around them. Partly expelled second polar bodies occur in the oviduct; they are elongated structures that lack organelles and have electron-dense nuclei. A small fertilization cone appears to form around the sperm tail at the time of sperm entry into the egg and an incorporation cone develops around the sperm head in the egg cytoplasm. In three fertilized eggs a small hole was seen in the zona, which was presumably formed by the spermatozoon during penetration. Cortical granules, present in ovarian oocytes, are not seen in fertilized tubal or uterine eggs; release of their contents probably reduces the chances of polyspermy, although at least one polyspermic fertilized egg was seen and several other fertilized eggs had spermatozoa within the zona pellucida. In the zygote the pronuclei come to lie close together, but there was no evidence of fusion. A "yolk mass," which becomes eccentric before ovulation, is extruded by the time the two-cell embryos are formed, but many vacuoles remain in the non-yolky pole of the egg. A shell membrane of variable thickness is present around all uterine eggs but its origin remains undetermined.     

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.     

Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs

Human spermatozoa were demembranated with Triton X-100 (TX) and injected into the mature eggs of Xenopus laevis. The nuclei of these spermatozoa decondensed and developed into pronuclei. Chromosomes did not appear in the eggs until the end of a 5-hr incubation period. When the demembranated human spermatozoa were further treated with dithiothreitol (DTT) before they were injected into the eggs, the sperm nuclear decondensation and pronuclear development took place considerably faster than in spermatozoa treated with the detergent alone. By the end of the 5-hr incubation period, decondensed chromatin threads or chromosome-like structures appeared, but none of the eggs cleaved. When human spermatozoa were injected into full-grown ovarian oocytes with intact germinal vesicle (GV) or oocytes which had matured without GV, the nuclei of a proportion of TX-treated and all TX-DTT-treated sperm decondensed but showed no sign of developing into pronuclei. Sperm nuclei injected into maturing oocytes formed condensed chromatin fragments as long as the oocytes were not activated, but they transformed into pronuclei when the oocytes were stimulated with electric shock. These results indicate that the cytoplasmic factors responsible for the decondensation of human sperm nuclei are present in egg cytoplasm independent of GV-materials. We also suggest that the factors controlling development of decondensed sperm nuclei into pronuclei are dependent on GV materials.     

Overexpressing sperm surface beta 1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions

Sperm surface beta 1,4-galactosyltransferase (GalTase) mediates fertilization in mice by binding to specific O-linked oligosaccharide ligands on the egg coat glycoprotein ZP3. Before binding the egg, sperm GalTase is masked by epididymally derived glycosides that are shed from the sperm surface during capacitation. After binding the egg, sperm- bound oligosaccharides on ZP3 induce the acrosome reaction by receptor aggregation, presumably involving GalTase. In this study, we asked how increasing the levels of sperm surface GalTase would affect sperm-egg interactions using transgenic mice that overexpress GalTase under the control of a heterologous promoter. GalTase expression was elevated in many tissues in adult transgenic animals, including testis. Sperm from transgenic males had approximately six times the wild-type level of surface GalTase protein, which was localized appropriately on the sperm head as revealed by indirect immunofluorescence. As expected, sperm from transgenic mice bound more radiolabeled ZP3 than did wild-type sperm. However, sperm from transgenic animals were relatively unable to bind eggs, as compared to sperm from wild-type animals. The mechanistic basis for the reduced egg-binding ability of transgenic sperm was attributed to alterations in two GalTase-dependent events. First, transgenic sperm that overexpress surface GalTase bound more epididymal glycoside substrates than did sperm from wild-type mice, thus masking GalTase and preventing it from interacting with its zona pellucida ligand. Second, those sperm from transgenic mice that were able to bind the zona pellucida were hypersensitive to ZP3, such that they underwent precocious acrosome reactions and bound to eggs more tenuously than did wild-type sperm. These results demonstrate that sperm-egg binding requires an optimal, rather than maximal, level of surface GalTase expression, since increasing this level decreases sperm reproductive efficiency both before and after egg binding. Although sperm GalTase is required for fertilization by serving as a receptor for the egg zona pellucida, excess surface GalTase is counterproductive to successful sperm-egg binding.     

Fertilization of parthenogenetically activated mouse eggs : I. Behaviour of sperm nuclei in the cytoplasm of parthenogenetically activated eggs

Oocytes from Swiss albino females were activated by heat-shock (44.5 °C) as described previously [8] and fertilized in vitro [10]. Time of insemination varied from 10 min to 3 h after activation. It has been found that spermatozoa may penetrate the zona pellucida and into the cytoplasm of the activated eggs. Sperm penetration may still occur as late as in the 3rd h after activation. The results indicate that the decondensation factor remains present in the activated eggs for at least 1.5 h after activation. Dispersion and transformation of the sperm chromatin into the early male pronucleus takes place at that time. In the pronucleus formed, no growth was registered. This may be caused by the fact that the processes of artificial activation precede those which accompany fertilization. The cytoplasm therefore loses the properties displayed in the course of the normal process of fertilization, when activation is the result of the penetrating spermatozoon.     

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

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.     

The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

Summary The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.     

Cell adhesion and fertilization: steps in oocyte transport,sperm-zona pellucida interactions,and sperm-egg fusion

Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte "pick-up," in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.     

Cortical Granules of Sea Urchin Eggs do not Undergo Exocytosis at the Site of Sperm-egg Fusion*

Microscopic observations of sea urchin egg fertilization (phase contrast, Nomarski and transmission electron microscope) reveal that the cortical granules in the area of sperm egg-fusion do not undergo exocytosis. These intact granules remain associated with the sperm, moving into the egg cytoplasm with the entering sperm. This sperm-cortical granule association occurs before the sperm centriole affects microtubule organization and the sperm-cortical granule association is not affected by cytochalasin D or griseofulvin. We discuss the possibility that a reorganization of the egg cytoplasm ensues from the sperm-egg interaction at the site of sperm-egg fusion. Other possibilities are that the retention of cortical granules is not related to egg reorganization, but is necessary for successful sperm incorporation or reflects an unrelated component of the activation process.