Early Events Of Fertilization Research Articles

Timing of sequential changes in chromosome configurations during the second meiotic division and cytoplasmic events of pig oocytes matured and fertilized in vitro

The present study determined the specifics of early events of fertilization of pig oocytes matured in vitro (IVM) and subsequently fertilized in vitro (IVF). The IVF system used for sperm capacitation, time of fertilization and fixation of eggs at 1–3 h intervals during the 30 h period after insemination precisely measured the timing of each critical stage of fertilization with precision. The first evidence of sperm penetration was observed 3 h after insemination. Attachment of the spermatozoa in the ooplasm caused resumption of meiosis (A 2/T 2 stage of second meiotic division), and was commonly observed from 4 to 5 h after insemination. The sperm heads of penetrating spermatozoa started to decondense, tails became detached and completion of the second meiosis (second polar body extrusion) occurred from 6 to 9 h. Further decondensation of the sperm head and female chromosomes took place from 9 to 12 h. Opposing and apposing pronuclei were frequently observed from 12 to 18 h and 18 to 24 h after insemination, respectively. Syngamy and prophase stage of first mitotic division, including first cleavage were observed from 24 to 30 h.

Cytoplasmic calcium and Fucus egg activation

ABSTRACT Eggs of the marine brown alga, Fucus serratus, exhibit small transient elevations of cytosolic Ca2+ of variable magnitude, corresponding to the onset of the fertilization potential. Microinjection of Ca2+ buffers (BAPTA (1-2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid)) at concentrations sufficient to block any global fertilization-associated Ca2+cyt elevation did not inhibit egg activation (monitored as exocytosis of cell wall) or subsequent development. However, egg activation could be inhibited with higher buffer concentrations. Br2BAPTA (Kd = 1.6 μM) was a more effective inhibitor of egg activation than BAPTA (Kd = 0.17 μM). Localized microinjection of Ca2+ produced only localized cell wall exocytosis at the injection site. Eggs injected with Br2BAPTA at intracellular concentrations, which blocked egg activation, exhibited prolonged fertilization potentials. 45Ca2+ influx across the plasma membrane increased during fertilization. Our results show that a large transient global elevation of Ca2+cyt is not necessary for Fucus egg activation but rather a localized elevation to micromolar levels results, at least in part, from increased Ca2+ influx across the plasma membrane. This is needed for early fertilization events, including the generation of the fertilization potential and cell wall secretion.

Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head.

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight approximately 20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization.

Fusion of membranes during fertilization. Increases of the sea urchin egg's membrane capacitance and membrane conductance at the site of contact with the sperm.

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.

Preparation of individual electrically and video-recorded eggs for integrated temporal and electron microscopic analyses.

A method for correlative studies of early fertilization events that integrates techniques of intracellular electrophysiological recording, video-imaging, and electron microscopy is described. A key feature of the method is its ability to identify the fertilizing sperm and to record the moment of egg excitation. Since the site of gamete interaction is recognizable throughout all stages of preparation, difficulties associated with locating the site of fertilization and determining specimen orientation for microtomy and electron microscopic examination are eliminated. Virtually all samples yield useful information. An example of interacting gametes fixed 4 sec after initiation of the fertilization potential and serial sectioned is described. The method is applicable to systems other than fertilizing eggs when functional, temporal, and spatial relationships of individual cells need to be correlated with changes in ultrastructure.

Sperm actin and calmodulin during fertilization in the hamster: An immune electron microscopic study

The distribution of actin and CaM in hamster spermatozoa was examined during the early events of fertilization using postembedding immunogold procedures. Actin was immunolocalized with a polyclonal antibody and two monoclonal antibodies. CaM was immunodetected with a polyclonal antibody. In epididymal sperm, actin labeling was found solely in the principal piece of the flagellum. CaM labeling was observed in the postacrosomal lamina, subacrosomal ring, and tip of the perforatorium. These distributions were not modified after capacitation and acrosome reaction. During the successive steps of sperm-egg fusion actin remained undetected in the sperm head whereas its location did not change in the flagellum. CaM distribution remained unmodified until the sperm head begins to decondense. At later stages of sperm head decondensation the postacrosomal lamina and its CaM labeling disappeared, whereas gold particles were still detected in the subacrosomal layer. The predominant location of actin into the egg cortex, particularly the microvillus-free area was confirmed. Except for the CaM labeling of the meiotic spindle, no special CaM location could be found throughout the egg. Thus, in hamster, a role for sperm actin in sperm-egg fusion appears unlikely. In contrast the CaM present in the Ca(2+)-rich postacrosomal lamina could be involved in the regulation of egg activation.

Time Sequence of Early Events in Fertilization in the Medaka Egg: (micropyle/exocytosis/sperm movement/calcium release/medaka egg).

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 108 /ml at 23°-25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5-16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.

Extreme Toxicity of Ethanol During Activation of Sea Urchin Eggs: (ethanol toxicity/ionophore A23187/parthenogenesis/Strongylocentrotus purpuratus/K+ -permeability).

We have examined the effects of ethanol on early fertilization events and later development in the sea urchin Strongylocentrotus purpuratus. Eggs can still be fertilized in ethanol concentrations as high as 480 mM (2.0%); egg cytolysis was rapidly observed postinsemination in 50% of the cells at 220 mM ethanol. Yet, sperm motility was essentially normal in 250 mM ethanol; 940 mM ethanol was required to affect a 50% reduction. To determine the effect of ethanol on K+ -efflux from eggs induced by fertilization, we used parthenogenetic activation induced by the Ca2+ -ionophore A23187. Surprisingly, ethanol at only 0.2 mM caused an abnormal K+ -efflux, but only when added between 1 and 3 min after induction of activation. The K+ -efflux rates of unfertilized eggs were not influenced by up to 730 mM ethanol. Finally, normal embryonic development through the mesenchyme blastula stage was observed in egg suspensions which were treated for 30 min with ethanol concentrations as high as 240 mM, but washed with normal seawater prior to insemination. Normal plutei were obtained from cultures which were continuously cultured in 24 mM ethanol from 15 min postinsemination. We conclude that an extreme ethanol sensitivity of embryogenesis is apparent only during the cortical reaction.

Calcium influx regulates antibody-induced glycoprotein movements within the Chlamydomonas flagellar membrane

The Chlamydomonas flagellar surface exhibits a number of dynamic membrane phenomena associated with whole-cell gliding locomotion and the early events in fertilization. Crosslinking of a specific population of flagellar surface-exposed glycoproteins with the lectin concanavalin A or an anti-carbohydrate mouse monoclonal antibody, designated FMG-1, results in a characteristic pattern of glycoprotein redistribution within the plane of the flagellar membrane. Recent evidence suggests that flagellar membrane glycoprotein movements are associated with both whole-cell gliding motility and the early events in mating. It is of interest to determine the transmembrane signaling pathway whereby crosslinking of the external domains of flagellar glycoproteins activates the intraflagellar machinery responsible for translocation of flagellar membrane glycoproteins. The redistribution of flagellar membrane glycoproteins requires micromolar levels of free calcium in the medium; lowering the free calcium concentration to 10(-7) M results in complete but reversible inhibition of redistribution. Redistribution is maximal in the presence of 20 microM free calcium in the medium. Redistribution is inhibited in the presence of 20 microM free calcium by the calmodulin antagonists trifluoperazine, W-7 and calmidazolium, the calcium channel blockers diltiazem, methoxyverapamil (D-600) and barium chloride, and the local anesthetics, lidocaine and procaine. The actions of all of these agents can be interpreted in terms of a requirement for calcium in the signaling mechanism associated with flagellar glycoprotein redistribution. In particular, the requirement for micromolar calcium in the external medium and the effects of specific calcium channel blockers suggest that flagellar membrane glycoprotein crosslinking may induce an increase in calcium influx, which may be the initial trigger for activating the flagellar machinery responsible for active movement of flagellar membrane glycoproteins.

Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins

The zona pellucida is a unique, oocyte-specific matrix that coats the surface of all mammalian eggs. Composed of three sulfated glycoproteins in the mouse (ZP1, ZP2, and ZP3), the zona pellucida facilitates early events in fertilization and protects the embryo during preimplantation development. Using DNA isolated from hamster-mouse somatic cell hybrids and from C57 BL 6 J × Mus spretus interspecific backcross progeny, Zp-2 was located on chromosome 7, 11.3 ± 3.2 cM distal to Tyr, and Zp-3 was located on chromosome 5, 9.2 ± 2.9 cM distal to Gus.

Monoclonal antibodies to a membrane glycoprotein induce the phosphorylation of histone H1 in sea urchin spermatozoa.

Two groups of mAbs reacting with external domains of a major sea urchin sperm membrane glycoprotein of 210 kD were isolated. Previous studies have shown that group I mAbs inhibit the acrosome reaction induced by egg jelly and also cause large increases in intracellular Ca2+ [( Ca2+]i). Group II mAbs, at comparable levels of cell surface binding, neither inhibit the egg jelly-induced acrosome reaction nor cause increases in [Ca2+]i. In this paper, we investigate the ability of these mAbs to induce the cAMP-dependent phosphorylation of sperm histone H1. Group I mAbs induce H1 phosphorylation to the same level and on the same peptide, as occurs upon treatment of sperm with egg jelly. These mAbs also activate adenylate cyclase to the same extent as egg jelly. Group II mAbs do not induce H1 phosphorylation and are only poor activators of adenylate cyclase. Group I mAbs compete with each other, but not with group II mAbs, for binding to the cell surface. These data indicate that the activation of adenylate cyclase is an initial event in the pathway leading from the binding of mAbs to a specific domain of the 210-kD protein at the cell surface, to the discrete phosphorylation of histone H1 in highly condensed sperm chromatin. The domain on the 210-kD protein recognized by group I mAbs plays a critical role in signal transduction during the early events of fertilization.

Monoclonal Antibodies to Porcine Zona Pellucida That Block the Initial Stages of Fertilization

Zona pellucida (ZP) is thought to be of utmost biological importance in the early stages of fertilization and implantation. Current hybridoma technology was used to produce monoclonal antibodies (MAbs) against specific antigens to porcine ZP. Two monoclonal antibodies (4F2 and 2D9) were raised that reacted against ZP antigens shared by human and porcine ZP. These antibodies were shown to block fertilization of human oocytes in in vitro fertilization (IVF). It is likely that MAb 4F2 recognized a protein epitope localized on the outer surface of ZP. These antibodies may be quite useful immunologic probes for studying the precise mechanisms of the early events of fertilization in mammals.

Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg: (microinjection/divalent cations/fertilization/medaka egg/membrane potential).

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes. Microinjection of either Ca2+ , Ba2+ or Sr2+ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca-Mg-free conditions, but microinjection of Mg2+ , Mn2+ or Co2+ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca-Mg-free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca2+ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca2+ , Mg2+ or K+ , the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca2+ . From these experiments, it was inferred that microinjected divalent cations such as Ca2+ , Ba2+ or Sr2+ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca2+ .

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg: (calcium/microinjection/calcium-uptake/Oryzias latipes/sperm penetration).

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca2+ . The effect of extracellular Ca2+ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca2+ . Verapamil significantly reduced the action of extracellular Ca2+ or Ba2+ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No 45 Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca2+ from the external medium is not required.

Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs.

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at approximately 23 s as measured by calcium-aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at approximately 51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.

Fertilization in a crab: I. Early events in the ovary, and cytological aspects of the acrosome reaction and gamete contacts

Early events in fertilization were studied in Carcinus maenas by in vitro experiments and ultrastructural analysis; some were found to occur in the lumen of ripe ovaries. The acrosome reaction generally conformed to the usual Reptantia Decapoda pattern. However, a prominent membrane system continuous with the nuclear envelope and located close to the base of the acrosome tubule characterized the type of spermatozoon observed in Carcinus maenas. Such complex anatomical connections linking the three parts of the reacted spermatozoon (acrosome tubule, membrane system and nucleus envelope) may be significant in relation to the membrane system's contribution to the acrosome reaction. The outer layer of the everted acrosomal vesicle was found to comprise tubular elements ending in bell-shaped corpuscles, deeply interdigitated with the oolemma microvilli during the establishment of the initial contacts between the reacted spermatozoon and the egg plasma membrane. At the site of contact, the oolemma formed a minute fertilization cone, locally depressed by the acrosome tubule. During these early fertilization events, the nucleus, like the other spermatozoon components, was seen to penetrate the egg coatings first, and later to be located near the oolemma.

The effects of taurine and hypotaurine on in vitro fertilization in the golden hamster

AbstractTaurine and hypotaurine were examined for their efficacy in replacing sperm motility factor (SMF), prepared from bovine adrenal cortex, for in vitro fertilization in the golden hamster. Combinations of these amino acids at concentrations of 0.001, 0.01, 0.1, and 1 mM together with 16 μM isoproterenol (a catecholamine β‐agonist) were added to the sperm incubations. After three hours of sperm preincubation, oviductal eggs were added to the sperm suspensions and examined for penetration and stage of fertilization after three or five hours of culture. At 0.001 mM, neither taurine or hypotaurine was capable of maintaining motility of hamster sperm for four to 4½ hours or of inducing fertilization. With all other concentrations, both amino acids were found to maintain motility of sperm as well as SMF. Hypotaurine stimulated motility to a greater extent than taurine and both required isoproterenol for the greatest motility. A low proportion of cumulus‐free ova were fertilized when sperm were preincubated with either amino acid alone over the range of 0.01 to 1 mM; however, over 80% fertilization was consistently obtained when isoproterenol was also present during sperm incubation. Proportions of ova fertilized with taurine or hypotaurine present during sperm preincubation were comparable to those achieved with SMF. The possibility that taurine or hypotaurine is the sperm motility factor is discussed.After three hours of sperm/egg incubation, a lag in the early events of fertilization was observed in experimental groups treated with one of the amino acids (0.01 mM) alone compared with groups treated with isoproterenol present. However, if sperm/egg incubation was extended from three to five hours, no increase in number of eggs penetrated was found. Therefore, the delay observed at three hours was considered a function of fewer numbers of capacitated sperm present in the absence of isoproterenol rather than of the need for an extended capacitation time.

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

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.

An ultrastructural analysis of the gametes and early fertilization in two bivalve molluscs, Chama macerophylla and Spisula solidissima with special reference to gamete binding.

An ultrastructural investigation of the gametes and their interaction during the early events of fertilization in molluscs has been performed. A gamete binding event involving large numbers of sperm has been identified and examined in detail. The surface of the oocyte is projected into numerous microvilli which extend through the vitelline envelope. Tufts of fibrillar material radiate from the tips of these microvilli, forming a layer external to the vitelline envelope. The acrosomal vesicle of the mature spermatozoon contains two major components, which function differently during fertilization. The vesicle is indented at its adnuclear surface, constituting a preformed acrosomal tubule. This tubule does not elongate during the acrosome reaction. Completion of the reaction results in the formation of an extracellular coat, derived from on component of the acrosomal vesicle, on the anterior surface of the sperm. Sperm-egg binding is accomplished by an association of the extracellular coat on the reacted sperm and the fibrous tufts on the tips of the microvilli of the oocyte. Evidence that gamete membrane fusion occurs by fusion of the acrosomal tubule and a microcillus is presented. These observations provide a generalized pattern of molluscan fertilization.