The Interactions of Sea Urchin Gametes During Fertilization

The interactions between sea urchin spermatozoa and ova duringfertilization usually exhibit a high degree of species specificity.Under natural conditions and reasonable gamete concentrations,most interspecific inseminations fail to yield zygotes. Macromoleculeson the external surfaces of the apposing gametes must surelybe responsible for successful gamete recognition, adhesion andfusion. Species specific recognition between surface componentsof sperm and egg could occur during at least three events comprisingthe fertilization process. The first event is the interactionof the sperm plasma membrane with the egg jelly coat. This inducesthe sperm acrosome reaction resulting in the exocytosis of the"bindin" -containing acrosome granule and also the extrusionof the acrosome process from the anterior tip of the sperm.The second event is the adhesion of the bindin-coated acrosomeprocess to glycoprotein "bindin receptors" on the external surfaceof the egg vitelline layer. The third event is the penetrationof the vitelline layer and the fusion of sperm and egg plasmamembranes. With the isolations of the component of egg jellywhich induces the acrosome reaction, sperm bindin from the acrosomevesicle and the egg surface bindin receptor from the vitellinelayer, there is hope of discovering the molecular basis of thismost interesting intercellular interaction which results inthe activation of embryonic development.     

Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.     

皱纹盘鲍受精过程的电镜观察

本文用透射电镜观察了皱纹盘鲍的受精过程。鲍卵子的胶膜使精子活化,并诱发了顶体反应,卵黄膜使顶体反应达到高潮。精子入卵后,卵发生皮层反应并形成受精膜开 减数分裂。此外,还观察到鲍的多精入卵现象。     

The Acrosome Reaction in Ciona intestinalis (Ascidia, Tunicata)

An acrosome reaction occurs by fusion between the acrosomal outer membrane and the plasmalemma enclosing the acrosome in Ciona intestinalis spermatozoa. The fusion seems to proceed along the peripheral margin of the acrosome, which causes vesiculation. The membrane bound vesicle formed by this process is probably shed by the sperm. The acrosomal inner membrane is exposed and becomes a part of the plasmalemma enclosing the anterior region of the sperm head. During this process, any acrosomal substance might be released through the opening formed by membrane fusion. The acrosome reaction most likely occurs in C. intestinalis spermatozoa, via vesiculation, in fundamentally the same way as observed in mammalian spermatozoa.     

Sperm plasma membrane breakdown during Drosophila fertilization requires sneaky, an acrosomal membrane protein

Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. We previously proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. Here we support this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. We further show that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), we show that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed us to follow the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, we find that the Drosophila acrosome is a paternally inherited structure. We provide evidence that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. We relate the characteristics of Drosophila Snky, acrosome function and sperm PMBD to membrane fusion events that occur in other systems.     

The jelly envelopes and fertilization of eggs of the newt, Notophthalmus viridescens

Fertilization in Notophthalmus viridescens is internal and involves passage of the sperm through five layers of egg jelly (J5-J1, from outermost to innermost), each of which is secreted by a discrete region of the oviduct. Polyspermy is normal. Passage of the sperm through the jelly and into the egg was studied by a technique of artificial insemination similar to natural insemination, in that undiluted fluid from the vas deferens was applied directly to eggs with various layers of jelly present, followed by flooding with water three to five minutes later. In general, successful fertilization increased as the number of jelly layers increased; jellyless coelomic eggs were not fertilizable. Sperm passage through the jelly and into the egg usually occurs within one to three minutes. Upon hydration of the jelly, barriers to sperm penetration develop in layers J5 and J3. Changes in the egg jelly thus seem to be involved in the restriction of polyspermy to a low level.     

Ultrastructural and experimental investigations of sperm-egg interactions in fertilization ofHydra carnea

Summary Fertilization in the freshwater hydrozoanHydra carnea has been examined by light, scanning and transmission electron microscopy. Sperm penetrate the jelly coat which covers the entire egg surface only at the site of the emission of the polar bodies. The egg surface exhibits a small depression, the so called fertilization pit at this site. Sperm-egg fusion takes place only at the bottom of the fertilization pit.Hydra sperm lack a structurally distinct acrosome and in most of the observed cases, fusion was initiated by contact between the membrane of the lateral part of the sperm head and the egg surfacce. Neither microvilli nor a fertilization cone are formed at the site of gamete fusion. The process of membrane fusion takes only a few seconds and within 1 to 2 min sperm head and midpiece are incorporated in the egg.Electron dense material is released by the egg upon insemination but cortical granule exocytosis does not occur and a fertilization envelope is not formed. The possible polyspermy-preventing mechanisms in hydrozoans are discussed. Hydra eggs can be cut into halves whereupon the egg membranes reseal at the cut edges and the fragments assume a spherical shape. Fragments containing the female pronucleus can be inseminated and exhibit normal cleavage and development. The observation that in such isolated parts the jelly coat will not fuse along the cut edges was used to determine its role in site-specific gamete fusion. These experiments indicate that site-specificity of gamete fusion can be attributed to special membrane properties at the fertilization pit.     

Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg

Morphological studies on the gametes and entry of the spermatozoan into the egg of the zebra danio, Brachydanio rerio, were conducted primarily with scanning electron microscopy. The spermatozoan showed a spherical head, which lacked an acrosome, a midpiece containing several mitochondria, and a flagellum. Observations of the unfertilized egg confirmed and extended prior studies showing a distinct cluster of microvilli on the plasma membrane, identified as the sperm entry site, beneath the inner micropylar aperture (Hart and Donovan, '83). The fertilizing spermatozoan attached to the sperm entry site within 5 seconds of the mixing of a gamete suspension. Binding to the egg microvilli appeared restricted to the equatorial surface of the spermatozoan. Fusion between the plasma membranes of the interacting gametes was followed by the formation of a distinct, nipple-shaped fertilization cone. The sperm head was partially incorporated into the fertilization cone cytoplasm by 60 seconds postinsemination. The incorporation of the entire sperm head, midpiece, and a portion of the flagellum occurred between 1 and 2 minutes. During this time, the fertilization cone shortened and was transformed into a massive, blister-like cytoplasmic swelling. Concurrently, upward movements of the ooplasm resulted in the gradual disappearance of the original depression in the egg surface containing the sperm entry site. The second polar body, fully developed by 10 minutes postinsemination, formed approximately 10-15 microns from the site of sperm penetration. Development of the fertilization cone, formation of the second polar body and exocytosis of cortical granules at the sperm entry site readily occurred in parthenogenetically activated eggs, indicating that these surface rearrangements do not require sperm binding and/or fusion.     

Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

Dynamics of the mammalian sperm plasma membrane in the process of fertilization

Sexual reproduction requires the fusion of sperm cell and oocyte during fertilization to produce the diploid zygote. In mammals complex changes in the plasma membrane of the sperm cell are involved in this process. Sperm cells have unusual membranes compared to those of somatic cells. After leaving the testes, sperm cells cease plasma membrane lipid and protein synthesis, and vesicle mediated transport. Biophysical studies reveal that lipids and proteins are organized into lateral regions of the sperm head surface. A delicate reorientation and modification of plasma membrane molecules take place in the female tract when sperm cells are activated by so-called capacitation factors. These surface changes enable the sperm cell to bind to the extra cellular matrix of the egg (zona pellucida, ZP). The ZP primes the sperm cell to initiate the acrosome reaction, which is an exocytotic process that makes available the enzymatic machinery required for sperm penetration through the ZP. After complete penetration the sperm cell meets the plasma membrane of the egg cell (oolemma). A specific set of molecules is involved in a disintegrin-integrin type of anchoring of the two gametes which is completed by fusion of the two gamete plasma membranes. The fertilized egg is activated and zygote formation preludes the development of a new living organism. In this review we focus on the involvement of processes that occur at the sperm plasma membrane in the sequence of events that lead to successful fertilization. For this purpose, dynamics in adhesive and fusion properties, molecular composition and architecture of the sperm plasma membrane, as well as membrane derived signalling are reviewed.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis, and Lytechinus variegatus, were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.     

Complexin I is required for mammalian sperm acrosomal exocytosis

Regulated exocytosis in many cells is controlled by the SNARE complex, whose core includes three proteins that promote membrane fusion. Complexins I and II are highly related cytosolic proteins that bind tightly to the assembled SNARE complex and regulate neuronal exocytosis. Like somatic cells, sperm undergo regulated exocytosis; however, sperm release a single large vesicle, the acrosome, whose release has different characteristics than neuronal exocytosis. Acrosomal release is triggered upon sperm adhesion to the mammalian egg extracellular matrix (zona pellucida) to allow penetration of the egg coat. Membrane fusion occurs at multiple points within the acrosome but how fusion is activated and the formation and progression of fusion points is synchronized is unclear. We show that complexins I and II are found in acrosome-intact mature sperm, bind to SNARE complex proteins, and are not detected in sperm after acrosomal exocytosis (acrosome reaction). Although complexin-I-deficient sperm acrosome-react in response to calcium ionophore, they do not acrosome-react in response to egg zona pellucida proteins and have reduced fertilizing ability, in vitro. Complexin II is present in the complexin-I-deficient sperm and its expression is increased in complexin-I-deficient testes. Therefore, complexin I functions in exocytosis in two related but morphologically distinct secretory processes. Sperm are unusual because they express both complexins I and II but have a unique and specific requirement for complexin I.     

Fertilization in Callochiton castaneus (Mollusca)

A fine-structural study of fertilization in Callochiton castaneus has revealed that the mechanism of sperm penetration into the egg is intermediate between the primitive condition found in members of the order Lepidopleurida and the more derived condition found in the Chitonida. C. castaneus sperm have the long needlelike nuclear filament and reduced acrosome that characterizes all Chitonida, but they have retained several plesiomorphic features such as an unspecialized mid-piece and a lack of flagellar reinforcement. As in some Lepidopleurida but unlike any Chitonida, the egg hull in this species comprises a thick, smooth jelly coat permeated by pores that permit sperm rapid access to the vitelline layer. The jelly coat is delicate and quickly dissolves when a sperm concentrate is used, suggesting that excess acrosomal enzymes may be responsible. Once the sperm have penetrated the vitelline layer, the long nuclear filament bridges the gap to cups in the egg membrane. However, once the fertilization membrane is raised, the perivitelline space exceeds the length of the nuclear filament, preventing other sperm from penetrating the egg. A fertilization cone forms around the nuclear filament of the penetrating sperm, but it does not appear to engulf the body of the sperm. Rather, the nuclear chromatin is injected into the egg as a long thread. The remaining sperm organelles are apparently abandoned on the egg surface. If this is the case, it would be a significant departure from fertilization in other molluscs and many other metazoans, in which sperm organelles, such as centrioles and mitochondria, enter the egg. New sperm and egg characters, as well as significant differences in fertilization, indicate that Callochitonidae are basal to all other members of the order Chitonida and may warrant separation as the sister taxon to the suborders Chitonina and Acanthochitonina.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. II. Ultrastructural changes associated with inhibition of the acrosome reaction.

Pretreatment of Strongylocentrotus purpuratus sperm with delta 9-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electron-dense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 microM THC. The electron-dense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.     

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.     

Mammalian sperm acrosome: formation, contents, and function

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

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.