Sperm-specific C-terminal processing of the proteasome PSMA1/α6 subunit

We previously reported that the ascidian sperm proteasome degrades the egg-coat protein extracellularly during fertilization. In order to explore an extracellular transport signal, we purified the proteasome from ascidian sperm and compared its subunit structure with egg and muscle proteasomes. The results showed that PSMA1/α6 subunit of the sperm proteasome is distinct from egg and muscle proteasomes. LC/MS/MS analysis revealed that the C-terminal 16 residues of sperm α6 subunit are processed. Whereas sperm-specific paralogous genes of α subunits are reported, its sperm-specific C-terminal processing is a newly discovered novel post-translational modification of the proteasome.     

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.     

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.     

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha

The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry. Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

A general solution for optimal egg size during external fertilization, extended scope for intermediate optimal egg size and the introduction of Don Ottavio 'tango'

Egg sizes of marine invertebrates vary greatly, both within and between species. Among the proposed causes of this are a trade-off between egg size, egg number and survival probability of offspring, and a selection pressure exerted by sperm limitation during external fertilization. Although larger eggs are indeed a larger target for sperm, producing larger eggs also implies making fewer of them. There has been discussion about whether sperm limitation can (theoretically) and does (in nature) select for larger egg size than under ad libitum sperm. In one specific model, based on a particular fertilization kinetics model and an empirically derived mortality function, the theoretical possibility of a negative shift in optimal egg size with sperm concentration was demonstrated. Here we present a generalized analytical model to explore the effects of survival and fertilization probabilities on optimal egg size. It is demonstrated that incorporating fertilization kinetics greatly increases the scope for intermediate optimal egg size, as opposed to eggs of minimal or maximal size. Second, we present a general analytical qualitative solution to the question whether optimal egg size depends on sperm concentration. It is shown that, under the condition that an intermediate optimal egg size exists, this qualitative outcome of the model (positive, negative or no relation between optimal egg size and sperm limitation) depends on the structure of the fertilization kinetics part of the model. Finally, we evaluate fertilization kinetics models with respect to the general solution, using two previously published kinetics models ('Don Giovanni' and 'Don Ottavio') and a novel alteration of one of them in which sperm concentration covaries with egg concentration (Don Ottavio 'tango'). For all three models the relationship between optimal egg size and sperm concentration is shown to be always negative. This paper thus shows how biologically realistic relationships between egg size on the one hand and survival and fertilization probability on the other hand predict optimal egg size to be intermediate, and that this optimum is in general expected to increase when sperm become more limiting.     

鳙鱼受精早期扫描电镜研究

镛鱼(Aristichthys nobilis)受精是精子通过卵膜孔附着于卵质膜表面精子穿入部的微绒毛,两者迅即发生融合,但未见到有明显的受精锥。授精一分钟,精子整个头部已与卵的质膜发生融合,并看到有精子整个尾部已被微绒毛包裹的情况。在受精精子附近有一尚未与卵完全分开的第一极体。本文还讨论了精子穿入部的功能。     

Glycobiology of sperm-egg interactions in deuterostomes

The process of fertilization begins when sperm contact the outermost egg investment and ends with fusion of the two haploid pronuclei in the egg cytoplasm. Many steps in fertilization involve carbohydrate-based molecular recognition between sperm and egg. Although there is conservation of gamete recognition molecules within vertebrates, their homologues have not yet been discovered in echinoderms and ascidians (the invertebrate deuterostomes). In echinoderms, long sulfated polysaccharides act as ligands for sperm receptors. Ascidians employ egg coat glycosides that are recognized by sperm surface glycosidases. Vertebrate egg coats contain zona pellucida (ZP) family glycoproteins, whose carbohydrates bind to sperm receptors. Several candidate sperm receptors for vertebrate ZP proteins have been identified and are discussed here. This brief review focuses on new information concerning fertilization in deuterostomes (the phylogenetic group including echinoderms, ascidians, and vertebrates) and highlights protein-carbohydrate interactions involved in this process.     

Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.     

Factors related to successful sperm microinjection of hamster eggs: The effect of sperm species, technical experience, needle dimensions, and incubation medium on egg viability and sperm decondensation following microinjection

A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.     

Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.     

Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg

The responses of the egg to insemination in a modified Fish Ringer's solution (FRS) were examined in eggs of the zebrafish ( Brachydanio rerio ) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5–8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3–4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.     

Mechanism of Ca2+ release at fertilization in mammals.

At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4, 5-trisphosphate (InsP(3)). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP(3). We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267-275, 1999.     

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.     

OBSERVATIONS ON SPERM PENETRATION IN THE RAT

The structural aspects of sperm penetration in the rat egg were investigated by electron microscopy. Eggs were recovered at intervals between 8 and 10:30 A.M. from females which had mated during the previous night. The oviducts were flushed with hyaluronidase and the eggs transferred into a 2 per cent osmium tetroxide solution, buffered at pH 7.8. After fixation, the eggs were mounted individually in agar, dehydrated in ethyl alcohol, and embedded in butyl-methyl methacrylate (3:1). The sperm penetrating the egg is covered by a plasma membrane which is present only on the side facing toward the zona pellucida; no membrane is visible on the side facing toward the vitellus. The sperm plasma membrane becomes continuous with the egg plasma membrane and forms a deep fold around the entering sperm. Cross-sections through the sperm midpiece in the perivitelline space show an intact plasma membrane. At the place of entrance, the plasma membrane of the sperm appears to fuse with the egg plasma membrane. After the sperm has penetrated the vitellus, it has no plasma membrane at all. The nuclear membrane is also absent. These observations suggest a new hypothesis for sperm penetration. After the sperm has come to lie on the plasma membrane of the egg, the egg and sperm plasma membranes rupture and then fuse with one another to form a continuous cell membrane over the egg and the outer surface of the sperm. As a result the sperm comes to lie inside the vitellus, leaving its own plasma membrane incorporated into the egg membrane at the surface of the egg.     

Effects of various enzymes on the ability of hamster egg plasma membranes to fuse with spermatozoa

To investigate which component of the plasma membrane of the hamster egg plays the central role in the sperm–egg fusion, the egg membrane was treated with a variety of proteolytic, carbohydrate-hydrolyzing, lipid-hydrolyzing, and other enzymes. The only enzyme that markedly effected the ability of the egg membrane to fuse with spermatozoa was phospholipase C. The lipid moieties of the egg plasma membrane (and possibly of the sperm membrane) must be of primary importance in sperm–egg fusion at fertilization.