Acrosome-reacted guinea pig spermatozoa become fusion competent in the presence of extracellular potassium ions

Guinea pig spermatozoa are able to undergo capacitation and the acrosome reaction in a K+-free (-deficient) medium. However, they are unable to fuse with eggs unless they are exposed to a millimolar concentration of extracellular K+ during or after the acrosome reaction. Apparently, the plasma membrane over the equatorial segment gains the ability to fuse with eggs in the presence of K+ during and/or after the acrosome reaction. Once it becomes fusible, the membrane retains its fusibility even in a K+-deficient medium. Rb+ is almost as effective as K+ in rendering the sperm membrane fusible. Li+ and Cs+ are less effective. The molecular mechanism by which K+ renders acrosome-reacted spermatozoa fusion competent is unknown, but it may involve K+-mediated efflux of H+ from the spermatozoa.     

Effects of polyamines upon capacitation and fertilization in the guinea pig

The effects of polyamines upon the ability of guinea pig spermatozoa to undergo the acrosome reaction and upon sperm-egg fusion were investigated. All of the polyamines (polyarginines and compound 48/80) stimulated the occurrence of the acrosome reaction, although differences in the duration of the preincubation period required were noted. The stimulatory effect of the polyarginines could not be reversed by washing, while that of 48/80 was abolished by its removal prior to induction of the acrosome reaction. Polyarginine-treated spermatozoa were fully capable of fertilizing intact guinea pig eggs. In contrast, 48/80 completely blocked sperm-egg fusion at only 5-10 micrograms/ml. This inhibition was reversible by washing either treated gamete. The ability of polyamines which may also be present at the time and site of fertilization, in vivo, to inhibit or stimulate gamete functions or interactions is discussed.     

Changing localizations of site-specific sperm surface antigens during sea urchin fertilization

Indirect immunofluorescence studies show that monoclonal antibody (mAb) J18/2 binds site-specifically to surface antigens localized over the acrosome and tail regions of mature Strongylocentrotus purpuratus spermatozoa. Within 5 min after induction of the acrosome reaction by exposure to egg jelly or ionophore A23187, these surface antigens become detectable over the lateral region of the head so that the entire surface of the spermatozoon is labeled. Polyspermically fertilized S. purpuratus eggs fixed at varying times after insemination and exposed to mAb J18/2 reveal that these surface antigens are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times, they undergo further dispersal so that by 45 min they are distributed over the entire surface of the egg. These results suggest that the sperm surface components recognized by mAb J18/2 gain the ability to disperse laterally during the acrosome reaction and proceed to do so in the egg plasma membrane after fertilization.     

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.     

Processes controlling sperm-egg fusion

Sperm interaction with the egg envelopes triggers the acrosome reaction. Indeed, sperm-egg fusion is accomplished by the fusion of the acrosomal process (or of the exposed inner acrosomal membrane in mammals) with the egg plasma membrane. Fusion must be preceded by the establishment of molecular contact between the two membranes. It is suggested that, as in the case of artificial phospholipid membranes, the two major obstacles to the establishment of molecular contact are electrostatic repulsion and the hydration barrier. It is argued that morphology of the acrosome is such as to favour the overcoming of such barriers. By analogy with the conditions governing fusion of artificial phospholipid membranes and cell fusion, it is proposed that the following processes play a role in sperm-egg fusion. The large calcium uptake accompanying the acrosome reaction may help fusion either through the known effect of calcium on fusion of phospholipid membranes or by shielding the surface charges of the acrosomal process. Fusogenic proteins at the surface of the acrosomal process are likely to play a role in the fusion of the acrosomal process with the egg plasma membrane. The activation of phospholipases in conjunction with the acrosome reaction may also be instrumental in sperm-egg fusion through the transient production of lysophosphatides. Clearance or translocation of intramembraneous proteins in the egg plasma membrane at the site of contact with the acrosomal process may also be required for fusion. Lastly it is suggested that a translocation or a conformational change of some proteins of the egg plasma membrane, which is required for fusion, may be induced by the depolarization of the egg plasma membrane that follows molecular contact with the acrosomal process.     

Possible involvement of CD81 in acrosome reaction of sperm in mice

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

Immunocytochemical localization of phospholipase A2 in hamster spermatozoa.

Antibodies raised against porcine pancreatic phospholipase A2 (PLA2) react in immunoblottings with both the antigen as well as with one protein band of about 14 kDa from hamster spermatozoa extracts. Immunoblottings of proteins extracted from spermatozoon head and tail fractions also show similar results. Anti-PLA2 purified IgGs were employed for light and electron microscopic immunocytochemistry in order to detect PLA2 in hamster cauda epididymal spermatozoa. When whole mount spread spermatozoa were used under light (employing the PAP complex) or electron microscopy (using anti-rabbit gold conjugated), the acrosomal area of the gametes shows a noticeable labelling; a characteristic which is not observed in samples treated with the pre-immune serum. Immunocytochemistry undertaken in ultrathin sections from spermatozoon samples embedded in Lowicryl, demonstrates that the antigen appears preferentially distributed in the acrosome. Besides, sperm tails showed a scattered distribution of gold granules in the mitochondria of the midpiece. Results suggest that the antibody used recognizes a PLA2 which is preferentially located in the acrosome and mitochondria. On the other hand, the presence of a surface PLA2 in the plasma membrane covering the acrosome is suggested. This surface PLA2 would be probably related to the acrosome reaction phenomenon that occurs in the spermatozoon before penetrating the oocyte.     

Immunocytochemical localization of phospholipase A2 in hamster spermatozoa

Summary Antibodies raised against porcine pancreatic phospholipase A2 (PLA2) react in immunoblottings with both the antigen as well as with one protein band of about 14 kDa from hamster spermatozoa extracts. Immunoblottings of proteins extracted from spermatozoon head and tail fractions also show similar results. Anti-PLA2 purified IgGs were employed for light and electron microscopic immunocytochemistry in order to detect PLA2 in hamster cauda epididymal spermatozoa. When whole mount spread spermatozoa were used under light (employing the PAP complex) or electron microscopy (using anti-rabbit gold conjugated), the acrosomal area of the gametes shows a noticeable labelling; a characteristic which is not observed in samples treated with the pre-immune serum. Immunocytochemistry undertaken in ultrathin sections from spermatozoon samples embedded in Lowicryl, demonstrates that the antigen appears preferentially distributed in the acrosome. Besides, sperm tails showed a scattered distribution of gold granules in the mitochondria of the midpiece. Results suggest that the antibody used recognizes a PLA2 which is preferentially located in the acrosome and mitochondria. On the other hand, the presence of a surface PLA2 in the plasma membrane covering the acrosome is suggested. This surface PLA2 would be probably related to the acrosome reaction phenomenon that occurs in the spermatozoon before penetrating the oocyte.     

Inhibition of in-vitro fertilization of intact and denuded hamster eggs by univalent anti-sperm antibodies.

Univalent (Fab) rabbit anti-hamster sperm antibodies added to an in-vitro fertilization system did not interfere with the sperm acrosome reaction or motility, but inhibited cumulus dispersion by the spermatozoa, sperm binding to and passage through the zona pellucida as well as sperm-egg fusion. Addition of the Fab preparations to the capacitated spermatozoa at various times before or up to 40-45 min after the sperm-egg mixing prevented penetration of spermatozoa through the zona pellucida. Detachment of the spermatozoa already bound as well as those partly inside the zona pellucida was achieved by a late addition of antibodies. In experiments with zona-free hamster eggs, addition of the Fab antibodies to the spermatozoa 10 min to 5 h before the introduction of unfertilized eggs reduced the rate of adhesion and fertilization to very low levels. These antibodies were not absorbed on hamster ovary, liver or kidney and had no direct effect on the fertilizability of zona-intact or zona-free eggs.     

Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction

The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M). When spermatozoa were incubated in a regular medium (containing 2 mM Ca²⁺) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca²⁺-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca²⁺-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca²⁺. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion. Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.     

Behavior of the spermatozoon during sperm-blastomere fusion and its significance for fertilization (Saccoglossus kowalevskii: hemichordata)

Summary Association of spermatozoa with blastomeres removed from the egg envelope was studied in order to investigate sperm membrane behavior under circumstances unlike those attending fertilization of normal envelope-enclosed eggs. Under the altered circumstances all parts of the surface of the sperm cell were afforded the opportunity of meeting blastomere plasma membrane. Nevertheless, in each case the spermatozoon first became activated, an acrosomal tubule formed, and it was this organelle alone which fused and established continuity with the blastomere plasma membrane. This behavior of the spermatozoon with respect to the denuded blastomere parallels the behavior of the sperm cell with respect to the envelope-enclosed egg at fertilization. Since the sperm cell behaves so similarly in two situations which are so different with respect to what the spermatozoon encounters, this behavior is considered to reflect the inherent nature of the spermatozoon, and analysis of this behavior in relation to a blastomere is considered to be valid also in relation to an egg during fertilization. It is concluded that membrane fusion, involving the acrosomal tubule of an activated spermatozoon, is the only means by which gametic union is established and that these are obligatory rather than fortuitous features of sperm-egg association in Saccoglossus. It is suggested that these features are probably obligatory also in other species which exhibit this pattern of sperm-egg association.This investigation was supported by Research Grant HD-00007 from the National Institutes of Health, United States Public Health Service.     

Ultrastructural studies of spermatozoa in three bivalve species with notes on evolution of elongated sperm nucleus in primitive spermatozoa

Sperm ultrastructure and spermiogenesis of the three bivalve species Musculus discors, Nucula sulcata, and Dreissena polymorpha have been studied. During spermatid differentiation in Musculus discors and Nucula sulcata the nucleus attains an elongated rod-like shape. The spermatozoon from Nucula sulcata was found to have a cup-shaped acrosome and five mitochondria surrounding two centrioles in the middle piece. The spermatozoa from Musculus discors has a long complex acrosome. From the distal centriole striated processes extend and attach to the plasma membrane. The spermatozoon of the fresh water species Dreissena polymorpha agrees in all main features with those of other invertebrate groups with external fertilization. It is thus of the primitive type with barrel-shaped nucleus and four to five mitochondria1 spheres in the middle piece. The acrosome is a prominant, complex structure at the apex of the mature spermatozoon. A comparison of sperm ultrastructure among bivalves indicates that there is a certain correlation between the evolution of the elongated sperm nucleus and large, yolk-rich eggs. In species with an elongated sperm nucleus the increased egg size has often led to a lecithotrophic or direct development. The elongated nucleus is a slight modification of the primitive type. There is a great variation in acrosome structure among bivalve spermatozoa, reflecting diverging functional demands at fertilization of the eggs.     

External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes

That a small amount of external calcium ions is requisite for the fertilization by spermatozoa with reacted acrosomes was found by some simple experiments using jelly-treated sperm of the sea urchin, Hemicentrotus pulcherrimus. When eggs were inseminated with the jelly-treated sperm in artificial seawaters containing calcium at various concentrations, the percentage of fertilization decreased concomitant with the reduction in the amount of external calcium ions, 50% at 40 μM calcium and almost 0% at less than 10 μM. On the other hand, it was observed that both the morphology of the reacted acrosome and the binding capacity of the jelly-treated spermatozoa to eggs were not influenced by the calcium deficiency. These results suggest that external calcium ions are indispensable even for the fertilization processes following sperm binding to eggs after the acrosome reaction, such as penetration of reacted spermatozoa through vitelline layer and/or membrane fusion between egg and spermatozoon.     

精子功能相关的蛋白质调控受精过程的研究进展

哺乳动物的受精过程涉及到精子一系列的功能活动,如精子在雌性生殖道的运行、精子的超活化与获能、顶体反应以及精卵融合等。在精子经历的这一系列过程中,精子功能相关的蛋白质发挥着不可或缺的作用,这些蛋白分子的正常与否与雄性个体的繁殖力高低密切相关,因此精子功能相关的蛋白质能够作为评定哺乳动物精液受精能力的生物标记。文章主要对哺乳动物精子功能相关的蛋白质进行了综述,以阐述相关蛋白分子对精子运动活力、精子获能、顶体反应、透明带穿入和精卵融合等方面的重要作用以及这些蛋白分子在家畜遗传改良上的潜在应用。     

REVERSIBLE INHIBITION OF SPERM-EGG FUSION IN THE HAMSTER BY LOW PH*

To study the effect of pH on sperm-egg fusion, hamster eggs were freed from egg investments (cumulus oophorus and zonal pellucida) and inseminated with acrosome-reacted spermatozoa in media with various pH values. One hundred percent of the eggs were penetrated by spermatozoa in phosphate buffered media with pH value higher than 7.1. The rate of penetration declined sharply below pH 7.0 and was 0 at pH 6.1. At pH 6.0–6.1, acrosome-reacted spermatozoa could bind to the egg plasma membrane, but were unable to fuse with it. Similar results were obtained with media buffered with Hepes and Bes. The block of sperm-egg fusion at low pH appeared to be reversible since the eggs that were not penetrated by spermatozoa at low pH were penetrated when they were returned to more alkaline media.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.     

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.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.