Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation,anti-polyspermy defense,and assisted reproduction

Perinuclear theca (PT) is the cytoskeletal coat of mammalian sperm nucleus that is removed from the sperm head at fertilization. PT harbors the sperm borne, oocyte-activating factor (SOAF), a yet-to-be-characterized substance responsible for triggering the signaling cascade of oocyte activation, thought to be dependent on intra-oocyte calcium release. The present article reviews the current knowledge on the biogenesis and molecular composition of sperm PT. Possible functions of sperm PT during natural and assisted fertilization, and in the initiation of embryonic development are discussed. Furthermore, evidence is provided that SOAF is transferred from the sperm PT to oocyte cytoplasm through the internalization and rapid solubilization of the post-acrosomal PT. It is shown that during natural fertilization the sperm PT dissolves in the oocyte cytoplasm concomitantly with sperm nuclear decondensation and the initiation of pronuclear development. SOAF activity is preserved in the differentially extracted sperm heads only if the integrity of PT is maintained. After intracytoplasmic sperm injection (ICSI), activation occurs only in those oocytes in which the injected spermatozoon displays complete or partial dissolution of PT. In the latter case, the residual PT of the sub-acrosomal and/or post-acrosomal sperm region may persist on the apical surface of the sperm nucleus/male pronucleus and may cause a delay or arrest of zygotic development. We propose that the sperm PT harbors SOAF in the post-acrosomal sheath, as this is the first part of the sperm cytosol to enter the oocyte cytoplasm and its disassembly appears sufficient to initiate the early events of oocyte activation. Dissolution of the sub-acrosomal part of the PT, on the other hand, appears necessary to insure complete DNA decondensation in the internalized sperm nucleus and initiate DNA synthesis of both pronuclei. The release of the SOAF from the sperm head into oocyte cytoplasm at fertilization ultimately leads to the activation of oocyte mechanism including the completion of the meiotic cell cycle, pronuclear development and anti-polyspermy defense.     

Localizations of intracellular calcium and Ca2+-ATPase in hamster spermatogenic cells and spermatozoa

Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37 degrees Celsius for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca(2+)-ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca(2+)-ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca(2+)-ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis.     

A study on the indexing method of the electron backscatter diffraction pattern assisted by the Kikuchi bandwidth

Bitumen is a widely used material employed as a binder in pavement engineering and as a surface sealant in construction. Its surface microstructure and microscale properties have been shown to be temperature-dependent, with effects manifesting themselves on surface composition and texture, including the formation of the visually striking catana ‘bee’-like structures. Despite the importance of a good performance of bitumen in subzero environments (<0°C), the behaviour of bitumen surface texture and composition at cold temperatures, affecting cracking, degradation and road icing, has received practically no attention. In particular, such knowledge is relevant to world regions experiencing long periods of subzero temperatures during the year. Employing advanced atomic force microscopy combined with infrared spectroscopy (AFM-IR) and an environmental chamber, we demonstrate the ability to characterise surface structure and composition with nanoscale precision for a broad range of temperatures. We show that cooling bitumen to subzero temperatures can have several interesting effects on its surface microtexture, nanotexture and composition, especially on its three surface domains, catana, peri and para. We found that the para domain coarsens and extends to form an interfacial transition domain (characterised by increasing surface roughness with peri domain composition) between the para and peri domains. We show that the catana and peri domains have a similar composition, but have different mechanical and chemical properties compared to the para domain. The essential findings of this work improve our understanding of the behaviour of bitumen in subzero environments, aiding us in our quest towards attaining better road and sealant performance.     

Galactosides in glycoconjugates of Xenopus laevis testis shown by lectin histochemistry

The implication of galactosides and other glycoconjugates on spermatogenesis has been previously reported. Glycans show such a complex structure that it makes them very difficult to analyze. Lectin histochemistry is a helpful tool for the study of glycan composition. Lectin histochemistry can be combined with deglycosylation pretreatments to explore the glycan type to which carbohydrates are linked. The aim of the present work was the localization of galactose (Gal)-containing glycoconjugates in the testis of Xenopus laevis, a species widely used in cell, molecular and developmental biology. Gal specific lectins BPL, PNA, BSI-B4, MAA-I, and RCA-I, were used in combination with deglycosylation procedures. Except for BPL, all the lectins were reactive for several testicular tissues. Some of the lectins showed a different reactivity depending on the stage of spermatogenic development, suggesting that cell glycoconjugates are modified during spermatogenesis. The surface of primary spermatocytes was strongly labeled with lectins from peanut (PNA) and castor bean (RCA-I), which agrees with the presence of galactosyl-glycolipids reported in the cell membrane of mammalian spermatocytes. The acrosome was unexpectedly negative to all the lectins tested, whereas the acrosome of mammals and other amphibians has shown a high expression of glycoconjugates, including galactosides. The results obtained after deglycosylation by β-elimination or incubation with PNGase F, which respectively remove O- and N-linked oligosaccharides, allowed us to elucidate the nature of the labeled glycans. The strong expression of galactosides at the cell surface of spermatocytes and spermatids suggests the involvement of these glycans in cell adhesion mechanisms during spermatogenesis.     

Contribution of epididymal secretory proteins for spermatozoa maturation

The final stages of sperm differentiation occur outside the gonad and are not under the genomic control of germ cells. Only sequential interactions with the medium surrounding the sperm are believed to induce the final steps of spermatogenesis. The epididymis, a long tubule with very active secretory and reabsorption functions, is able to create sequential changes in the composition of luminal fluid throughout its length. The chronologies of the changes, which occur on/in the sperm with those in their surrounding environment, are described. Correlations between the highly regionalized epididymal activities and sperm characteristics linked to their survival and fertility potential are presented in this review.     

Fertilization-Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self-assembly of an extracellular matrix

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self-assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick-frozen, deep-etched, and rotaryshadowed eggs, are illustrated.     

Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation

The mammalian acrosome is a highly specialized organelle overlying the anterior part of the sperm nucleus and contains a variety of proteins, including hydrolytic enzymes and matrix molecules. Functionally, the anterior acrosome is involved in the acrosome reaction or sperm-zona pellucida interaction, while the equatorial segment (posterior acrosome) is involved in sperm-egg fusion. The acrosome is formed during spermiogenesis, during which associated molecules are transported from the Golgi apparatus and organized. Many of the molecules thus arranged gradually become compartmentalized during sperm passage through the epididymis. Some of them are further modified during the fertilization process. The findings indicate that acrosomal molecules are not only restricted to a specific region (domain) of the acrosome but also undergo ongoing relocation in a stage-specific manner during sperm maturation in the testis and epididymis. Such maturation-associated modifications are considered essential for sperm molecules to reach the correct or final site before fertilization. This review focuses on the organization and modifications of the acrosomal molecules as well as their compartmentalization within the acrosome.     

From fertilization to cancer: the role of centrosomes in the union and separation of genomic material

Centrosomes play crucial roles in the union of sperm and egg nuclei during fertilization and in the equal separation of genomic material during cell division. While many studies in recent years have focused on the molecular composition of centrosomes, this article focuses on the structural behavior of centrosomes and on factors that play a role in centrosome functions under normal, artificially altered, and abnormal conditions. We review here how studies in the classic sea urchin egg model have contributed to our knowledge on the centrosome cycle within the cell cycle, on compaction and decompaction of centrosomal material, and on the contributions of maternal and paternal centrosomes during fertilization. Centrosome material is activated in unfertilized eggs by increasing pH with ammonium and by increasing calcium with the ionophore A23187, which are conditions that are normally induced by sperm. D(2)O and taxol also induce centrosome aggregation in the unfertilized egg. Maternal and paternal centrosome material both contribute to the formation of a functional centrosome but the formation of a bipolar centrosome requires material from the paternal centrosome. Fertilization of taxol-treated eggs reveals that the male centrosome possesses the capability to attract maternal centrosome material. When pronuclear fusion of the male and female pronuclei is inhibited with agents such as the disulfide reducing agent dithiothreitol (DTT) a bipolar mitotic apparatus is formed from the paternal centrosome. Furthermore, one centrosome of the bipolar mitotic apparatus is capable of organizing an additional half spindle that attaches to the female pronucleus indicating a functional and perhaps structural connection between centrosomes and chromatin. Sea urchin eggs are also useful to study centrosome abnormalities and consequences for the cell cycle. While classic studies by Theodor Boveri have shown that dispermic fertilization will result in abnormal cell division because of multiple centrosomes contributed by sperm, abnormal cell division can also be induced by chemical alterations of centrosomes. Compaction and decompaction of centrosome structure is studied using chloral hydrate or the chaotropic agent formamide, which reveals that centrosomes can be chemically altered to produce mono- or multipolar abnormal mitosis and unequal distribution of genomic material upon release from formamide. The patterns of abnormal centrosome reformations after recovery from formamide treatment resemble those seen in cancer cells which argues that structural defects of centrosomes can account for the formation of abnormal mitosis and multipolar cells frequently observed in cancer. In summary, the sea urchin model has been most useful to gain information on the role of centrosomes during fertilization and cell division as well as on adverse conditions that play a role in centrosome dysfunctions and in disease.     

Perivitelline space: does it play a role in blocking polyspermy in mammals?

The perivitelline space of mammalian oocytes changes in size and composition during preimplantation development. Often overlooked in the past, this space contains a hyaluronan-rich extracellular matrix prior to fertilization and a cortical granule envelope following release of the cortical granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is well developed in some species such as opossums and humans but is scant in rodents including the hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit membrane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be negotiated by the fertilizing sperm. Following fertilization, the cortical granule envelope forms as the cortical granules disperse, thereby causing the PVS to increase significantly in size. Calcium is important in the dispersion of the cortical granules following exocytosis. Once formed, the cortical granule envelope in some species is about the same thickness as the zona pellucida, but it is not readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS until the time of blastocyst hatching. Although little work has been done on the functions of the cortical granule envelope, several studies are consistent with the idea that it plays a role in blocking polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in human smokers have not been characterized, but could be addressed in human in vitro fertilization labs.     

Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis

The acrosome of the mature spermatozoon functions as a regulated secretory vesicle which performs several critical functions in mammalian fertilization. Acrosome assembly occurs throughout spermiogenesis and continues during posttesticular sperm maturation in the epididymis, resulting in a structurally polarized membrane-bounded organelle that contains an assortment of hydrolases and a stable infrastructure termed the acrosomal matrix. The role of stable acrosomal matrix assemblies in acrosomal biogenesis and function are poorly understood. This article presents ultrastructural, immunocytochemical, and biochemical data on the remodeling of the hamster acrosomal matrix during spermiogenesis and posttesticular sperm maturation in the epididymis. Specific posttranslational modifications of the major acrosomal matrix protein are evident in late, step 16, spermatids and matrix protein processing continues within specific acrosomal subdomains of caput epididymal spermatozoa. At the completion of sperm maturation, the acrosomal matrix consists of two structurally distinct domains which are adherent to the outer acrosomal membrane and exhibit a localized distribution pattern. Coincident with acrosomal matrix differentiation, a paracrystalline cytoskeletal complex is assembled onto the outer acrosomal membrane of epididymal spermatozoa. This cytoskeletal network appears to establish transmembrane structural interactions with the acrosomal matrix and may maintain attachment of the acrosomal cap to the sperm head during the early steps of the acrosome reaction.     

Ubiquitin-dependent proteolysis in mammalian spermatogenesis,fertilization, and sperm quality control: killing three birds with one stone

Ubiquitin and ubiquitin-like proteins control the degradation of substrates as diverse as cyclins, viral envelope proteins, plasma membrane receptors, and mRNAs. The ubiquitinated substrates are targeted towards the lysosomal or proteasomal degradation sites. The number and position of ubiquitin molecules bound to substrates' lysine residues and the number and position of ubiquitin molecules in polyubiquitin chains determine the astonishing substrate specificity of ubiquitin-mediated proteolysis. Ubiquitin is likely to be expressed in mammalian gametes and embryos at any given developmental step, but the information on ubiquitin dependence of gametogenesis and fertilization is sketchy. Ubiquitin ligases E1, E2, E3, and UBC4 are active in the testis. Ubiquitin and proteasomal subunits can be detected in the human sperm centrosome that undergoes dramatic reduction during spermatid elongation. Spermatid histones are ubiquitinated as they are being transiently replaced by transitional proteins and permanently by protamines. Ubiquitin tagging of the sperm mitochondrial membranes may serve as a death sentence for paternal mitochondria at fertilization, thus promoting the maternal inheritance of mitochondrial DNA (mtDNA) in mammals. The defective spermatozoa become surface-ubiquitinated during sperm descent down the epididymis. Finally, new evidence suggests the involvement of ubiquitin-proteasome pathway in the zona penetration by the acrosome-reacted spermatozoon. Such differential patterns of ubiquitination in the testis and epididymis, and inside the egg, may be necessary for reproductive success in humans and animals. Deciphering and eventually manipulating the ubiquitin-dependent proteolysis in the reproductive system could allow us to redirect the mode of mtDNA inheritance after cloning and ooplasmic transplantation, provide germ line therapy in some cases of male infertility, develop new contraceptives, manage polyspermia during in vitro fertilization, and establish objective markers for infertility diagnostics, semen evaluation, and prediction of future fertility.     

Polyspermy prevention in marine invertebrates

In marine invertebrates, as in most other organisms, normal development requires that only one sperm nucleus joins with the egg nucleus at fertilization. The principal mechanisms employed are (1) prevention of sperm-egg plasma membrane fusion and (2) modifications of the egg extracellular coat to prevent sperm binding and/or penetration. In a third strategy, fertilization is polyspermic, but only one sperm nucleus fuses with the egg nucleus. Other factors such as gamete density during spawning, chemotaxis, and localized sites for sperm entry may also affect the numbers of sperm reaching the egg.     

Egg envelopes of Armadillidium vulgare (Latreille, 1804) (Crustacea,Isopoda Oniscidea): Ultrastructure and lectins binding

The ultrastructural study carried out on (a) oocytes of Armadillidium vulgare during vitellogenesis, (b) mature eggs taken from the ovaries during the parturial moult of the posterior half of the body, and (c) fertilized eggs collected within a few hours of their release into the brood pouch, has clearly demonstrated that before the fertilization the chorion is the only envelope present in the egg of oniscidean isopods. In the mature eggs, the chorion appears as a uniformly electron‐dense lamina, about 0.4–0.5 µm thick, which does not show any specialized area. A second envelope, described by other authors as vitelline envelope, is formed above the oolemma only right after fertilization and appears separated from the chorion by a space full of liquid. The ways in which the genesis of this envelope is realized are not yet clear; it could be interpreted rather as a fertilization membrane. The investigations carried out with the aid of a battery of FITC‐lectins have highlighted the presence at the chorion surface of unfertilized eggs of various saccharide residues distributed in uniform way. No significant change was observed in the pattern of lectins binding to the chorion of eggs taken from the brood pouch, thus demonstrating how, after the fertilization, no significant rearrangement in the distribution of saccharide residues present on the egg surface occurs in A. vulgare. The ways in which, therefore, the recognition, the binding and the entry of the peculiar sperm of oniscidean isopods into the egg occur, still remain all to be deciphered. Microsc. Res. Tech. 79:792–798, 2016. © 2016 Wiley Periodicals, Inc.     

Glycan modifying enzymes in luminal fluid of rat epididymis: are they involved in altering sperm surface glycoproteins during maturation?

Testicular spermatozoa undergo morphological and biochemical alterations, collectively termed epididymal maturation, in the intraluminal environment of epididymis. As a result of these modifications, the spermatozoon becomes a motile and functionally competent cell capable of undergoing capacitation and binding to the zona pellucida, the extracellular coat that surrounds the mammalian oocyte. Although details of all the changes are not fully known, several studies provide evidence suggesting that sperm plasma membrane undergoes extensive biochemical changes, including organization and modification of surface glycoproteins as spermatozoa transit from the proximal to the distal epididymis. In this article, I have attempted to summarize results with two sets of glycoprotein (glycan)-modifying enzymes, namely, glycohydrolases (hydrolytic enzymes) and glycosyltransferases (synthetic enzymes) present in the epididymal luminal fluid (LF). The in vitro experimental approaches described in this report demonstrate that: 1) a PNA-positive glycoprotein(s) (containing O-linked glycan) of 135-150 kDa subunit molecular mass which is present on the surface of caput (but not the cauda) spermatozoa can be degalactosylated by the enzymatic digestion with LF beta-D-galactosidase; and 2) an N-linked glycan chain(s) which is present on a sperm surface glycoprotein (apparent subunit molecular mass of 86 kDa) can be fucosylated in vitro when distal caput sperm (or sperm plasma membrane-rich fractions) are incubated in the presence of a nucleotide sugar (GDP[(14)C]fucose). Combined, these results strongly suggest a role for the glycan-modifying enzymes in degalactosylation and fucosylation of sperm surface glycoproteins during epididymal transit.     

Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.     

Junctional complexes and cell polarity in the urinary tubule

In this review, we demonstrate how differentiated membrane domains can be detected in epithelial cells using conventional light and electron microscopy, freeze-fracture electron microscopy and the immunoand cytochemical detection of membrane components. Using specific examples from the kidney, we show how the polarized insertion of these components into either apical or basolateral plasma membrane regions on either side of the tight junction barrier is related to specific functions of principal and intercalated cells in the collecting duct. In addition, distinct basal and lateral membrane domains have been revealed in some cells that are maintained in the absence of a tight junctional barrier in the plane of the membrane. This suggests that other factors, possibly related to cytoskeletal elements, may be involved in the functional segregation of these membrane areas. We propose that epithelial cell plasma membranes should be subdivided into apical, lateral and basal regions, and that the term “basolateral” may be an oversimplification.     

Synaptic membrane proteins form stable microdomains in early endosomes

In the plasma membrane, membrane proteins are frequently organized in microdomains that are stabilized both by protein‐protein and protein‐lipid interactions, with the membrane lipid cholesterol being instrumental for microdomain stability. However, it is unclear whether such microdomains persist during endocytotic membrane trafficking. We used stimulated emission‐depletion microscopy to investigate the domain structure of the endosomes. We developed a semiautomatic method for counting the individual domains, an approach that we have validated by immunoelectron microscopy. We found that in endosomes derived from neuroendocrine PC12 cells synaptophysin and several SNARE proteins are organized in microdomains. Cholesterol depletion by methyl‐β‐cyclodextrin disintegrates most of the domains. Interestingly, no change in the frequency of microdomains was observed when endosomes were fused with protein‐free liposomes of similar size (in what constitutes a novel approach in modifying acutely the lipid composition of organelles), regardless of whether the membrane lipid composition of the liposomes was similar or very different from that of the endosomes. Similarly, Rab depletion from the endosome membranes left the domain structure unaffected. Furthermore, labeled exogenous protein, introduced into endosomes by liposome fusion, equilibrated with the corresponding microdomains. We conclude that synaptic membrane proteins are organized in stable but dynamic clusters within endosomes, which are likely to persist during membrane recycling. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

TNF alpha and the TNF receptor superfamily: structure-function relationship(s)

Tumour Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis. The protein is also important for resistance to infection and cancers. TNF alpha exerts many of its effects by binding, as a trimer, to either a 55 kDa cell membrane receptor termed TNFR-1 or a 75 kDa cell membrane receptor termed TNFR-2. Both these receptors belong to the so-called TNF receptor superfamily. The superfamily includes FAS, CD40, CD27, and RANK. The defining trait of these receptors is an extra cellular domain comprised of two to six repeats of cysteine rich motifs. Additionally, a number of structurally related "decoy receptors" exist that act to sequester TNF molecules, thereby rescuing cells from apoptosis. The crystal structures of TNF alpha, TNF beta, the extracellular domain of TNFR-1 (denoted sTNFR-1), and the TNF beta sTNFR-1 complex have been defined by crystallography. This article will review the structure/function relationships of the TNF alpha and the TNF receptor superfamily. It will also discuss insights as to how structural features play a role in the pleiotropic effects of TNF alpha.     

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.