P26h and dicarbonyl/L-xylulose reductase are two distinct proteins present in the hamster epididymis

We have previously identified a 34 kDa protein (P34H) on the human sperm surface covering the acrosome. Using the hamster, we have also described a sperm protein, P26h, which is acquired by spermatozoa during epididymal transit. Both P34H and P26h belong to the carbonyl reductase family. Using molecular tools derived from P34H, we searched in the hamster epididymis for another protein related to the human sperm protein. Cloning and sequencing of P31h cDNA revealed 100% homology with the kidney DCXR (Dicarbonyl/L-Xylulose reductase). Northern Blot experiments revealed a single mRNA that was more expressed in the caput than in the corpus and cauda segment of adult epididymides. In situ hybridization was performed on sexually mature hamsters showing that the mRNA was localized in the principal cells throughout the epididymis. Using an anti-P34H antibody we have identified a P34H related protein named P31h (for 31 kDa). This protein showed 2D-electrophoretic behavior different from P26h and was detectable all along the epididymis (caput, corpus, and cauda) by Western Blot analysis. Immunohistochemistry techniques showed that P31h was localized in the perinuclear region of the principal cells of the epididymal epithelium within the three sections, both in sexually mature and immature animals. Results are discussed with regards to the potential function of DCXR in the epididymis.     

Hamster sperm antigen P26h is a phosphatidylinositol‐anchored protein

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the egg's zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High‐salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose‐dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol‐anchored protein and that prostasomes may be implicated in this process. Mol. Reprod. Dev. 52:225–233, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of a hamster sperm 26-kilodalton dehydrogenase/reductase that is exclusively localized to the mitochondria of the flagellum

Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.     

Human DCXR – another ‘moonlighting protein’ involved in sugar metabolism,carbonyl detoxification,cell adhesion and male fertility?

Dicarbonyl/l ‐xylulose reductase (DCXR; SDR20C1), a member of the short‐chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of α‐dicarbonyl compounds and monosaccharides. Its role in the metabolism of l ‐xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as ‘sperm surface protein P34H’, it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non‐catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS‐21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non‐primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm–zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry.     

Activation of mouse sperm phosphatidylinositol-4,5 bisphosphate-phospholipase C by zona pellucida is modulated by tyrosine phosphorylation

Many cellular responses to the occupancy of membrane receptors include the hydrolysis of phosphatidylinositol-4,5 bisphosphate (PIP₂) by phospholipase C (PLC) and the subsequent generation of inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). In the gamete interaction system, sperm respond to binding to the egg's extracellular matrix, the zona pellucida (zp), by exocytosis of the acrosome in a process known as the acrosome reaction (AR). Under physiological conditions, zp binding stimulates ARs only after sperm have undergone a final maturation phase, known as capacitation. One of the zp glycoproteins, ZP3, serves as the ligand for sperm plasma membrane receptors and as the trigger for this regulated exocytosis. Both phosphoinositide-linked and tyrosine kinase-mediated pathways participate in the signalling cascade triggered by sperm-zp interaction. This paper reports that stimulation with solubilized zp increased PIP₂-PLC enzymatic activity from mouse sperm. ZP3 is the zp component responsible for this stimulation. The effect was abolished by tyrphostin, suggesting that zp activation of PLC was mediated by tyrosine phosphorylation and that γ was the PLC isoform involved. We show the presence and distribution of PLCγ1 in mouse sperm. Immunostaining studies indicate that PLCγ1 is restricted to the sperm head. Sperm capacitation induced translocation of PLCγ1 from the soluble to the particulate fraction. These data suggest that PLCγ1 constitutes a component in the cascade that couples sperm binding to the egg's extracellular matrix with acrosomal exocytosis, a regulated secretory response upon which fertilization depends absolutely. © 1996 Wiley-Liss, Inc.     

Bull subfertility is associated with low levels of a sperm membrane antigen

During epididymal transit, mammalian spermatozoa acquire new surface antigens that may participate in gamete interaction. We have previously described a 26 kDa (P26h) epididymal hamster sperm protein that is proposed to be involved in fertilization. We have also identified its human homolog, P34H. Variability in the amount of P34H on spermatozoa from fertile and idiopathic infertile men provides strong evidence that this protein is a potential marker of male fertility. Since these sperm antigens constitute a family of proteins with common antigenicity, we have investigated the presence of a related protein in bovine sperm. In the present study, a P26h antiserum recognized two bull sperm proteins of 21 kDa and 25 kDa (MW) on SDS‐PAGE. We showed that P25b could be extracted with detergent as a surface protein, whereas the P21b was associated with non‐soluble intracellular structures. Sonication of whole sperm cell suspensions and subsequent Percoll gradient centrifugation revealed that P21b may be a flagellar protein whereas the P25b may be located in the head region. Western blot analysis was used to determine the amount of P25b and P21b proteins present on spermatozoa obtained from fertile and subfertile bulls. P21b protein levels were similar in fertile and subfertile bulls, but P25b protein levels were variable. Thus, all bulls with high Non‐Return Rates (fertile bulls) demonstrated high amounts of P25b, whereas P25b levels were decreased in semen from subfertile bulls. We conclude that the protein P25b is a potential fertility marker in the bull and consequently may provide an invaluation tool for the evaluation of bull fertility. Mol. Reprod. Dev. 52:57–65, 1999. © 1999 Wiley‐Liss, Inc.     

Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein

The binding of the spermatozoon to the zona pellucida is a species-specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26-kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species-specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti-P26h antibodies in a sperm-zona pellucida assay using hamster and mouse gametes. Anti-P26h IgGs inhibit, in a dose-dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti-26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm-zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti-P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley-Liss, Inc.     

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Molecular cloning,expression and tissue distribution of hamster diacetyl reductase. Identity with l-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced l-xylulose as well as diacetyl, and slowly oxidized xylitol. The K_m values for l-xylulose and xylitol were similar to those reported for l-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with l-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Sperm Proteome Maturation in the Mouse Epididymis

In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.     

Enzymatic characteristics and subcellular distribution of a short-chain dehydrogenase/reductase family protein, P26h, in hamster testis and epididymis

A hamster sperm 26 kDa protein (P26h) is strikingly homologous with mouse lung carbonyl reductase (MLCR) and is highly expressed in the testis, but its physiological functions in the testis are unknown. We show that recombinant P26h resembles NADP(H)-dependent MLCR in the tetrameric structure, broad substrate specificity, inhibitor sensitivity, and activation by arachidonic acid, but differs in a preference for NAD(H) and high efficiency for the oxidoreduction between 5alpha-androstane-3alpha,17beta-diol (k(cat)/K(M) = 243 s(-1) mM(-1)) and 5alpha-dihydrotestosterone (k(cat)/K(M) = 377 s(-1) mM(-1)). The replacement of Ser38-Leu39-Ile40 in P26h with the corresponding sequence (Thr38-Arg39-Thr40) of MLCR led to a switch in favor of NADP(H) specificity, suggesting the key role of the residues in the coenzyme specificity. While the P26h mRNA was detected only in the testis of the mature hamster tissues, its enzyme activity was found mainly in the mitochondrial fraction of the testis and in the nuclear fraction of the epididymis on subcellular fractionation, in which a mitochondrial enzyme, isocitrate dehydrogenase, exhibited a similar distribution pattern. The enzyme activity of P26h in the two tissue subcellular fractions was effectively solubilized by mixing with 1% Triton X-100 and 0.2 M KCl, and enhanced more than 10-fold. The enzymes purified from the two tissue fractions exhibited almost the same structural and catalytic properties as those of the recombinant P26h. These results suggest that P26h mainly exists as a tetrameric dehydrogenase in mitochondria of testicular cells and plays a role in controlling the intracellular concentration of a potent androgen, 5alpha-dihydrotestosterone, during spermatogenesis, in which it may be incorporated in mitochondrial sheaths of spermatozoa.     

SERPINE2, a serine protease inhibitor extensively expressed in adult male mouse reproductive tissues, may serve as a murine sperm decapacitation factor

SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.     

Epididymal SPAM1 is a marker for sperm maturation in the mouse

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

Fertilization of hamster eggs in vitro at sperm:egg ratios close to unity

Hamster epididymal spermatozoa were washed and preincubated at extremely low sperm concentrations (100/ml or less) in a culture medium containing naturally-occurring sperm motility-stimulating substances. These substances were partially purified "sperm motility factor" (SMF) derived from hamster adrenal glands and catecholamines (epinephrine or isoproterenol). After preincubation for three hours, a small number (5 or less) of washed, cumulus-free hamster eggs was added to each sperm suspension. Many of these eggs were undergoing fertilization when examined two to three hours later. Fertilization was accomplished in vitro at sperm:egg ratios approaching 1:1, a situation comparable to that believed to exist in vivo. It appears that this demonstration will considerably enhance the potential of in vitro fertilization studies for providing useful information on mammalian gamete interactions.