Developmental changes in localization of the main ganglioside during sea urchin embryogenesis

Ganglioside M5 (NeuGcalpha2-6Glcbeta1-1'Cer), the main ganglioside in sea urchin and sand dollar eggs, exists mainly in the endoplasmic reticulum and yolk granules in unfertilized eggs. To study the localization of ganglioside M5 after fertilization, early embryos were stained with an anti-ganglioside M5 monoclonal antibody. Using immunofluorescent and immunoelectron microscopy, intense label was observed in the outer surface and cytoplasm of embryos. These results indicate that ganglioside M5 was secreted during embryogenesis and localized in the extracellular matrix (ECM). When living embryos were incubated in sea water containing 7-nitrobenz-2-oxa-1,3-diazole labeled-ganglioside M5 (NBD-M5), the ECM and plasma membrane were strongly stained. Since the localization of NBD-M5 in the ECM was similar to that of extracellular M5, NBD-M5 was likely to be useful to examine the fate of extracellular ganglioside M5. Interestingly, NBD-M5 was incorporated in subcortical vesicles during embryogenesis, suggesting that the extracellular ganglioside M5 is transported into the cytoplasm. When fertilized eggs were incubated with NBD-M5 and tetramethylrhodamine dextran (a marker dye for endocytotic vesicles), colocalization of the dyes was observed in the vesicles. Thus, it was concluded that NBD-M5 in the ECM and/or plasma membrane was internalized in the cells by endocytosis, suggesting that extracellular M5 is transported from the ECM to endocytotic vesicles.     

Distributional Change of Membrane-associated Calcium in Sea Urchin Eggs during Fertilization

The "source and sink" for the intracellular calcium released during fertilization were examined in sea urchin eggs, Hemicentrotus pulcherrimus , with chlortetracycline as a fluorescent chelate probe. In order to distinguish the differential distribution of membrane-associated calcium in various compartments in cytoplasm, eggs were stratified by centrifugation before or after fertilization. Only the layer containing mainly mitochondria exhibited the chlortetracycline-fluorescence in unfertilized eggs. After fertilization, a new fluorescent band emerged in the membrane-rich clear layer of stratified eggs. Chlortetracycline-fluorescence in the clear layer was gradually redistributed surrounding the prophase nucleus and then incorporated into the mitotic apparatus. From these observations, we postulate that the major source(s) of released free calcium ions at fertilization is in the mitochondira layer and membranes in the clear layer are newly activated as the calcium sequestering system after fertilization.     

Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.     

Disulfide formation in bovine zona pellucida glycoproteins during fertilization: evidence for the involvement of cystine cross-linkages in hardening of the zona pellucida

The time for solubilization of the bovine zona pellucida in a hypotonic buffer containing 5% (v/v) beta-mercaptoethanol and 7 mol urea l-1 increased by 10% after fertilization. Coupling with a specific fluorescent thiol probe, monobromobimane (mBBr), was markedly greater in the zona pellucida of ovarian eggs compared with fertilized eggs, indicating that the cysteine residues in the zona pellucida of unfertilized eggs are oxidized to cystines during fertilization. After endo-beta-galactosidase digestion to remove N-acetyllactosamine repeats of the carbohydrate chains, three zona pellucida glycoproteins (ZPA, ZPB and ZPC) coupled with the fluorescent bimane groups were fractionated efficiently by reverse-phase HPLC. Estimation of bimane groups in the three components and SDS-PAGE revealed that intramolecular disulfide bonds in ZPA and intra- and intermolecular disulfide bonds in ZPB were formed during fertilization, but oxidation of cysteine residues in ZPC was low. Specific proteolysis of ZPA during fertilization was also observed. These results indicate that the formation of disulfide linkages together with specific proteolysis result in the construction of a rigid zona pellucida structure, which is responsible for hardening of the zona pellucida.     

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina , the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O₂ consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0–4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [¹⁴C]glucose for 10min, considarable ¹⁴C-radioactivity accumulated in the glycogen fraction. The rate of [¹⁴C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.     

Evidence for cell surface and internal phospholipase activity in ascidian eggs

Upon fertilization, ascidian eggs release a cell surface glycosidase used in the block to polyspermy and undergo cortical contractions resulting from increased intracellular calcium levels. The glycosidase is released by fertilization, calcium ionophores or added phospholipase C (PLC) activity. The PLC inhibitor D609 blocks glycosidase release. Intact Ascidia ceratodes eggs cleave 4-methylumbelliferyl-phospho-choline when it is added to seawater. This yields highly fluorescent 4-methylumbelliferone. Authentic phospholipase C but not phospholipase D can cleave this substrate. Thus, the authors believe that cleavage of the substrate is specific for PLC activity. Eggs incubated in the fluorogenic substrate after having been washed and detergent extracted were not fluorescent. Therefore the substrate failed to enter intact cells. Glycosidase release and PLC activity were stimulated by ionomycin. Octylglucoside or Triton X-100 extracts of ascidian eggs had two forms of phospholipase activity as shown by ion affinity chromatography: PL1 eluting at 0.25 mol/L NaCl and PL2 eluting at 0.6mol/L NaCl. The PL1 appeared to be isolated as a single protein. When surface proteins were labeled with non-penetrating biotin and were subsequently reacted with streptavidin, half of the PLC activity bound. This demonstrates that half the ascidian egg PLC activity is located on the surface of either the egg or follicle cell, and half is located within the egg.     

Localization of an Exogastrula-Inducing Peptide (EGIP) in Embryos of the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing peptides (EGIPs) are present in the unfertilized eggs and embryos of the sea urchin Anthocidaris crassispina . They induce exogastrulation when added exogenously to the embryos. The localization of EGIP-D during embryogenesis has been explored using polyclonal antibodies against EGIP-D. Immunofluorescent staining revealed that EGIP-D is stored in the cytoplasm of immature oocytes and is concentrated into vesicles in unfertilized eggs. At fertilization, the vesicles containing EGIP-D (EGIP-vesicles) migrate to the cortical surface of the zygotes and are distributed in a ring-like pattern at the apical surface of blastomeres, disappearing from basal surfaces and those adjacent to neighboring cells, during development from cleavage stages to larval stages. Mesenchyme cells also contain the vesicles but no such polarized distribution of vesicles is apparent. Acidic vesicles with a similar polarized distribution were examined by staining with acridine orange, which revealed that acidic vesicles were in close proximity to the surface of eggs at fertilization and were then distributed in a ring-like pattern at the apical surface of blastomeres as are the EGIP-vesicles. Furthermore, immunoelectron microscopy revealed that EGIP-D is present in vesicles that are located at the apical surface of blastomeres. The significance of the localized distribution of EGIP-D is discussed in relation to its function.     

Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs

To identify guanine nucleotide binding proteins (G-proteins) in sea urchin eggs and to investigate their role in signal transduction at fertilization, we used cholera toxin (CTX) and pertussis toxin (PTX), which catalyze the specific ADP-ribosylation of G-proteins. Cell surface complex, consisting of plasma membranes and adhering cortical vesicles, was prepared from eggs of Lytechinus variegatus and incubated with 32P-labeled NAD in the presence of CTX or PTX. CTX catalyzed the ADP-ribosylation of a 47-kDa polypeptide, whereas PTX catalyzed the ADP-ribosylation of a 40-kDa polypeptide. Microinjection of approximately 30 micrograms/ml whole CTX or approximately 20 micrograms/ml CTX subunit A into intact eggs caused exocytosis of cortical vesicles. However, if the eggs were first injected with EGTA (0.6-1.4 mM), injection of CTX did not cause exocytosis. Eggs injected with 0.8-2.8 mM cAMP or 1.0-4.0 mM adenosine 3':5'-monophosphotioate cyclic Sp-isomer (cAMP-S), a hydrolysis-resistant analog of cAMP, did not undergo exocytosis. These results suggest that a CTX-sensitive G-protein is involved in regulating Ca2+ release and exocytosis of cortical vesicles in sea urchin eggs.     

Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. II. Bindin induces the fusion of mixed-phase vesicles that contain phosphatidylcholine and phosphatidylserine in vitro

Bindin from sea urchin sperm associates with gel-phase phospholipid bilayers (Glabe, C. G., 1985, J. Cell Biol., 100:794-799). Bindin also interacts with phospholipid vesicles containing both gel-phase and fluid-phase domains and thereby induces their aggregation. Association of bindin with vesicles containing gel-phase domains of dipalmitoylphosphatidylcholine (DPPC) and fluid-phase domains of brain phosphatidylserine (PS) was found to result in the fusion of the vesicles. After incubation with bindin, these mixed-phase vesicles were much larger as determined by gel filtration chromatography and electron microscopic observations of negatively stained samples. The average diameter of the vesicles after incubation was 190 +/- 109 nm compared with 39 +/- 20 nm for vesicles incubated in the absence of bindin. Resonance energy transfer studies also indicated that bindin induces the fusion of vesicle bilayers. Two fluorescent probes (NBD-PE and Rh-PE) were incorporated into the membrane of mixed-phase DPPC:PS vesicles at a density of 0.5 mol%, where efficient energy transfer occurs between the probes. The efficiency of energy transfer was proportional to the concentration of the fluorescence energy acceptor in the bilayer. The fluorescent vesicles were mixed with an excess of unlabeled target vesicles to quantify fusion. After bindin addition, there was a significant decrease in the efficiency of energy transfer compared with controls incubated in the absence of bindin. Although bindin induced the fusion of vesicles in the absence of calcium, the rate of fusion in the presence of 2 mM calcium was three-fourfold higher. In the presence of calcium, approximately half of the vesicles in the population had fused with another vesicle after incubation with bindin for 20 min. Bindin did not induce the fusion of gel-phase DPPC vesicles or mixed-phase vesicles of DPPC and dioleoylphosphatidylcholine, which suggests that the fusagenic activity of bindin requires specific phospholipids. Electron microscopic observations of DPPC:PS vesicles incubated in the presence of bindin suggest that the outer leaflets of bindin-aggregated vesicles are in close apposition. This is believed to be an important initial event for membrane fusion. These observations suggest that bindin may play a dual role in fertilization: Bindin mediates the attachment of sperm to glycoconjugate receptors of the egg surface and may also participate in the fusion of the sperm and egg plasma membranes.     

Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus)

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 10(6)-10(7) spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4',6'-diamidino-2-phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1-3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

Changes in Enzyme Activities and Lipid Content of Echinoderm Egg Membranes, at Maturation and Fertilization

Membrane enriched fractions of eggs show changes in relativelipid and acylglyceride fatty acid content at maturation andfertilization, when many critical membrane changes occur. Manyof the changes in lipids seen at fertilization in sea urchinsare reversed from those seen at maturation in starfish. Maybea special permissive lipid content appears at maturation anddisappears at fertilization. Mycostatin alters the lipid contentof unfertilized eggs, prevents fertilization, blastula cellaggregation and normal development, with decreasing susceptibilitywith age, suggesting altered cell surfaces during development.Membrane enriched fractions also show changes in enzyme specificactivity at fertilization. Attempts were made to localize theseenzymes in various surface components (membrane or associatedcoats or adherent cortex) and several fractions were separated.Egg surfaces are complex and therefore difficult to study. DTTor pronase treatment, for example, changes the activity of G6PDin the surface of whole eggs, with further changes occurringin such eggs following fertilization. Increases in G6PD activityin ghosts and supernate of fertilized eggs appear due to isoenzymechanges instead of one enzyme changing compartment. Isozymepatterns of G6PD extracted from pellets differ from those ofsupernates. Patterns also differ following fertilization orpartial activation by ammonia or calcium ionophore. Some isozymesseem to be breakdown products of larger forms, and can be producedby treatment with papain, DTT, or urea. Isozyme studies supporta dynamic model of the egg surface. Surface changes may occurenzymatically, by insertion of vesicles, or by release of materialfrom vesicles.     

Distribution of fluorescently labeled actin in living sea urchin eggs during early development

Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.     

Sperm Penetration and in vitro Fertilization of the Egg of the House Cricket Acheta domesticus

Summary

A study of sperm penetration of the egg of the house cricket, Acheta domesticus, using a fluorescent microscope technique, showed that sperm penetration of the chorion, prior to fertilization, is not restricted to a specialised area of the egg surface, such as the micropyle. An acrosomal filament is seen on penetrating sperm. Polyspermy (multiple sperm attachment) is seen under normal conditions.

Eggs fertilized in vitro developed to the 4 day (pre-katatrepsis) stage, but did not undergo katatrepsis. Development was confirmed by cytogenetic studies. The percentage of eggs showing cleavage nuclei (i.e. initial development) was 59% after in vitro fertilization and 5% in ovarian eggs incubated in a hypotonic medium.     

Regulation of ooplasmic sodium and potassium activities in developing locust eggs

Ooplasmic activities of potassium and sodium were measured with ion sensitive microelectrodes before and during the period of maximal water uptake which occurs 3–5 days after oviposition for eggs incubated at 37°C. Potassium activity increased from 84 mM in eggs before fertilization at 118 mM in eggs 1 day after fertilization (d1). Sodium activity increased from 8 mM to 29 mM over the same period. These changes exceeded those predicted from the decrease in water content (8%) during the first day after oviposition. Between d1 and d3, potassium and sodium activities decreased to values predicted on the basis of the 88% increase in egg water content. Although water content increased an additional 46% between d3 and d5, ooplasmic sodium activity remained constant at 11 mM and potassium activity increased from 64 mM to 74 mM during this time. Declines in concentrations of sodium and potassium measured in whole eggs by atomic absorption spectrometry mirrored the increase in egg water content. The results suggest that regulation of ooplasmic sodium and potassium activities is accomplished by release of these ions from internal stores, possibly the york spheres. © 1992 Wiley-Liss, Inc.     

Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin

Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.     

Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice.

Purified mouse sperm receptor, a zona pellucida glycoprotein called mZP3, binds to plasma membrane overlying acrosome-intact sperm heads (P.M. Wassarman, 1999, Cell 96, 175-183). Some evidence suggests that mZP3 binds to sp56, a protein reported to be associated peripherally with the plasma membrane of acrosome-intact sperm heads (J.D. Bleil and P.M. Wassarman, 1990, Proc. Natl. Acad. Sci., USA 87, 7215-7219; A. Cheng et al., 1994, J. Cell Biol. 125, 867-878). Here, we report that membrane vesicles prepared from acrosome-intact sperm contain sp56. When these vesicles are incubated with eggs they inhibit binding of sperm to eggs in vitro (ID50 approximately 50-100 microg protein/ml). On the other hand, a monoclonal antibody directed against sp56 relieves the inhibition of binding of sperm to eggs by membrane vesicles. As expected, incubation of intact sperm with the antibody directed against sp56 inhibits binding of the sperm to eggs. Results of immunoprecipitation of sperm extracts incubated with mZP3, by either a polyclonal antibody directed against mZP3 or a monoclonal antibody directed against sp56, suggest that mZP3 is specifically associated with sp56. Results of laser scanning confocal microscopy of fixed sperm probed with antibodies directed against either sp56 or a approximately 155 kDa acrosomal protein, suggest that the two proteins are present in the acrosome, but with different distributions. Furthermore, confocal images of sperm, fixed after exposure to purified mZP3 and probed with antibodies against mZP3 and sp56, reveal overlap between mZP3 and sp56 at the surface of the sperm head. The possible implications of these results are discussed in the context of mammalian fertilization.     

Impaired transport and fertilization in vivo of calcium-treated spermatozoa from +/+ or congenic tw32/+ mice.

To determine whether calcium alters processes important for fertilization in vivo, mouse (+/+) spermatozoa were incubated in medium with 1.0-1.7 mM calcium prior to artificial insemination (AI) into the cervix of hormonally primed females. Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium prior to AI. Spermatozoa from mice of both genotypes incubated in calcium-containing medium fertilized significantly fewer eggs after AI than did spermatozoa incubated in calcium-deficient medium. In addition, calcium-treated spermatozoa from tw32/+ mice fertilized significantly fewer eggs than calcium-treated +/+ spermatozoa. Pretreatment with calcium also reduced the number of spermatozoa in the oviducts 0.5-4.5 h after AI, and the oviducts of females inseminated with calcium-treated spermatozoa from tw32/+ mice contained significantly fewer spermatozoa than those of females inseminated with calcium-treated +/+ spermatozoa. These results suggest that preincubation in millimolar levels of calcium changes the physiology of epididymal spermatozoa in such a way as to impair sperm transport to the oviduct and fertilization in vivo.