Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

Relationship between the association of rat epididymal protein "DE" with spermatozoa and the behavior and function of the protein

Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

Immunochemical localization of epididymal protein DE on rat spermatozoa: Its fate after induced acrosome reaction

The localization of rat epididymal protein DE on cauda epididymis spermatozoa was studied with a specific antibody and the peroxidase antiperoxidase (PAP) immunocyto-chemical reaction. At the light microscopic level, all spermatozoa appeared to be labeled over the dorsal portion of the head, whereas tails were negative. This observation was confirmed using scanning electron microscopy. A large number of particles were seen on the external surface of the plasma membrane covering the acrosomal region and a smaller number on the postacrosomal portion. Flagella appeared free of particles. Sperm suspensions were incubated in conditions that induce capacitation and the acrosome reaction, and, in this instance, the permanence of protein DE on the vesicles and the postacrosomal region of the membrane were observed. The localization of this epididymal protein on the sperm surface is compatible with a role in the gamete interaction process.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.     

Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa.

The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.     

Effects of in vitro incubation on a zona binding site found on murine spermatozoa

Murine cauda epididymal sperm possess a site, the acceptor, on the plasma membrane over the apical cap region of the acrosome which recognizes both a proteinase inhibitor of seminal vesicle origin and homologous zonae. The acceptor site may participate in both capacitation and zona binding. This presentation explores the effect of in vitro incubation in a medium known to induce capacitation on the binding capabilities of this site. Approximately 80% of fresh cauda epididymal sperm will bind the seminal inhibitor in vitro. Incubating sperm, pretreated with inhibitor for 2 hr in a medium (M199-M) known to support capacitation, reduces by 60% the number of sperm showing evidence of the inhibitor. No such decrease is seen when sperm are incubated in a medium (M199) that does not support capacitation. During the 2-hr incubation in either medium, 60-70% of the sperm retain two diverse components on the plasma membrane over the acrosome: a receptor for the Fc portion of IgG and an epitope recognized by a monoclonal antibody to the acceptor site. These observations suggest that the plasma membrane in the acrosome region of the cell remains structurally intact during incubation. Furthermore, sperm retain the ability to bind the seminal inhibitor during incubation. After a 2-hr incubation in M199-M, sperm pretreated with heat-solubilized zonae no longer bind the inhibitor. These sperm, however, retain the plasma membrane over the acrosomal cap region. When the sperm are incubated in M199, no decrease in inhibitor binding due to zona treatment is noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro

Glycosaminoglycans (GAGs) were reported to induce acrosome reactions (AR) in epididymal and ejaculated bovine sperm (4,5). The GAGs chondroitin sulfate A (CS-A) and heparin were tested on ejaculated bovine sperm for their ability to increase in vitro fertilization (IVF) frequencies. Regardless of treatment, a sperm-egg incubation time of 18 hr was sufficient to achieve maximal rates of fertilization. The IVF frequency of sperm incubated 6 hr with 10 mug/ml heparin (116 173 , 67%) was increased (P<0.05) above control levels (56 181 , 31%); however, 10 mug/ml CS-A (56 164 , 34%) was without effect (P>0.05). In contrast to previous reports, CS-A did not (P>0.05) induce AR in ejaculated (9.5-hr incubation) or epididymal sperm (22.5-hr incubation). Linear increases in fertilization frequency (40% to 81%; P=0.001) and AR (9% to 32%; P相似文献   

Release of acrosomal enzymes following in vitro capacitation of ejaculated rabbit spermatozoa

Significant release of the acrosomal enzymes arylsulfatase, β-N-acetylhexosaminidase and hyaluronidase was observed following the treatment of ejaculated rabbit spermatozoa for 12 hours in 20% rabbit serum for inducing in vitro capacitation, and these sperm were capable of in vivo fertilization; however, the treatment of sperm for 15 minutes in high ionic strength (380 mOsm/kg) or low ionic strength medium (305 mOsm/kg) for in vitro capacitation did not result in any significant release of the above enzymes nor were the sperm capable of in vivo fertilization. Serum-treated spermatozoa remained significantly motile following the 12 hour treatment, 51% underwent the acrosome reaction and were capable of fertilizing 66% of the ova in vivo. Identical serum treatment of lysosomes from rabbit liver resulted in a comparable release of the lysosomal enzymes. Serum treatment for in vitro capacitation resulted in vesiculation of the anterior margin of half the spermatozoa, but left their inner acrosomal membranes and equatorial segments intact. A biochemical relationship between the release of acrosomal enzymes and capacitation is suggested.     

Semen-coagulating protein, SVS2, in mouse seminal plasma controls sperm fertility

Mammalian seminal plasma is known to contain a decapacitation factor(s) that prevents capacitation and thus, the fertility of sperm. This phenomenon has been observed in experiments conducted in vitro that assessed the inhibition of epididymal sperm fertility by seminal plasma or by the purified decapacitation factor. However, the phenomenon of decapacitation has not yet been characterized in vivo. In the present study, we demonstrate that seminal vesicle protein secretion 2 (SVS2), which is a 40-kDa basic protein and a major component of the copulatory plug, enters the uterus and interacts with ejaculated sperm heads after copulation. The SVS2-binding region of sperm changed from the postacrosomal region to the equatorial segment, while the sperm migrated through the uterus and finally disappeared in the oviduct. Furthermore, SVS2 reduced the fertility of epididymal sperm. The sperm treated with SVS2 decreased the percentage of fertilized oocytes from 60% to 10%. The capacitation state was assessed by protein tyrosine phosphorylation and the comprehensiveness of the acrosome reaction. SVS2 functioned to maintain sperm in the uncapacitated state and to reverse capacitated sperm to the uncapacitated state. We found that the fertility of ejaculated sperm is associated with SVS2 distribution in the female reproductive tract. These results indicate that SVS2 functions as a decapacitation factor for mouse sperm.     

Rabbit epididymal secretory proteins. II. Immunolocalization and sperm association of REP38

Polyclonal antibody was used to partially characterize REP38, a major rabbit epididymal secretory protein. Western blot analyses and immunohistochemistry indicated that REP38 is only expressed in regions 5 and 6 of the epididymis (corpus epididy-midis) and is localized in the supranuclear region and microvilli of the principal cells in these regions. It was not expressed in other tissues of the body. In region 8 (cauda epididymidis), REP38 was detected in the luminal border and cytoplasm of scattered principal cells, indicating that it may be reabsorbed in this region. This protein accumulated on the sperm plasma membrane downstream of region 5 and was localized predominantly over the acrosomal and postacrosomal regions of the head and the middle piece. Although tightly bound to epididymal sperm, REP38 migrated to the equatorial segment under conditions in vivo that would promote capacitation. When tested in vitro, anti-REP38 IgG reduced the percentage of ova fertilized in a concentration-dependent manner, apparently by blocking sperm-egg fusion.     

Sperm surface changes during the acrosome reaction as observed by freeze-fracture

The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze-fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle-free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea-pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.     

Bicarbonate is required for migration of sperm epididymal protein DE (CRISP-1) to the equatorial segment and expression of rat sperm fusion ability

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.     

In vitro capacitation of goat spermatozoa

Goat epididymal and ejaculated spermatozoa were incubated in Krebs-Ringer bicarbonate buffer containing pyruvate and lactate as energy source. A 3 hr incubation for epididymal and 4 hr for ejaculated spermatozoa was required for the capacitation and acrosome reaction to take place. Calcium is an essential requirement which was needed for motility maintenance/activation and for the initiation of acrosome reaction. A 2-fold increase in cAMP content was measured over 3 hr period of incubation of goat epididymal spermatozoa which was not seen when calcium ions were either omitted or chelated with EGTA. There is thus a definite involvement of Ca2+ ions and cAMP in capacitation and acrosome reaction of goat spermatozoa.     

Effects of angiotensin II on the acrosome reaction in equine spermatozoa

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.     

Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.     

Quantitative changes of Ricinus communis agglutinin I and Helix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit.

During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1 M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections using Ricinus communis agglutinin I (RCA I) or Helix pomatia lectin (HPL) to detect D-galactose- and N-acetyl-D-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per microns 2 of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p less than 0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p less than 0.01). No change was found between the initial segment and caput epididymidis. Controls showed a virtual absence of labeling. These results suggest that in addition to a multitude of changes occurring to spermatozoa during epididymal transit, there are also significant quantitative changes in the glycoconjugate content within the acrosome.     

The effect of moderate epididymal aging on the kinetics of the acrosome reaction and fertilizing ability of hamster spermatozoa

Spermatozoa within one sample do not appear to complete the capacitation process at the same rate. This could reflect a population composed of cells of different absolute age and thus of different maturational state, or the existence of inherent programmed differences among subpopulations of spermatozoa in regard to their capacitation potential. To explore this question, hamster spermatozoa aged within the range of their viable life in the cauda epididymidis were assessed, in comparison with controls, for onset of the acrosome reaction (AR) and hyperactivation as a function of capacitation time in vitro. Aging spermatozoa in the cauda epididymidis beyond the normal period did not synchronize onset of the AR among them. Such aging merely suppressed the ability of a major (late-reacting) segment of the motile population to react and, coincidentally, reduced their fertilization performance in vivo. Experiments using the calcium ionophore A 23187 indicate that these subtle epididymal aging effects reflected in the function--but not motility--of spermatozoa probably are exerted on the regulation of ion channels in the plasmalemma. We conclude that the asynchrony of onset of the AR and hyperactivation during capacitation probably reflects inherent or programmed differences among spermatozoa. The fact that moderate aging tends to suppress the ability of many motile spermatozoa to react and to fertilize in vivo suggests that an optimal functional state can persist in the cauda for not much longer than spermatozoa remain there before normal replacement by younger cells.     

Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment

Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.