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

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

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.     

Molitity and acrosomal changes in ionophore-treated bovine spermatozoa and their relationship with in vitro penetration of zone-free hamster oocytes

Frozen-thawed spermatozoa from Friesian bulls held at stud in Ireland were used to assess the effect of ionophore on motility, acrosome reaction and heterologous in vitro fertilization. Bovine spermatozoa penetrated zone-free hamster oocytes following treatment with calcium ionophore in the absence of bovine serum albumin (BSA) and in the presence of 10 mM caffeine. Sperm velocity was stimulated in concentrations of caffeine <2.5 mM following dilution with medium containing BSA. Sperm attachment to the plasmalemma showed no association with penetration rates of zona-free hamster oocytes. Penetrated oocytes in regimens with >0.1 mM ionophore did not progress through Meiosis II. Increasing concentrations of ionophore induced the acrosome reaction more rapidly, although this was associated with reduced motility. Hyperactive motility was observed in calcium ionophore-treated spermatozoa which were capable of penetrating zona-free hamster oocytes. Sperm velocity remained unchanged. whereas the track:vector ratio, a measurement of curvilinear movement, was reduced. This work may have important implications for the assessment of bovine fertility and cytogenetic analysis of bovine sperm.     

Monoclonal antibody that recognizes an epitope of the sperm equatorial region and specifically inhibits sperm-oolemma fusion but not binding.

Balb/c mice were immunized with purified hamster sperm heads for induction of antisera and the production of monoclonal antibodies that recognize preferentially the equatorial segment. Twenty-six hybridoma clones secreted monoclonal antibodies with strong affinity for spermatozoa. The supernatants of 16 clones contained antibodies against the equatorial segment, of which 11 were specific to this region. Five supernatants (M1-M5) containing antibodies that bind to various regions of the sperm head were selected and assessed for the ability to inhibit hamster fertilization in vitro using intact and zona-free oocytes. All the supernatants inhibited fertilization compared with the control. However, M1 supernatant specifically inhibited sperm-egg fusion in a concentration-dependent manner, while sperm-oolemma binding and sperm motility remained unaffected. M1 supernatant recognized an epitope that is exclusive to the equatorial segment and expression of this epitope increased after capacitation and the acrosome reaction. Preliminary immunoblot analysis indicated that M1 monoclonal antibody recognized two protein bands of 37.5 and 34.0 kDa.     

Fate of postacrosomal perinuclear theca recognized by monoclonal antibody MN13 after sperm head microinjection and its role in oocyte activation in mice

Monoclonal antibody (mAb) MN13 labels mouse sperm head postacrosomal perinuclear theca (PT), which is possibly involved in oocyte activation during fertilization. The antigenic site is expressed after mild sonication followed by treatment with dithiothreitol (DTT) or heat (45 degrees C), and is visible as a thick band in the postacrosomal region. The presence of protease inhibitors in the sonication medium suppresses the exposure of MN13 epitope (MN13p), suggesting the involvement of a proteolytic reaction in this process. Spermatozoa do not express MN13p after the induction of acrosome exocytosis by Ca(2+) ionophore, zona binding, or during zona penetration, a strategy that ensures safe delivery of postacrosomal PT proteins to oocytes after fusion. MN13 labeling was not detectable during fertilization by zona-free in vitro fertilization, suggesting that the antigenic site does not react with proteolytic enzymes during sperm-oocyte fusion and the antibody does not recognize the nascent epitope. Microinjection of sperm heads prepared by sonication and DTT treatment led to the activation of metaphase II oocytes. The oocyte activating function of such sperm heads was significantly diminished after labeling with MN13 prior to intracytoplasmic sperm injection (ICSI), but labeling with antiequatorin antibody MN9 activated oocytes with a frequency similar to that of unlabeled sperm heads. The sperm heads in inactive oocytes formed premature chromosome condensations (PCCs), which were invested by independent metaphase-like spindles. These observations indicate that the postacrosomal PT recognized by mAb MN13 is involved in oocyte activation. MN13p is dissociated from sperm heads during the early stages of decondensation after ICSI. In activated oocytes, MN13-labeled fine granules were redistributed in the midzone spindle region, whereas in inactive oocytes they formed a ring around the polar regions of the metaphase II and PCC spindles.     

Subcellular localization of beta1,4-galactosyltransferase on bull sperm and its function during sperm-egg interactions

The process of sperm-oocyte recognition is a complex interaction between the plasma membrane of sperm and the extracellular matrix of the oocyte. The best studied mammalian system is the mouse, in which sperm plasma membrane receptors recognize specific oligosaccharides on the egg coat glycoprotein ZP3. A well-defined ZP3 receptor on mouse sperm is beta1,4-galactosyltransferase (GalT). In this study, we investigated the possibility that GalT is present on bull sperm, and that it may participate during bovine sperm-oocyte binding. Using Western immunoblotting, bull sperm were found to have a protein of molecular weight similar to mouse GalT at approximately 60 kDa. Immunogold low voltage scanning electron microscopy reveals that GalT epitopes are confined to the anterior cap of fresh or capacitated bull sperm. To investigate the function of bovine sperm GalT, fresh bull sperm were pretreated with either preimmune or anti-GalT antibody and added to in vitro-matured bovine oocytes. Sperm exposed to preimmune serum fertilized 82.7% (153 of 185) of the oocytes, whereas sperm exposed to anti-GalT antiserum fertilized only 42.3% (202 of 478) of the oocytes. We determined whether the inhibition of fertilization resulted from a direct inhibition of sperm-oocyte binding. The number of sperm bound to eggs was determined by low voltage scanning electron microscopy following pretreatment with preimmune or anti-GalT antibody. An average of 25.3+/-2.2 (mean +/- SEM) sperm bound per half-oocyte when treated with preimmune serum. In contrast, exposure of sperm to anti-GalT antiserum significantly lowered (P<0.001) the frequency of sperm binding to 9.9+/-0.8 bound per half-oocyte. These results show that GalT is present on the anterior cap of the bovine sperm head, where it participates in fertilization by facilitating sperm-oocyte binding. The function of GalT in both the murine and bovine systems suggests that it may serve as a generalized gamete receptor in mammals.     

Characterization of glycolysis and pentose phosphate pathway activity during sperm entry into the mouse oocyte

Studying the events that occur during gamete fusion and sperm decondensation in the oocyte remains difficult because sperm-oocyte fusion and subsequent sperm decondensation represent a short part of the fertilization process, and their exact timing is difficult to determine. There is therefore a need for greater understanding of the events that occur during this period. The main purpose of this study was to examine the metabolic aspects of this time frame by characterizing glucose metabolism (glycolytic and pentose phosphate pathway [PPP] activities) during sperm fusion and decondensation into zona-free oocytes in mice. The metabolism of glucose through both glycolysis and the PPP was measured in ovulated MII oocytes, free of cumulus cells, and the levels of glucose metabolized were found to be low. Upon sperm entry, both glycolytic and PPP activity increased substantially. To determine whether this elevation in glucose metabolism was part of the activation process, the metabolism of parthenogenetically activated oocytes was measured, and no increase in metabolism was observed. The characterization of glucose metabolism during sperm fusion and decondensation into the oocyte, and comparison to parthenogenetically activated oocytes, showed that the fertilizing sperm is responsible for an increase in both glycolytic and PPP activity during fusion and/or decondensation. The significance of this observation during the fertilization process and for the developing embryo is as yet unclear and warrants further investigation.     

Isolation and characterisation of two sperm membrane proteins recognised by sperm-associated antibodies in infertile men

Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.     

Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos

The duration of sperm-oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm-oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1-3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P<0.05), and the highest rate of normal fertilization was observed for 5h culture (73.4%; P<0.05). The sex ratio was significantly (P<0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P<0.05) and decreased the proportion of HOST positive sperm (65.8-48.6%; P<0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5-10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1h (treatment 5) or 5h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P<0.05), and the 1h co-incubation significantly skewed (P<0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P<0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly (P<0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus-sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.     

Effect of low temperatures on in-vitro matured bovine oocytes

This research concerned effects of cooling in vitro matured bovine oocytes on subsequent fertilization and development in vitro. Oocytes were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C or 0 degree C for 5, 10, or 20 min, then fertilized and cultured in vitro for 7 d. The proportion of fertilized oocytes that cleaved and developed to the morula/blastocyst stage was compared between different treatments. Duration of exposure had no effect on the results. Fertilization rate was higher (P < 0.05) for oocytes maintained at 39 degrees C (73.2%) than for oocytes cooled at 20 degrees C (58.6%), 10 degrees C (47.3%), or 0 degree C (36.9%). Cleavage rates were 58.3, 45.3, 15.7 and 7.0% for 39 degrees C, 20 degrees C, 10 degrees C and 0 degree C, respectively (P < 0.05). The lowest development rate to the blastocyst stage was obtained with oocytes cooled to 10 degrees C (0.0%) or 0 degree C (0.9%), followed by 20 degrees C (7.1%) and 39 degrees C (16.5%; P < 0.05). In a second experiment, the zona pellucida was removed after cooling but prior to fertilization (zona-free) from a portion of the in vitro- matured bovine oocytes in each treatment. When sperm penetration rates of zona-free oocytes were compared (percentage of oocytes exhibiting > or = 2 pronuclei), there was no difference (P > 0.05) between oocytes cooled at 0 degree C (59.7%) or 10 degrees C (67.9%). However, penetration rates in these 2 groups were lower (P < 0.05) when compared to zona-free oocytes cooled at 20 degrees C (83.1%) or those maintained at 39 degrees C (83.1%). Zona-free oocytes had higher penetration rates (P < 0.05) when cooled at 0 degree C (59.7%) or 10 degrees C (67.9%) than zona-intact oocytes cooled at 0 degree C (37.3%) or 10 degrees C (47.2%). However, there was no difference in the penetration rate when zona-free and zona-intact oocytes were cooled at 20 degrees C or maintained at 39 degrees C. These data demonstrate that cooling in vitro-matured bovine oocytes decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro. Moreover, at least part of the decrease in fertilization following oocyte cooling is due to effects on the zona pellucida.     

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida

An assay to determine the binding of pig spermatozoa to the zona pellucida (ZP) of pig oocytes was developed using conditions compatible with in-vitro fertilization of pig eggs and with pig sperm penetration of zona-free hamster ova. These conditions were used to define which of the pig oocyte ZP components were involved in sperm binding by a competitive inhibition approach. Assay variables that were optimized included: the method of sperm preparation; sperm preincubation time; sperm-oocyte coincubation time; sperm concentration and temperature; and methods for the separation of free from oocyte-bound spermatozoa. Inclusion of solubilized ZP in the sperm preincubation medium inhibited sperm binding approximately 50%. Both the 55K and 90K components inhibited sperm binding although the 55K component was more effective. The two polypeptides derived from chemical deglycosylation of the 55K families did not inhibit sperm binding. Of several monoclonal antibodies to the ZP components tested, only one directed against the 55K alpha glycoprotein family inhibited sperm binding. Sperm binding to pig oocyte ZP is therefore dependent on the carbohydrate moiety of the glycoproteins and appears to involve more than a single ZP glycoprotein.     

Pentoxifylline improves in vitro fertilization and subsequent development of bovine oocytes

The purpose of this study was to examine whether pentoxifylline improves in vitro fertilization and developmental rates of bovine oocytes. In the first experiment, we examined the effects on the fertilization rate of various concentrations of pentoxifylline (0-7.5 mM) combined with heparin (10 IU/mL). In the second experiment, we examined fertilization cleavage and blastocyst rates after frozen-thawed spermatozoa, obtained from four different bulls, were incubated with heparin (10 IU/mL) with or without caffeine (5 mM) or pentoxifylline (5 mM). In the first experiment, a significantly higher fertilization rate was obtained in heparin containing 5 mM pentoxifylline compared to that in heparin alone or in heparin containing 7.5 mM pentoxifylline (86% vs 60% vs 64%, respectively). The percentage of monospermy in 5 mM pentoxifylline (81%) was significantly higher than in heparin alone (57%). In the second experiment, the interactions among Bulls A, B, C, and D; between treatments (pentoxifylline-with-heparin, caffeine-with-heparin and heparin alone), and between bulls and treatments were analyzed for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts. Among bulls, there was a significant difference in the number of oocytes penetrated (P < 0.01), monospermic oocytes (P < 0.05), cleaved oocytes (P < 0.001), and blastocysts (P < 0.001). Between treatments, there was a significant difference in the number of oocytes penetrated (P < 0.001), monospermic oocytes (P < 0.01) and cleaved oocytes (P < 0.001). Interaction between bulls and treatments was observed for the number of oocytes penetrated (P < 0.05). Individually, for Bulls A, C and D, the numbers of oocytes penetrated and monospermic oocytes in pentoxifylline-with-heparin were significantly higher than in heparin alone. For Bull D, significantly higher results were obtained for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts in pentoxifylline-with-heparin compared to caffeine-with-heparin and heparin alone (P < 0.05). These results suggest that treating sperm with 5 mM pentoxifylline in combination with heparin is effective for bovine in vitro fertilization and it that this treatment is effective even for bulls that produce low fertilization and blastocysts after sperm treatment with caffeine-with-heparin or heparin alone.     

Behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm-zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm-egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 microg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm-oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.     

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.     

Role of guinea-pig sperm autoantigens in sperm binding to the zona pellucida and oocyte penetration

In vitro, binding of acrosome-reacted spermatozoa to the zona pellucida of mature guinea-pig oocytes was inhibited by guinea-pig sperm anti-T IgG and antibodies. Anti-P IgG antibodies prevented oocyte penetration without interfering with sperm-zona binding. The fusion of acrosome-reacted spermatozoa with zona-free oocytes was prevented by anti-T IgG and it was diminished by anti-P IgG. In the same conditions anti-S antibodies had no effect in these in-vitro fertilization events. Immunization of female guinea-pigs with P antigen resulted in a significant decrease of the number of tubal cleaved eggs. T antigens were less clearly implicated in fertilization in vivo. This study provides evidence that well characterized autoantigenic molecules of guinea-pig spermatozoa are involved in fertilization events.