Effects of semen plasma from different fractions of individual ejaculates on IVF in pigs

We applied IVM/IVF techniques to investigate effects of preincubation of sperm with different fractions of semen plasma harvested from fresh ejaculates on in vitro penetration and fertilization of in vitro matured oocytes. Three fractions of semen plasma were separated from the complete ejaculate of three Landrace boars and used to coincubate sperm obtained from the first sperm-rich fraction of the same ejaculates. After 14 to 16 h coincubation at room temperature, sperm were preincubated in capacitation medium and then inseminated into fertilization medium containing porcine oocytes matured in vitro. The semen plasma used for coincubation affected penetration rate (P < 0.001); Sperm coincubated with Fraction 1 semen plasma had a higher penetration rate compared with sperm coincubated with Fraction 2 (P < 0.05), but not with Fraction 3. Boar affected male pronucleus formation rates after insemination (P < 0.05), but no difference among boars was found in monospermy rate, average number of sperm penetrating into each fertilized oocyte, or the average number of sperm attached. No boar by fraction interaction was found for any parameters studied.     

Effect of short periods of sperm-oocyte coincubation during in vitro fertilization on embryo development in pigs

The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus-oocyte complexes from five replicates were matured in vitro and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6h. After 6h, the oocytes from each group were cultured in embryo culture medium for another 6h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34.1 +/- 1.7, 46.8 +/- 2.8, 62.8 +/- 3.8 and 139.5 +/- 6.1 for 10, 30, 60 min and 6h, respectively, P < 0.02), the penetration rate was not significantly different among groups (61.3-68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0.04) as the coincubation time was increased (26.6 +/- 2.9%, 29.0 +/- 4.4%, 39.5 +/- 6.2%, and 49.3 +/- 3.0% for 10, 30, 60 min and 6h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5-25.5%). These results demonstrate that although a sperm-oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm-oocyte coincubation does not improve the efficiency of in vitro pig embryo production.     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

Effects of extracellular potassium concentrations on acrosome reaction and polyspermy during in vitro fertilization and subsequent development in vitro in the pig

Potassium (K(+)) concentration in the mammalian oviduct and uterus is particularly interesting due to its unusually high concentration (12 to 25 mM) compared with that in the blood stream (3 to 6 mM). In this study we examined the effects of various K(+) concentrations in the fertilization medium on polyspermy and subsequent in vitro development of porcine oocytes. In the absence of K(+) in the fertilization medium, sperm penetration was not observed. The incidence of polyspermy was significantly higher in the fertilization medium that contained 6 or 12 mM K(+) as compared with 3 mM K(+). The mean number of sperm penetrated in oocytes in medium with 6 and 12 mM K(+) was higher than in medium with 3 mM K(+). The addition of 3, 6 or 12 mM K(+) to the fertilization medium did not significantly affect the proportion of zygotes that developed to the blastocyst stage (14.1, 12.4 and 15.0%, respectively). Chlorotetracycline (CTC) analysis was used to determine the capacitation and acrosome reaction of spermatozoa incubated for 3 h with various concentrations of K(+). The number of acrosome reacted spermatozoa decreased with increasing K(+) concentration. These results suggest that extracellular K(+) in the fertilization medium affects sperm acrosome reaction which may be related to the sperm penetration.     

In vitro fertilization of pig oocytes after different coincubation intervals

The effects of gamete coincubation time on porcine in vitro fertilization for 4, 6 or 8 hours in Trial 1 or for 1, 2, 3 or 4 hours in Trial 2 were studied. The 876 oocytes were recovered from oviducts of prepubertal gilts. Ovulation was induced. Excessive spermatozoa attached to zona pellucida were removed by pippeting after coincubation (1 to 8 hours). Optimum results were obtained after 3 to 4 hours of coincubation, when 23 to 29% of the oocytes were fertilized by a single spermatozoa. Shorter intervals of coincubation were characterized by reduced percentages of fertilized oocytes and longer intervals of coincubation by increased rates of polyspermic fertilization. The employed sperm concentration was deliberately high (2 x 10(6) sperm/ml). The results show that a coincubation time of 4 hours may be optimal for pig in vitro fertilization. Under these assay conditions, modification of other factors such as sperm concentration, medium volume and physiological environment may increase the percentage of monospermic fertilization.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Birth of piglets by in vitro fertilization of zona-free porcine oocytes

The present experiments were conducted to optimize in vitro fertilization conditions for zona pellucida-free (ZP-free) oocytes and their subsequent development. The results demonstrated that: (1) maximal fertilization efficiency was achieved at 200 spermatozoa per ZP-free oocyte. At this sperm dose, there were no significant differences in penetration rates and polyspermy rates from controls (zona-intact oocytes with 1000 spermatozoa/oocyte), indicating that ZPs of in vitro matured pig oocytes failed to block polyspermy during in vitro fertilization. (2) In vitro development of zygotes from ZP-free oocytes showed that there was no difference in cleavage rates. The blastocyst rate was slightly lower in the ZP-free group than the control. However, there was no difference in cell number per blastocyst between the control and the ZP-free group. (3) Examination of acrosome status by a specific fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining procedure revealed that frozen-thawed pig spermatozoa could undergo acrosome reaction and penetrate oocytes without induction by ZP. These data suggested that there are alternative mechanistic pathways for acrosome reaction induction during the fertilization process than the widely accepted sperm-zona receptor models. Finally, the viability of ZP-free derived embryos was demonstrated by full-term development and the delivery of healthy piglets following embryo transfer. In conclusion, the present experiments showed for the first time in farm animals, that normal embryos could be produced by in vitro fertilization of ZP-free oocytes in optimized conditions and that they could develop normally to full-term.     

Chondroitin sulfate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization

Bovine epididymal spermatozoa were incubated for 22 h in a modified Tyrode's medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. The addition of chondroitin sulfate A (CS-A) significantly increased the incidence of acrosome reaction. When observed by electron microscopy, acrosome-reacted sperm had undergone vesiculation of the plasma and outer acrosomal membranes. Sperm incubated in the presence of CS-A demonstrated a significantly higher incidence of vesiculation when compared to the controls. Additionally, rates of in vitro fertilization of bovine oocytes were significantly elevated when sperm and ova were exposed to CS-A. These results suggest that glycosaminoglycans in the female reproductive tract may be responsible for some of the biochemical changes associated with fertilization, and a light microscope procedure can be used to assess occurrence of the acrosome reaction.     

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.     

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.     

At least half of capacitated, motile mouse sperm can fertilize zona-free mouse oocytes.

The percentage of individual sperm capable of fertilizing zona pellucida-free mouse oocytes was investigated by placing motile sperm near zona-free oocytes with a micromanipulator. Incubation with one or two capacitated sperm per oocyte resulted in 50% and 70% fertilization, respectively, compared to 88% for cumulus intact (10(5) sperm/ml) and 87% for zona-free (2 x 10(3) sperm/ml) control oocytes. When sperm were treated with .1 microM calcium ionophore A23187 to facilitate the acrosome reaction, fertilization rates for single motile sperm were markedly lower than for capacitated, nontreated single sperm (4% and 35%, respectively). Similar fertilization rates resulted when one sperm was incubated per two ova (4% and 48% per sperm for A23187-treated and controls, respectively). When a lower dose of A23187 (.001 microM) was used to treat sperm, 7% of oocytes incubated with single sperm were fertilized. These experiments demonstrate that at least half of motile, capacitated mouse sperm are capable of fertilizing zona-free mouse oocytes in vitro, and that motile, A23187-treated mouse sperm resulted in poor fertilization rates.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

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.     

In vitro assays for vesper mice (Calomys laucha) sperm using heterologous substrates from nonrodent species

Small vesper mice (Calomys laucha) may be considered as an animal model for in vitro fertilization studies, but limited data about in vitro evaluations of their sperm quality and fertility are available. The in vitro penetration (IVP) assay is used to estimate potential sperm fertility for many mammal species, but it still requires reduction in cost and labor. This study tested improvements in the IVP assay for C. laucha sperm using swine oocytes and perivitelline layers (PVL) of chicken eggs as substrates, and evaluated associations among C. laucha sperm quality, IVP, and in vivo fertility after natural mating. In the IVP assay, gametes coincubation was carried out flat-bottomed wells with M2, in water bath at 37°C for 2 hr. C. laucha sperm presented motility, normal morphology, membrane integrity, and acrosome integrity equal to 90.6 ± 5.6, 90.2 ± 6.6, 88.7 ± 9.6, and 90.5 ± 11.5%, respectively. The IVP rate was 39.8% in swine oocytes and 87.5% in the inner PVL. Considering in vivo fertility as the gold standard, the IVP assay in swine oocytes presented a sensitivity of 16.0% and specificity of 83.3%. The sensitivity of the IVP assay in the inner PVL was 84.0%, but the specificity was not determined because there were no true negative results. Sperm membrane integrity was correlated with parturition after natural mating (r = 0.38, P<0.01) and litter size (r = 0.54; P<0.0002).The IVP assay using swine oocytes as substrates can be performed in nearly 2 hr without gametes' coincubation in CO(2).     

Effects of cumulus cells on sperm penetration of bovine oocytes in protein-free medium

The present study was conducted to examine the effects of cumulus cells on sperm capacitation, acrosome reaction and penetration of bovine oocytes in vitro in a protein-free medium. In vitro matured oocyte-cumulus complexes (OCCs) and denuded oocytes were co-incubated with spermatozoa in the medium with or without bovine serum albumin (BSA). Higher fertilization rates were obtained in the OCCs (92 and 89%, respectively) than denuded oocytes (57 and 6%, respectively) in the medium with or without BSA (P<0.01). Higher proportion of the denuded oocytes were fertilized in the medium with BSA (57%) than without BSA (6%; P<0.01). These results suggest that the cumulus cells are more effective for increasing fertilization rate than BSA (P<0.05). Both the percentages of capacitated and acrosome-reacted spermatozoa incubated for 4 h with isolated cumulus cells were not significantly different in the medium without cumulus cells in the presence or absence of BSA. The denuded oocytes were inseminated with isolated cumulus cells taken from OCCs matured with or without hormones, follicle stimulating hormone (FSH) and estradiol-17beta (E(2)), and from immature OCCs in a protein-free medium. Presence of the cumulus cells matured with hormones enhanced sperm penetration of denuded oocytes more effectively (81%) than either of the cells matured without hormones (41%) or the immature cells (26%; P<0.01). The conditioned medium of cumulus cells matured with hormones was not effective for sperm penetration of denuded oocytes (2%), while a high proportion (82%) of the oocytes were fertilized when they were inseminated with isolated cumulus cells (P<0.01). In conclusion, the presence of cumulus cells matured with FSH and E(2) was effective for sperm penetration but not for sperm capacitation or acrosome reaction.     

Estrous sheep serum as a potent agent for ovine IVF: effect on cholesterol efflux from spermatozoa and the acrosome reaction

The aim of the present study was to evaluate the effect of heat-inactivated estrous sheep serum (ESS) on sheep IVF. When the capacitation and the fertilization media contained 20% ESS, a fertilization rate of 85% was achieved. The beneficial effect of ESS on sheep IVF was further demonstrated since the fertilization rate was null when ESS was omitted during sperm capacitation and fertilization. Estrous sheep serum supported both sperm capacitation and fertilization as shown by the results of experiments in which it was omitted during one of these steps: sperm capacitation in serum-free medium resulted in delayed sperm-oocyte penetration, while fertilization in serum-free medium significantly decreased the percentage of fertilized oocytes. To investigate the influence of serum on sperm ability to undergo the acrosome reaction, salt-stored follicular sheep oocytes were inseminated, and the acrosomal status of spermatozoa attached to zonae was examined by electron microscopy after a 4-h period of coincubation. Quantitative analysis on thin sections demonstrated that fewer acrosome-reacted spermatozoa were observed when the capacitation and insemination steps were carried out in DM-H medium without serum than in DM-H-SS supplemented with 20% ESS (0.08, [0; 0.34], (median, range)/100mum zona vs 1.32, [0.90; 2.28]/100mum zona; P < 0.01). Since a higher number of spermatozoa attached to the zona surface in DM-H medium, the proportion of acrosome-reacted spermatozoa was much lower (0.7%, [0%; 2.2%], (median, range) vs 54%, [25%; 100%]; P < 0.01) in the absence of serum. These results indicate that in our IVF system the development of the acrosome reaction depended on serum. Sperm cholesterol efflux during in vitro capacitation was measured on [(3)H] cholesterol labeled spermatozoa resuspended in DM-H or DM-H-SS medium. A time-dependent cholesterol removal was observed in the presence of serum (60 +/- 5%, mean +/- SD, after 5 h), whereas it was limited to 14 +/- 3 % in DM-H medium; hence addition of serum to the capacitation medium efficiently supports cholesterol efflux, which is thought to be a key-event in the capacitation process.     

Maturation conditions and boar affect timing of cortical reaction in porcine oocytes

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.     

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.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

Distribution of α-D-mannose residues on zona pellucida and their role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cumulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost completely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4,spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.