Medium effects on capacitation and sperm penetration through the zona pellucida in inbred BALB/c spermatozoa

Inbred BALB/c mice are one of the most difficult inbred strains to fertilize in vitro. In this study we examined the abilities of various media used for mouse in vitro fertilization (IVF) to support capacitation and sperm penetration through the zona pellucida (ZP) of inbred BALB/c spermatozoa. Media examined were TYH, M16, CZB, mWhitten medium, T6, modified Tyrode's solution (mTyrode's), mKSOM, MEM and TCM199. Modified human tubal fluid (mHTF) was used as a control medium. When sperm were capacitated and inseminated in the same medium, mHTF showed the best fertilization (approximately 80%) scored by male pronuclear formation (<26%) at 5h post-insemination (PI). When sperm were capacitated in various media and inseminated in mHTF, sperm capacitated in TYH solution (93%) but no other media (<45%) showed a significantly higher level of sperm nuclear decondensation (SND) than mHTF at 2 h PI (approximately 65%). When sperm were capacitated in mHTF and inseminated in various media, only mTyrode's (52%) was not significantly lower than mHTF (66%) in terms of SND at 2h PI (<49%). Sperm capacitation also was examined by chlortetracycline (CTC) staining. Sperm capacitated in TYH solution showed a significantly higher percentage of capacitation (46%) than those treated in HTF (28%) and other media (<24%). These results indicate that the best approach for IVF in the BALB/c strain is capacitation in TYH and insemination in mHTF. Poor fertilization of BALB/c may result from suboptimal conditions of sperm capacitation and insemination, and overall IVF success may differ depending on strains used.     

In vitro fertilization in inbred BALB/c mice II: effects of lactate, osmolarity and calcium on in vitro capacitation

To elucidate requirements for in vitro sperm capacitation in inbred BALB/c mice, osmolarity, calcium and lactate were optimized using modified simplex optimization medium (mKSOM). Modified human tubal fluid (mHTF), a capacitation-supporting medium, was used as a control. In the first series of experiments, the effects of calcium and osmolarity were studied in the presence of lactate. Although preincubation with >or=5 mM CaCl2 improved fertilization after insemination significantly, it was still significantly lower than incubation with mHTF. To obtain fertilization at the equivalent levels to that of mHTF, isotonic osmolarity (305 mOsmol) was required. Trehalose, an osmotic reagent, could substitute for NaCl partially. In the second series of experiments, the effects of lactate were examined using a concentration of 5 mM calcium and isotonic osmolarity. Preincubation with 75%), as well as the percentages of B (capacitated) pattern sperm (>or=40%) in chlortetracycline (CTC) staining, as compared with incubation in mHTF (46% and 28%, respectively; p<0.05). In the third series of experiments, the effects of osmolarity and calcium in the absence of lactate were examined. An increase in osmolarity during sperm preincubation increased both fertilization and B-pattern sperm significantly in a dose-dependent manner. Trehalose, sucrose and choline chloride could substitute for NaCl. An increase in CaCl2 concentration during preincubation had no effect on fertilization, but this increase reduced the percentages of B-pattern sperm. In vitro capacitation of inbred BALB/c mice is sensitive to lactate and osmolarity, but that sensitivity for calcium varies depending on the presence or absence of lactate.     

Immunolocalization of proacrosin/acrosin in bovine sperm and sperm penetration through the zona pellucida

The aim of the present work was to immunolocalize acrosin in bull spermatozoa incubated for up to 6 h in capacitating culture medium (TALP-heparin), in order to study the kinetics of its release during the acrosome reaction and in vitro sperm penetration. Six replicates from semen of one bull were used. Acrosin was localized by the silver-enhanced immunogold technique using anti-bovine acrosin monoclonal antibody ACRO-C2E5. Spermatozoa thus showed the presence of acrosin only at the acrosomal region. Four different patterns were seen: (1) no labeling: (2) intense labeling on the rim of the portion of the acrosome; (3) diffuse label over the entire acrosomal region; and (4) intense label over the entire acrosomal region. Spermatozoa incubated in capacitating medium for 4 h showed that unlabeled (pattern 1) spermatozoa decreased from 72% to 28% difference that was found to be significant (p<0.05). Patterns 3 and 4 increased from about 10% to 20-29%, (p<0.05). With further incubation (4-6 h), pattern 1 increased while patterns 3 and 4 decreased differences were not significant (p0.05). The incidence of pattern 2 did not change through the whole incubation period. Sperm penetration through the zona pellucida of in vitro matured bovine oocytes (57%) or empty zonae pellucida (70.5%) increased (p<0.05) as a function of sperm incubation time in capacitating medium. The presence of acrosin, as determined by the silver-enhanced immunogold technique, was highly correlated with sperm penetration of in vitro mature bovine oocyte (r=0.98) and cryopreserved zonae pellucidae (r=0.93) (p<0.01).     

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1-10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p<0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p<0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P<0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.     

Mouse sperm antigens that participate in fertilization. II. Inhibition of sperm penetration through the zona pellucida using monoclonal antibodies

To dissect the process of mammalian sperm interaction with the egg at a molecular level, we have generated monoclonal antibodies (mAbs) to mature mouse sperm using syngeneic mouse testis as the immunogen. In this paper, we report upon three members of a mAb family, all of which displayed identical immunofluorescence patterns on cauda epididymal mouse sperm. Each of these mAbs, termed M42, M5, and M41, localized to a restricted region of plasma membrane overlying the acrosome. When tested for an effect on the fertilization process in vitro, two of the mAbs, M42 and M5, demonstrated significant inhibition. The inhibitory capacity was dependent upon the presence of the zona pellucida; neither M42 nor M5 was capable of blocking fertilization when zona pellucida-free mouse eggs were used. Identification of the antigens recognized by this group of mAbs was achieved by immunologic detection of sodium dodecyl sulfate-extracted sperm components separated via electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels followed by transfer to nitrocellulose. M42, which blocked fertilization, recognized a high molecular weight cluster of bands with Mr of approximately 220,000 to 240,000. M5, which also prevented fertilization, specifically recognized a sperm component with subunit molecular weight of approximately 54,000. M41, which did not interfere with fertilization, did not interact with any high molecular weight components, but recognized components with Mr of approximately 60,000, 35,000, and 21,000. Taken together with the work presented in a companion paper (Saling, Irons, and Waibel, this issue), we have demonstrated that it is possible to describe particular cellular regions of mammalian sperm with respect not only to location and function, but also to the molecules that are candidates for a role in that function.     

Sperm concentration influences fertilization and male pronuclear formation in vitro in pigs

To study the effect of sperm concentration on the results of pig in vitro fertilization (IVF), 313 oocytes recovered from oviducts of prepubertal gilts after induction of ovulation were used. After capacitation, the number of live spermatozoa in the fertilization dishes was adjusted to 3 x 10(5), 6 x 10(5) and 12 x 10(5) cell/ml. After 4 hours of co-culture in TCM-199, the oocytes were pippeted to remove cumulus cells and the excess spermatozoa around the zona pellucida, and were transferred to fresh TCM-199 for another 12 14 hours . The results showed that 6 x 10(5) spermatozoa/ml is the optimum concentration for this system; the percentage of fertilized ova (71.6%) was not different from the best (76.8%), that was obtained with the highest concentration, and the percentage of monospermy (62.3%) was not different from the best (68.1%), that was obtained with the lowest concentration. The percentage of spermatozoa that reached the pronuclear stage increased while sperm concentration was decreased. The percentage of spermatozoa at the decondensed stage was decreased when the sperm concentration increased.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

Immunocontraception is induced in BALB/c mice inoculated with murine cytomegalovirus expressing mouse zona pellucida 3

Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.     

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.     

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

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

Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

The purpose of the present study was to investigate the optimal concentration of osmolarity, calcium and bicarbonate for sperm penetration and formation of pronuclei (PN), and to investigate the time required for capacitation, penetration across the zona pellucida and formation of PN in bovine cumulus-free oocytes matured in vitro. Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro. Bovine sperm penetrated the zona pellucida in medium containing 240 to 440 mOsm, whereas PN formation was observed in a narrow range of osmolarities, from 280 to 360 mOsm. Maximal penetration by spermatozoa and PN formation was obtained in the medium with 2.5 mM calcium. High rates of spermatozoa penetration were observed in the medium with 37 to 49 mM NaHCO3. However, PN were formed regardless of the concentration of NaHCO3. The times required for sperm capacitation and penetration through the zona pellucida were 260 and 50 min, respectively. The first development of PN was recorded at 120 min after sperm penetration. Therefore, our study suggests that fertilization ability of spermatozoa in vitro appears to be more stable in high concentrations of NaCI. Oocytes are more sensitive to osmotic stress than spermatozoa. Calcium is required for both sperm penetration and PN formation in cumulus-free oocytes, but bicarbonate may be needed mainly for the penetration of spermatozoa.     

Zona-free sperm penetration assay and inducers of the acrosome reaction: A model for sperm microinjection under the zona pellucida

In order to minimize the percentage of false-negative results in the zona-free sperm penetration assay (SPA), a wide range of substances and/or physical agents capable of inducing the acrosome reaction (AR) have been incorporated in the incubation medium. These agents can also be used for treatment of severe male infertility using the technique of sperm microinjection under the zona pellucida (SMUZ). In the present review, the percentages of acrosome-reacted spermatozoa induced by several physiological, biochemical or physical agents published in the literature are compared in order to find the most efficient method(s) of inducing the AR In human sperm as a previous requirement for optimizing the technique of SMUZ. A working estimate of the level of efficiency of a given AR inducer is calculated by adding up its range score in each of three different arrangements from the highest to the lowest value of percentages of AR and differences in percentages of AR and penetration indexes between treated and control groups in SPA. The agents able to induce the AR by nonphysiological (electropermeabilization, lysophosphatidyl choline, and freezing-thawing) have better positions in this hierarchical system than those ones which require the active participation of sperm membrane receptors or second messenger systems (progesterone, zona pellucida, and stimulators of protein kinase A). Electropermeabilization appears to be the most efficient AR inducer. However, more possibilities need to be explored to enhance the relatively low percentages of acrosome-reacted spermatozoa shown by infertile men. © 1993 Wiley-Liss, Inc.     

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.     

Properties of the first and second factors released after hamster sperm make contact with the zona pellucida prior to fertilization in vitro

An investigation was made as to the nature of two of the factors, termed S1, released within the first 30 minutes after contact is made between capacitated hamster sperm and the zona pellucida in vitro. Previous studies showed that these S1 factors were detected two and 20 to 25 minutes after the gametes were combined and that, based on filtration studies, the former possessed a molecular weight of less than 5,000 daltons. The present results show that the quantity of the 20–25-minute S1 factor released into the supernatant increased linearly as a function of the sperm concentration. This factor passed unimpeded through a filter with a 5,000 molecular weight cutoff but only 42% of the activity traversed a filter with a cutoff of 2,000 daltons. The two-minute S1 factor, in the virtual total absence of cells, was stable for 10 to 15 minutes, but lost significant activity upon longer incubation. Under the same conditions, the 20–25-minute factor lost approximately 25% of its activity within 15 minutes, but remained stable at this level for at least 45 minutes of incubation. Both S1 factors were not affected by a mixture of glycosidases, but were inactivated by subtilisin, trypsin, and leucine aminopeptidase which was contaminated with endopeptidases. The activity of the two-minute S1 factor appeared more susceptible to the action of the proteases than that of the 20–25-minute S1 factor. In contrast to previous results obtained with the two-minute S1 factor, the release of the 20–25-minute S1 factor was not inhibited by the inclusion of soybean trypsin inhibitor a t concentrations which are known to inhibit penetration of the zona by the sperm. The results suggest that the two- and 20–25-minute S1 factors are peptides which are not identical.     

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

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

Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: effects on fertilization and early development

During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.