scholarly journals Fertilization of Pronase-Treated Mouse Ova in Vitro

1981 ◽  
Vol 34 (2) ◽  
pp. 245 ◽  
Author(s):  
P Quinn ◽  
JD Stanger
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Relative Effectiveness ◽  
Fertilization Rate ◽  
Enzyme Treatment ◽  
Perivitelline Space ◽  
Sperm Binding ◽  
Mechanical Removal ◽  
Better Than

In order to obtain consistently a large number of zona-free mouse ova for studies of sperm-egg interactions, a study was made of the relative effectiveness of removing the zona pellucida from ova by mechanical or enzymatic treatments. Ova exposed to pronase before mechanical removal of the zona pellucida in medium devoid of pronase had similar fertilization rates in vitro compared with ova mechanically denuded in the absence of pronase. Ova with pronase-weakened zonae were easier to denude and survived the mechanical manipulations better than the ova denuded by vigorous aspiration in narrow-bore pipettes. However, exposure to pronase did significantly lower the incidence of polyspermy in the naked ova, indicating that some of the enzyme may have diffused across the perivitelline space and damaged sperm-binding sites on the vitelline plasma membrane. The enzyme treatment also reduced the fertilization rate of zona-intact ova.

Evaluation of the function of fresh and frozen - thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay

2010 ◽  
Vol 22 (4) ◽  
pp. 710 ◽  
Author(s):  
J. R. Clulow ◽  
G. Evans ◽  
W. M. C. Maxwell ◽  
L. H. A. Morris
Keyword(s):  
Zona Pellucida ◽  
Binding Assay ◽  
Binding Ability ◽  
Treatment Groups ◽  
Functional Integrity ◽  
Sperm Binding ◽  
Heterologous Sperm ◽  
Bovine Oocytes ◽  
Better Than

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen–thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen–thawed non-sorted spermatozoa; and (4) frozen–thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed ‘attached’ or ‘bound’ depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen–thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen–thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen–thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status.

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

scholarly journals Roles of Resveratrol in Improving the Quality of Postovulatory Aging Oocytes In Vitro

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1132 ◽  
Author(s):  
Yan-Li Sun ◽  
Shou-Bin Tang ◽  
Wei Shen ◽  
Shen Yin ◽  
Qing-Yuan Sun
Keyword(s):  
Binding Sites ◽  
Aging Process ◽  
Oocyte Quality ◽  
Reproductive Technology ◽  
Early Embryo ◽  
Normal Morphology ◽  
Sperm Binding ◽  
Postovulatory Aging

After ovulation, mammalian oocytes will undergo a time-dependent process of aging if they are not fertilized. This postovulatory aging (POA) seriously affects the oocyte quality and then impairs the subsequent fertilization and early embryo development, which should be avoided especially in assisted reproductive technology (ART). Resveratrol is an antioxidant substance that can scavenge free radicals and is effective in improving ovary functions. Here, mouse oocytes were used to investigate the effects and mechanisms of resveratrol on POA oocytes in vitro. With 1.0 µM resveratrol treatment during aging process, the rates of fertilization and blastocyst in POA oocytes increased significantly compared with those in the POA group. Resveratrol can reduce the loss of sperm binding sites by stabilizing Juno. Resveratrol can maintain the normal morphology of spindle and mitochondrion distribution and alleviate the levels of ROS and early apoptosis. Additionally, resveratrol can reduce the changes of H3K9me2. Therefore, resveratrol can significantly improve the quality of POA oocytes in vitro to enhance the rates of fertilization and blastocyst, which may be very helpful during the ART process.

The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes

2016 ◽  
Vol 28 (8) ◽  
pp. 1223 ◽  
Author(s):  
K. Schmidt ◽  
A. Clark ◽  
A. Mello ◽  
C. Durfey ◽  
A. Buck ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Zona Pellucida ◽  
Glucuronic Acid ◽  
In Vitro Fertilisation ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Blastocyst Formation ◽  
Space Thickness ◽  
Intracellular Glutathione

High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48 h and 144 h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (P < 0.05) the perivitelline space thickness and significantly lowered the incidence (P < 0.05) of polyspermy. GlcNAc supplementation significantly increased (P < 0.05) intracellular glutathione concentrations. Supplementation with 0.005 mM glucuronic acid plus 0.005 mM GlcNAc during oocyte maturation produced significantly higher rates (P < 0.05) of cleavage and blastocyst formation by 48 and 144 h after IVF compared with all other groups. These results indicate that supplementing with 0.005 mM glucuronic acid and 0.005 mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.

scholarly journals Effect of immunization of hamsters against recombinant P26h on fertility rates

Reproduction ◽  
2002 ◽  
pp. 307-313 ◽  
Author(s):  
C Gaudreault ◽  
L Montfort ◽  
R Sullivan
Keyword(s):  
Zona Pellucida ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Fertilization Rate ◽  
Active Immunization ◽  
Antigenic Determinants ◽  
Binding Assays ◽  
Contraceptive Vaccine

Despite the various contraceptive methods available, an effective and inexpensive method remains to be established. Immunocontraception may help to achieve this goal. P26h has been proposed as a candidate for the development of a male contraceptive vaccine. P26h, a hamster sperm protein, interacts with the zona pellucida. Furthermore, in vivo fertilization can be blocked completely by active immunization of male hamsters against P26h. Maltose binding protein (MBP)-P26 shares antigenic determinants with the native P26h present on cauda epididymal spermatozoa. The aim of the present study was to reproduce the immunocontraceptive properties of native P26h by immunizing male hamsters against a recombinant P26h fused with a maltose binding protein (MBP). Active immunization of male hamsters with the MBP-P26h showed that specific anti-P26h circulating IgGs could be generated. Mating of immunized male hamsters with superovulated females resulted in a significant decrease, 20-25%, in the fertilization rate. This result is in agreement with results from in vitro sperm-zona pellucida binding assays. Indeed, the anti-recombinant P26h IgGs showed lower inhibitory properties when compared with anti-native P26h IgG. Despite the high anti-P26h IgG titres generated in hamsters, histological studies showed that active immunization has no pathological sequelae to the reproductive tissues. The potential of P26h as a candidate for a contraceptive vaccine is discussed.

Zona pellucida characteristics and sperm-binding patterns of in vivo and in vitro produced porcine oocytes inseminated with differently prepared spermatozoa

2005 ◽  
Vol 63 (2) ◽  
pp. 352-362 ◽  
Author(s):  
Detlef Rath ◽  
Edda Töpfer-Petersen ◽  
Hans-Wilhelm Michelmann ◽  
Peter Schwartz ◽  
Silja Ebeling
Keyword(s):  
Zona Pellucida ◽  
Sperm Binding

Alkaline phosphatase activity in bovine oocytes and preimplantation embryos as affected by removal of the zona pellucida and culture medium constituents

2003 ◽  
Vol 15 (5) ◽  
pp. 285 ◽  
Author(s):  
J. L. Edwards ◽  
A. M. Powell ◽  
C. E. Rexroad Jr
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Zona Pellucida ◽  
Bovine Serum ◽  
Culture Medium ◽  
Preimplantation Embryos ◽  
Perivitelline Space ◽  
Mechanical Removal ◽  
Bovine Oocytes

The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.

The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 75-83 ◽  
Author(s):  
R. D. Moreno ◽  
M. S. Sepúlveda ◽  
A. de Ioannes ◽  
C. Barros
Keyword(s):  
Zona Pellucida ◽  
Active Site ◽  
Enzyme Activation ◽  
Binding Domain ◽  
Present Evidence ◽  
Sperm Binding ◽  
Mammalian Sperm ◽  
Hydrolysis Of

SummaryMammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellcida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization

2001 ◽  
Vol 114 (22) ◽  
pp. 4127-4136
Author(s):  
Elizabeth Howes ◽  
John C. Pascall ◽  
Wolfgang Engel ◽  
Roy Jones
Keyword(s):  
Zona Pellucida ◽  
Solid Phase ◽  
Binding Assays ◽  
Vitro Fertilization ◽  
Sperm Binding ◽  
Secondary Receptor ◽  
Specificity Of Binding ◽  
Solid Phase Binding ◽  
Complementary Binding

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

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