Development of a Zona Pellucida Sperm Binding Assay for the Assessment of Stallion Fertility1

Oocyte cryopreservation has been used to restore fertility problems in women undergoing oocyte-toxic chemotherapy and also to salvage the genetic potential of endangered/valuable species. In domestic cats, knowledge regarding the cryopreservation technique of feline oocytes is rather limited. We examined the effect of OPS vitrification on zona pellucida ultrastructure and also the developmental competence of immature cat oocytes. Immature cat oocytes were vitrified using different CPA and CPA exposure techniques: (1) 2-step DMSO, n = 71; (2) 4-step DMSO, n = 78; (3) 2-step EG, n = 73; (4) 4-step EG, n = 117; (5) 2-step EG + DMSO, n = 67 and (6) 4-step EG + DMSO, n = 88. A combination of EG and DMSO or EG alone using 4-step equilibration technique yielded the highest maturation rates (28/88: 31.8% and 44/117: 37.6%, respectively). This was, however, significantly lower than nonvitrified controls (36/59, 61% maturation rate). We further examined the effect of OPS vitrification on ultrastructure of the zona pellucida using scanning electron microscopy and also to test the quality of the zona pellucida using sperm binding assay. Our modified OPS protocols did not significantly affect the appearance of zona pellucida of vitrified oocytes and also the number of tightly bound sperm as compared with controls (80.8 ± 63.8 v. 81.6 ± 70.4, respectively). Vitrification of immature oocytes significantly reduced the number of oocytes reaching cleavage and blastocyst stages compared with nonvitrified controls (24.8 and 7.5% v. 62.5 and 30.8% for cleavage and blastocyst formation rates, respectively). It is concluded that modification of OPS vitrification can be successfully applied for immature cat oocytes, in terms of zona pellucida ultrastructure, ability to bind sperm, and also their developmental competence. This study was financially supported by MRG4980108, CHE-TRF Senior Research Scholars RTA-5080010, The Thailand Research Fund, and The Research Unit of Reproductive and Biological Science, Chulalongkorn University.

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Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Reproduction ◽

10.1530/reprod/119.1.77 ◽

2000 ◽

pp. 77-83 ◽

Cited By ~ 15

Author(s):

B Strom Holst ◽

B Larsson ◽

C Linde-Forsberg ◽

H Rodriguez-Martinez

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Zona Pellucida ◽

Binding Assay ◽

Binding Capacity ◽

Storage Conditions ◽

Deep Freezing ◽

Sperm Binding ◽

Significant Difference ◽

Scanning Electron

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.

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Fertilization and early embryology: The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Human Reproduction ◽

10.1093/oxfordjournals.humrep.a019429 ◽

1996 ◽

Vol 11 (7) ◽

pp. 1516-1519 ◽

Cited By ~ 15

Author(s):

Y.Q. Yao ◽

W.S.B. Yeung ◽

P.C. Ho

Keyword(s):

Zona Pellucida ◽

Binding Assay ◽

Factors Affecting ◽

Sperm Binding

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Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae

Reproduction ◽

10.1530/rep-11-0099 ◽

2012 ◽

Vol 143 (5) ◽

pp. 577-585 ◽

Cited By ~ 12

Author(s):

Marco A Coutinho da Silva ◽

George E Seidel ◽

Edward L Squires ◽

James K Graham ◽

Elaine M Carnevale

Keyword(s):

Zona Pellucida ◽

Binding Assay ◽

Skim Milk ◽

Milk Proteins ◽

Sodium Caseinate ◽

Sperm Binding ◽

Threefold Increase ◽

Series Of Experiments ◽

Semen Extender ◽

Positive Effect

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm–ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

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

Reproduction Fertility and Development ◽

10.1071/rd09033 ◽

2010 ◽

Vol 22 (4) ◽

pp. 710 ◽

Cited By ~ 26

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.

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Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽

10.1530/reprod/119.1.15 ◽

2000 ◽

pp. 15-23 ◽

Cited By ~ 15

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.

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