In vitro fertilization of pig oocytes matured in vitro

Betancourt, Miguel; Fierro, Reyna; Ambriz, D.

doi:10.1016/0093-691x(93)90286-e

Cited by 17 publications

(12 citation statements)

References 12 publications

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“…They correlated well with published data, according to which fertilization rates average around 70%, with high incidence of polyspermy [21,[23][24][25][26]. We did not use native proacrosin as a competitor because we know that when incubated with the ZP, it influences fertilization rates by causing proteolytic modification of the ZP rather than by blocking acrosin binding sites (unpublished results).…”

Section: Role Of ␤-Acrosin In Sperm Penetrationsupporting

confidence: 84%

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Effect of Recombinant Boar β-Acrosin on Sperm Binding to Intact Zona Pellucida During In Vitro Fertilization1

Crosby

Barros

1999

Biology of Reproduction

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In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.

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Section: Role Of ␤-Acrosin In Sperm Penetrationsupporting

confidence: 84%

“…Pig oocyte in vitro maturation followed published protocols [20][21][22][23]. Briefly, groups of 20 oocyte complexes, each with a compact cumulus, were cultured in maturation medium with hormonal supplement for 24 h at 39ЊC in an atmosphere of 5% CO 2 in air.…”

Section: Maturation Of Pig Oocytes In Vitromentioning

confidence: 99%

Effect of Recombinant Boar β-Acrosin on Sperm Binding to Intact Zona Pellucida During In Vitro Fertilization1

Crosby

Barros

1999

Biology of Reproduction

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“…Harrison et al [9,10] found that bicarbonate=CO 2 induces rapid and reversible changes of the sperm membrane lipid architecture, increases Ca 2 þ influx and stimulates a capacitation signal transduction pathway. Although bicarbonate=CO 2 is known to function at temperatures above 30 C, it may induce its sperm membrane destabilizing action at 16 C. Maxwell and Johnson [16] found higher percentages of capacitated sperm when semen was resuspended in a bicarbonate-containing solution (Tyrode) as compared to a bicarbonatefree solution (PBS) at 15 C. Reading extender has 80% of the bicarbonate content of Tyrode [3,18], which may explain the structural membrane changes observed. Further studies must be done to determine the effect of bicarbonate on sperm membrane stability at low temperature.…”

Section: Discussionmentioning

confidence: 99%

“…The supernatant was removed and the pellet resuspended in 1 mL TALP-Hepes capacitation medium supplemented with BSA (6 mg=mL) and 1 mM sodium pyruvate at pH 7.4. Aliquots containing 15 Â 10 6 sperm were placed in a 4-well plastic dish (Nunc-Denmark) and incubated at 39 C for 4.5 h in a humid atmosphere with 5% CO 2 [3].…”

Section: In Vitro Capacitationmentioning

confidence: 99%

Membrane Status and in Vitro Capacitation of Porcine Sperm Preserved in Long-Term Extender at 16°c

Conejo-Nava

Fierro

Gutiérrez³

et al. 2003

Archives of Andrology

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“…Oocytes were obtained and matured in vitro [3]. Approximately 15 to 20 oocytes were incubated per 100 ml of maturation medium: TM-199 (Microlab, Mexico City, Mex), supplemented with 10% heat treated fetal calf serum (Microlab), 2 IU-ml LH and FSH (Serono, Mexico City, Mex), 1 mg-ml b-estradiol (Sigma, St. Louis, MO, USA), 1 mg-ml glucose (Baker, Mexico City, Mex), 0.25 mM sodium pyruvate (Sigma) and 50 mg-ml gentamycin (Scheramex, Mexico City, Mex) and incubated 48 h at 39 C.…”

Section: Oocyte Collection/maturationmentioning

confidence: 99%

INHIBITION OF SPERM-ZONA PELLUCIDA BINDING BY A 55 kDa PIG SPERM PROTEININ VITRO

Zayas-Pérez

Casas

Bonilla

et al. 2005

Archives of Andrology

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This study was conducted to evaluate the role of a 55 kDa pig sperm protein on the oocytesperm binding process, and its location in situ. For this purpose, in vitro matured oocytes were incubated with isolated and purified protein, and incubated with capacitated spermatozoa. In addition, capacitated sperm were incubated with anti-55 kDa antiserum and later with mature oocytes. Immunolocalization assays were performed using non-capacitated, capacitated and acrosome reacted sperm, which were incubated independently with anti-55 kDa protein antibodies and analyzed under fluorescence light microscopy. The 55 kDa protein concentrations correlated negatively with the amounts of sperm bound to the zona pellucida (ZP); the presence of the anti-55 kDa protein totally inhibited this binding. The immunolocalization assays revealed that fluorescence was located preferentially at the apical edge of the head in capacitated sperm, but not in acrosome reacted sperm. It would appear that the 55 kDa protein binds specifically to the oocyte ZP, and that it may be responsible for primary gamete binding during fertilization.

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In vitro fertilization of pig oocytes matured in vitro

Cited by 17 publications

References 12 publications

Effect of Recombinant Boar β-Acrosin on Sperm Binding to Intact Zona Pellucida During In Vitro Fertilization1

Effect of Recombinant Boar β-Acrosin on Sperm Binding to Intact Zona Pellucida During In Vitro Fertilization1

Membrane Status and in Vitro Capacitation of Porcine Sperm Preserved in Long-Term Extender at 16°c

INHIBITION OF SPERM-ZONA PELLUCIDA BINDING BY A 55 kDa PIG SPERM PROTEININ VITRO

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