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Effects of the addition of glutathione during maturation on in vitro fertilisation of prepubertal goat oocytes

Published online by Cambridge University Press:  28 November 2001

P. Mayor
Affiliation:
Departament Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, E-01893 Bellaterra, Barcelona, Spain.
M. López-Béjar
Affiliation:
Departament Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, E-01893 Bellaterra, Barcelona, Spain.
E. Rodríguez-González
Affiliation:
Departament Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain.
M.T. Paramio
Affiliation:
Departament Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain.

Abstract

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is a ubiquitous intracellular free thiol that improves development of the male pronucleus at fertilisation and has also been implicated in promoting the development of preimplantation embryos. The objective of this study was to evaluate the effects of adding GSH or cysteine to the in vitro maturation medium on intracellular GSH amounts after in vitro maturation and fertilisation of prepubertal goat oocytes. Oocytes were matured in TCM199 medium supplemented with 10% bovine fetal serum, 1 mg/ml 17β-estradiol, 10 μg/ml o-FSH, 10 μg/ml LH and 50 mg/ml gentamicin. In vitro maturation medium was completed with two independent treatments: GSH at different concentrations (0, 0.25, 0.50 and 1.00 mM) and L-cysteine at different concentrations (0, 150, 300, 600 and 900 μM). After 27 h of culture at 38.5 °C in 5% CO2 in air, the nuclear stage was evaluated. Simultaneously, another sample of oocytes was frozen and the intracellular GSH level was evaluated with spectrophotometric methodology. Oocytes were inseminated with fresh semen (2-3 × 106 sperm/ml) in TALP medium supplemented with 1 mg/ml hypotaurine. Oocytes were fixed at 20 h post-insemination to evaluate the in vitro fertilisation. Oocytes matured in 1.00 mM GSH-supplemented medium exhibited higher amounts of intracellular GSH (3.23 pmol per oocyte). The percentage of normal fertilisation (17-27%) was similar for the treatment groups. In conclusion, the addition of 1.00 mM GSH to the maturation medium could be a useful method for increasing the intracellular GSH levels of prepubertal goat oocytes. However, this increase was not associated with a higher normal fertilisation rate of prepubertal goat oocytes.

Type
Research Article
Copyright
2001 Cambridge University Press

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