Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development.
The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.
DISCIZIA, Faculty of Veterinary Medicine, Federico II University, Via Delpino 1, 80137 Naples, Italy. bgasparr@unina.it
Bianca
B
Gasparrini
Lucia
L
Boccia
Joelle
J
Marchandise
Rossella
R
Di Palo
Fabienne
F
George
Isabelle
I
Donnay
Luigi
L
Zicarelli
D000818:Animals; D002020:Buffaloes / Q000196:embryology* / Q000254:growth & development; D002467:Cell Nucleus / Q000187:drug effects; D002970:Cleavage Stage, Ovum / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D003543:Cysteamine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology*; D004526:Efficiency / Q000187:drug effects; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D005306:Fertilization / Q000187:drug effects; D005307:Fertilization in Vitro / Q000379:methods; D005978:Glutathione / Q000032:analysis / Q000096:biosynthesis* / Q000187:drug effects*; D008297:Male
D003470:Culture Media; D003553:Cystine; D003543:Cysteamine; D005978:Glutathione