Glutathione content and glutathione peroxidase expression in in vivo and in vitro matured equine oocytes.
The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte complexes (COCs) benefit from the addition of cysteamine during IVM, (2) to compare the GSH content of oocytes after in vivo maturation and IVM, (3) to assess whether cysteamine administration during IVM of equine oocyte enhances early embryonic developmental capability following ICSI, (4) to study the glutathione peroxidase (GPX) mRNA level in COCs. In vivo matured COCs were collected by aspiration from preovulatory follicles, and analyzed at collection. Immature COCs were collected in vivo or from slaughterhouse ovaries and matured in culture media supplemented or not with 100 microM cysteamine. After nuclear stage assessment, oocytes were analyzed for GSH concentration and both oocytes and cumulus cells were analyzed for GPX and GAPDH mRNA. Our data showed that the maturation capability was similar in both in vivo aspirated oocytes and in those isolated from slaughterhouse ovaries. Moreover, the addition of cysteamine during IVM affected neither GSH content nor maturation rate. At the time of collection, intra-oocyte GSH content was not influenced by the chromatin status. GSH concentration was similar in in vivo and in vitro matured metaphase II (MII) stage oocytes, and was significantly higher in MII than immature germinal vesicle stage oocytes. Moreover, the presence of serum inhibited whereas its absence stimulated the accumulation of GSH within oocytes during IVM. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 hr of culture. Cumulus cells expressed GPX mRNA, while GPX transcript was absent in both immature and mature oocytes. Cumulus expression of GPX mRNA was significantly higher when analyzed at collection than after IVM. Taken together, our results demonstrate that in equine oocytes, GSH increases during IVM but the relative intra-oocyte content of this thiol does not affect maturation and early development efficiency after fertilization. We hypothesize that factor(s) other than GSH/GPX are responsible for the limited in vitro early developmental capability of equine oocytes.
Luciano
Alberto M
AM
Institute of Anatomy of Domestic Animals, Histology and Embryology, Faculty of Veterinary Medicine, University of Milan, Milan, Italy. alberto.luciano@unimi.it
Goudet
Ghylène
G
Perazzoli
Federica
F
Lahuec
Cecile
C
Gérard
Nadine
N
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002970:Cleavage Stage, Ovum / Q000187:drug effects / Q000378:metabolism; D003543:Cysteamine / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000032:analysis / Q000378:metabolism*; D006736:Horses; D008540:Meiosis / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D018384:Oxidative Stress / Q000187:drug effects; D011837:Radiation-Protective Agents / Q000494:pharmacology
D011837:Radiation-Protective Agents; D003543:Cysteamine; D005979:Glutathione Peroxidase; D005978:Glutathione