294 EFFECT OF CYSTEAMINE ADMINISTRATION DURING EQUINE OOCYTE MATURATION ON GLUTATHIONE CONTENT, NUCLEAR MATURATION, AND DEVELOPMENTAL CAPABILITY AFTER INTRACYTOPLASMIC SPERM INJECTION

Abstract

In the recent years, assisted reproduction methods have produced only limited success in equine species in comparison with other domestic mammals. A major factor affecting oocyte viability during in vitro culture is oxidative stress. Oxidative modifications could be responsible for oocyte-defective in vitro maturation and consequently compromise subsequent fertilization and embryonic development. Low-molecular-weight thiol compounds such as cysteamine, added during in vitro culture of bovine, porcine, and ovine oocytes, increase intracellular glutathione (GSH) synthesis, which prevents oxidative damages and consequently improves in vitro maturation and embryo development. The present study was aimed at investigating whether equine oocyte maturation efficiency and embryonic developmental capability following ICSI benefit from the addition of cysteamine during in vitro maturation (IVM). Cumulus oocyte complexes (COCs) were collected from slaughtered ovaries and cultured for 30 h at 38.5°C in 500 μL of control medium (TCM199 + 0.4% BSA + 0.1 IU/mL rhFSH + 50 ng/mL EGF) either supplemented with 100 μM cysteamine or not. After culture, nuclear stage was assessed by Hoechst 33342 staining after cumulus cell removal, and MII oocytes were analyzed for GSH content (Baker MA et al. 1990 Anal. Biochem. 190, 360–365). Groups of COCs matured under the same conditions were denuded with hyaluronidase and only oocytes with a visible polar body were fertilized by ICSI. The number of embryos that reached the 2–4 cell stage was assessed by nuclear staining with propidium iodide after 72 h of culture in SOF supplemented with 5% calf serum at 38.5°C in a modified atmosphere (5% CO₂, 5% O₂, and 90% N₂). Our data indicated that oocytes cultured in the presence of cysteamine had a nuclear maturation rate similar to those cultured in control medium (Table 1). Intraoocyte GSH content increased during IVM, and the addition of cysteamine induced a significant GSH accumulation in matured oocytes. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 h of culture. The results of this study demonstrate that the addition of cysteamine to the IVM medium increases GSH content in equine oocytes. However, this affects neither the maturation rate nor the capability to reach the early embryonic development after ICSI. We hypothesize that factor(s) other than GSH content are responsible for the limited in vitro developmental capability of equine oocyte.

Table 1.
Effect of cysteamine administration on maturation rate, oocyte GSH content (pmol/oocyte), and early embryonic development after ICSI

This work was supported by a 2003 UniMi Grant.