In vitro developmental potential of macaque oocytes, derived from unstimulated ovaries, following maturation in the presence of glutathione ethyl ester

Abstract

Background: The inadequacies of oocyte in vitro maturation (IVM) systems for both non-human primates and humans are evidenced by reduced fertilization and poor embryonic development, and may be partly explained by significantly lower glutathione (GSH) contents compared with in vivo matured (IVO) oocytes. As this influence has not been fully explored, this study investigated the effect of the GSH donor, glutathione ethyl ester (GSH-OEt), on the IVM and development of macaque oocytes as a model of human oocyte IVM.

Methods: Macaque oocytes derived from unstimulated ovaries were cultured in mCMRL-1066 alone or supplemented with 3 or 5 mM GSH-OEt. In vitro matured oocytes were subjected to the GSH assay, fixed for the assessment of spindle morphology or prepared ICSI. Embryo development of zygotes cultured in mHECM-9 was assessed up to Day 9 post-ICSI. RESULTS Supplementation of the maturation medium with GSH-OEt significantly increased oocyte maturation and normal fertilization rates compared with control oocytes, but only 5 mM GSH-OEt significantly increased the oocyte and cumulus cell GSH content. Confocal microscopy revealed significant differences in the spindle morphology between IVO and control in vitro matured metaphase II oocytes. Oocytes matured with 5 mM GSH-OEt exhibited spindle area and spindle pole width similar to that seen in the IVO oocyte. While no significant differences were observed in blastocyst rates, addition of 3 mM GSH-OEt during IVM significantly increased the proportion of embryos developing to the 5-8 cell stage while 5 mM GSH-OEt significantly increased the proportion of morula-stage embryos compared with controls.

Conclusions: Supplementation of the IVM medium with GSH-OEt promotes better maturation and normal fertilization of macaque oocytes compared with non-supplemented medium. However, further improvement of the primate oocyte IVM culture system is required to support better blastocyst development of oocytes derived from unstimulated ovaries.

Figure 1

Representation of normal metaphase II spindle measurements on a confocal projection of an in vitro matured macaque oocyte; (a) Measurement of spindle dimensions (tubulin; green): SpL, spindle length; SpW, spindle width; P, pole width. (b) Measurement of chromatin dimensions (blue): ChL, chromatin length; ChW, chromatin width. (c) Merged image of spindle and chromatin. Bar = 5µm.

Figure 2

GSH content of cumulus cells collected from in vitro matured macaque oocytes. Bars with different letters are significantly different (P < 0.05). GSH data are expressed as mean ± SEM from 3–4 replicate experiments. GSH-Oet, mCMRL-1066 medium supplemented with GSH-OEt at the indicated concentration; BSO, mCMRL-1066 medium supplemented with BSO (5 mM).

Figure 3

Three-dimensional isosurfaces produced from the confocal z-series of abnormal MII spindles in in vitro matured macaque oocytes. Spindle tubulin (green) and chromatin (blue) (a) Abnormal chromatin, displaced chromosome(s) indicated by an arrowhead (▸); (b) Tripolar spindle, spindle poles indicated by arrows (→). Bar = 5 µm.

Figure 4

Three-dimensional isosurfaces produced from the confocal z-series of normal MII spindles of in vitro and in vivo matured macaque oocytes. Spindle tubulin (green) and chromatin (blue) (a) in vitro matured control; (b) GSH-OEt-treated (5 mM); (c) BSO-treated (5 mM); (d) in vivo matured. Bar = 5 µm.