Abstract:
Ovarian fragments were exposed to 0.5 M sucrose
and 1 M ethylene glycol (freezing solution; FS) with or
without selenium or Trolox. Histological and ultrastructural
analyses showed that the percentages of normal follicles in
control tissue and in tissue after exposure to FS+50 μM
Trolox were similar. Trolox prevented endoplasmic reticulum
(ER)-related vacuolization, which is commonly observed in
oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1)
was up-regulated in ovarian tissue exposed to FS+10 ng/ml
selenium. Ovarian fragments were subsequently frozenthawed
in the presence of FS with or without 50 μM Trolox,
followed by in vitro culture (IVC). Antioxidant capacity in
ovarian fragments decreased after freeze-thawing in Troloxfree
FS compared with FS+50 μMTrolox. Although freezing
itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of
viable follicles (67 %), even after IVC (61 %). Furthermore,
stress markers SOD1 and ERp29 were up-regulated in ovarian
tissue frozen-thawed in Trolox-free medium. Relative mRNA
expression of growth factors markers was evaluated after
freeze-thawing followed by IVC. BMP4, BMP5, CTGF,
GDF9 and KL were down-regulated independently of the
presence of Trolox in FS but down-regulation was less pronounced
in the presence of Trolox. Thus, medium supplementation
with 50 μMTrolox prevents ER stress and, consequently,
protects ovarian tissue from ER-derived cytoplasmic
vacuolization. ERp29 but not ERp60, appears to be a key
marker linking stress caused by freezing-thawing and cell
vacuolization.
