MiPsummer Programme pdf - Mitochondrial Physiology Society

72
Abstract # 41
Superoxide dismutase 2 and thioredoxin reductase 2 in the adult rat ovary during follicular
development
Li Meng, Katja Teerds., Jaap Keijer
Human and Animal Physiology, Animal Sciences, Wageningen University, Wageningen, The
Netherlands. li.meng@wur.nl
Background Reactive oxygen species (ROS) and their scavenging systems play important
physiological roles in the ovary[1]. How these processes are exactly regulated during follicular
development and atresia is still unclear. Superoxide dismutase 2 (SOD2), located in the
mitochondria, metabolizes superoxide radicals to hydrogen peroxide, which is further detoxified to
water and oxygen by catalase or glutathione peroxidase. Although SOD2 has been localized in the
human and bovine ovary, its localization in the rat and mouse ovary during follicular development
and atresia is less clear. Thioredoxin reductase 2 (TXNRD2), a reductase of thioredoxin2 (TXN2),
provides a major line of defence against mitochondrial ROS, nevertheless, not much is known about
the localization of TXNRD2 in the mammalian ovary[2].
Objectives To identify the localization of SOD2 and TXNRD2 in ovaries of adult rats in order to
understand their role in normal follicle development and atresia.
Methods SOD2 and TXNRD2 protein levels were measured in ovaries from 100-day-old adult rats,
using western-blotting on homogenates of whole ovaries. The cellular localization of SOD2 and
TXNRD2 was determined by immunohistochemistry using 5 µm thick paraffin sections. The
sections were stained using a rabbit polyclonal antibody against SOD2 (diluted 1:1000, Abcam) and
TXNRD2 (diluted 1:200, Novus). Staining was performed in ovaries of at least 6 animals. For
western blotting the same antibodies were used at dilutions of 1:5000 and 1:500 for SOD2 and
TXNRD2, respectively.
Results In the western blots, specific bands for SOD2 and TXNRD2 were detected at the expected
molecular weight of 25 kD and 56 kD, respectively.
Immunohistochemical analysis showed the prominent presence of SOD2 in healthy preantral and
preovulatory follicles (staining in theca and granulosa cells, and oocyte) and in the luteal cells of the
corpora lutea (CL). Staining was faint to absent in granulosa cells of atretic preantral and antral
follicles. TXNRD2 immunostaining was observed in the granulosa and theca cells of healthy
preantral and antral, and conspicuously present in the steroidogenic cells of the CL. TXNRD2
staining was faint to absent in granulosa and theca cells of atretic preantral and antral follicles.
Conclusion SOD2 and TXNRD2 are present in granulosa and theca cells of developing healthy
follicles in the adult rat ovary, but staining is faint to absent in atretic follicles. It is possible that the
levels of these proteins in granulosa cells of atretic follicles are not high enough to scavenge
superoxide radicals, resulting in increased superoxide radical levels which may cause cell death
leading to follicular atresia.
1. Devine PJ, Perreault SD, Luderer U. Roles of reactive oxygen species and antioxidants in ovarian toxicity. Biol
Reprod 2012; 86:27
2. Jones ML, Mark PJ, Lewis JL, Mori TA, Keelan JA, Waddell BJ. Antioxidant defenses in the rat placenta in late
gestation: increased labyrinthine expression of superoxide dismutases, glutathione peroxidase 3, and uncoupling
protein 2. Biol Reprod 2010; 83:254-260