Repeated Superovulation via PMSG/hCG Administration Induces 2-Cys Peroxiredoxins Expression and Overoxidation in the Reproductive Tracts of Female Mice

Abstract

Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.

Keywords: 2-Cys peroxiredoxins; exogenous gonadotropin; female mice; oxidative stress; reproductive organ environment.

Fig. 1.

Histological changes in female reproductive tracts (ovary, oviduct, uterus) of mice following treatment with PMSG/hCG. Whole reproductive tract sections from untreated mice (normal) as well as from mice intraperitoneally (ip) injected with 5 IU PMSG / 5 IU hCG once (PMSG/hCG X1) or three times (PMSG/hCG X3) stained with Hematoxylin and eosin (H&E). O, oocyte; F, follicle at any developmental stage; CL, corpus luteum; E, endometrium; EC, epithelial cells; EE, endometrial epithelial cells; L, lumen. Scale bar =100 μm.

Fig. 2.

Representative micrographs of immunohistochemistry staining in the ovary, oviduct and uterus of mice. (A) Peroxiredoxin 1-, (B) Peroxiredoxin 2-, and (C) Peroxiredoxin 3-stained sections from untreated mice (normal) as well as from mice ip injected with 5 IU PMSG/5 IU hCG once (PMSG/hCG X1) or three times (PMSG/hCG X3). Scale bar = 100 μm. (D) The Relative immunoreactivity levels of Prx1-3 in the selected cells of reproductive organ region were assigned -, not detectable; +, weak staining; ++, moderate staining; +++, strong staining.

Fig. 3.

Expression of the 2-Cys Peroxiredoxins and 2-Cys Peroxiredoxins-SO_2/3 proteins in the ovaries of mice following stimulation with PMSG/hCG. (A) Western blot analysis of the protein expression levels of β-actin (loading control), Prx1, Prx2, Prx3, as well as Prx1-, 2-, and 3-SO_2/3 in the ovaries of ICR mice subjected to single (×1) or repeated (×3) PMSG/hCG injections. Ovaries were harvested 12 h after the final dose of PMSG/hCG. Tissue collection of the untreated mice (normal) was restricted to the estrus/metestrus stage of the natural estrous cycle.(B) Quantification of the relative protein expression levels of Prx1, 2, and 3 as well as Prx1-, 2-, and 3-SO_2/3 normalized to β-actin expression. Data in the bar graphs represent means ± SEM of three independent experiments. ^**p < 0.01, ^***p < 0.001 compared with normal.

Fig. 4.

Expression of the 2-Cys Peroxiredoxins and 2-Cys Peroxiredoxins-SO_2/3 proteins in the oviducts of mice following stimulation with PMSG/hCG. (A) Western blot analysis of the protein expression levels of β-actin (loading control), Prx1, Prx2, Prx3, as well as Prx1-, 2-, and 3-SO_2/3 in the oviducts of ICR mice subjected to single(×1) or repeated (×3) PMSG/hCG injections. Oviducts were harvested 12 h after the final dose of PMSG/hCG. Tissue collection of the untreated mice (normal) was restricted to the estrus/metestrus stage of the natural estrous cycle.(B) Quantification of the relative expression levels of Prx1, 2, and 3 as well as Prx1-, 2-, and 3-SO_2/3 normalized to β-actin expression. Data in the bar graphs represent means ± SEM of three independent experiments. ^*p < 0.05, ^**p < 0.01 compared with normal.

Fig. 5.

Expression of the 2-Cys Peroxiredoxins and 2-Cys Peroxiredoxins-SO_2/3 proteins in the uteri of mice following stimulation with PMSG/hCG. (A) Western blot analysis of the protein expression levels of β-actin (loading control), Prx1, Prx2, Prx3, as well as Prx1-, 2-, and 3-SO_2/3 in the uteri of ICR mice subjected to single or repeated (3×) PMSG/hCG injections. Uteri were harvested 12 h after the final dose of PMSG/hCG. Tissue collection of the untreated mice (normal) was restricted to the estrus/metestrus stage of the natural estrous cycle. (B) Quantification of the relative expression levels of Prx1, 2, and 3 as well as Prx-1, 2-, and 3-SO_2/3 normalized to β-actin expression. Data in the bar graphs represent means ± SEM of three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with normal.