Figure 1.
Expression of PRDX4 protein was mainly located in granulosa cells of human ovaries.
Cellular localization of PRDX4 expression was evaluated in normal ovaries and PCOS ovaries. Five samples of PCOS ovaries and five samples of control group were fixed in 4% Paraformaldehyde for 24 h, replaced in 70% ethanol, dehydrated and embedded in paraffin. The paraffin-embedded samples were used for pathology and immunohistochemical staining. Sections were incubated with anti-PRDX4 antibody. A, B, C were normal ovarian tissues. D, E, F were PCOS ovarian tissues. Panel A, D were negative control slides,using IgG as primary antibody. Expression of PRDX4 protein was most prominent in granulosa cells in both two groups. Arrows indicated positive immunoreactive signals in granulosa cells visualized as brown staining. Scale bar = 50 µm.
Figure 2.
Expressions of PRDX4 mRNA and protein in normal ovaries and PCOS ovaries.
Total RNA and total protein were individually extracted from five separate ovarian tissues of normal and PCOS groups. Expression level of PRDX4 mRNA in control group were twice as high as that in PCOS group (p<0.05) (A). Consistent with above results, the Western blot results indicated that expression level of PRDX4 protein was overall lower in PCOS group compared to the control group (p<0.05) (C). And three representative Western blot results of each group were presented (B). The experiments were repeated four times. Data were presented as mean ± SEM. *, p<0.05.
Figure 3.
Different expression of PRDX4 protein and mRNA in LFs (mature follicles) and SFs (immature follicles).
Expression level of PRDX4 mRNA in granulosa cells were detected by qRT-PCR (A). Sixteen GCs samples from PCOS group and Sixteen from control group were used, every four samples were pooled to get enough cells. Expression level of PRDX4 mRNA in LFs was much higher than that in SFs, almost two fold in both normal group (A, Normal LFs vs. Normal SFs) and PCOS group (A, PCOS LFs vs. PCOS SFs) (p<0.05). To detect the change of expression of PRDX4 mRNA during IVM, granulosa cells from sixteen IVM patients were obtained. Expression level of PRDX4 mRNA was incresed after IVM (A, PCOS IVM vs. PCOS SFs), the same chang was found after in vivo maturation (A, PCOS IVM vs. PCOS LFs). Ten samples of control group were used in Western blot, every two samples were pooled to get enough cells. The representative data were showed in this figure (B). GAPDH was used as the standardized reference. PRDX4 was differently expressed in the granulosa cells of normal ovarian LFs and SFs. Expression level of PRDX4 protein in granulosa cells of LFs was almost twice as high as that in SFs (p<0.05) (C). The experiments were singly repeated four times. Data were presented as mean ± SEM. *, p<0.05; **, p<0.01.
Figure 4.
Schematic model illustrated the possible roles of PRDX4 in PCOS ovarian follicles.
PRDX4, as an antioxidant, mainly located in granulosa cells, can partly counteract ROS in follicles. The lowered expression of PRDX4 in PCOS ovaries may contribute to the oxidative stress related to granulosa cell apoptosis and follicular development abnormality.