Oxidized Oils and Oxidized Proteins Induce Apoptosis in Granulosa Cells by Increasing Oxidative Stress in Ovaries of Laying Hens

Abstract

The storage and preparation of corn for animal feed inevitably lead to lipid and protein peroxidation. Granulosa cells play an important role in follicular development in the ovaries, and hen laying productivity is likely to be dependent on follicle health and number. We hypothesized that oxidized oil and protein induce apoptosis via oxidative stress in laying hen granulosa cells. A sample of 360 38-week-old Lohmann commercial laying hens was used in a 2 × 2 factorial design for 8 weeks. Dietary treatments included dietary oil (fresh corn oil (FO) or oxidized corn oil (OO)) and corn gluten meal (fresh corn gluten meal (FP) or oxidized corn gluten meal (OP)). Productivity, ovarian histology, granulosa cell apoptosis, and indicators of oxidative stress were evaluated in all groups. Both dietary OO and OP decreased egg production and the average daily feed intake (ADFI) of laying hens. Flow cytometry, TUNEL, and real-time PCR revealed that both dietary OO and OP induced granulosa cell apoptosis in prehierarchical and hierarchical follicles. Furthermore, dietary OO and OP caused oxidative stress in prehierarchical and hierarchical follicles, as indicated by the downregulation of antioxidant-related-gene expression. Moreover, forkhead box O1 (FoxO1), extracellular regulated protein kinase (ERK), and c-Jun NH₂ kinase (JNK) are involved in potential apoptosis regulation pathways in the granulosa cells of laying hens fed OO and OP, as indicated by the upregulation of FoxO1 expression and downregulation of ERK/JNK expression. These results indicate that OO and OP induce granulosa cell apoptosis via oxidative stress, and the combined use of OO and OP aggravates the adverse effects of oxidative stress in laying hens.

Figure 1

Effect of dietary oxidized oil and oxidized protein on ovary histology of laying hens. Images of the ovaries from each treatment were captured using a camera. Black arrows indicated atretic follicle (a, bar = 1 cm). Ovary histology was from ovarian tissue in which small yellow follicles and hierarchical follicle were excluded. Hematoxylin and eosin stain shows the appearance of ooplasm (bar = 100 μm). The enclosed box in each panel shows the overall images (b). CON: control; OP: oxidized protein; OO: oxidized oil; Combination: oxidized oil+oxidized protein.

Figure 2

Effect of dietary-oxidized oil and dietary-oxidized protein on prehierarchical follicle (a–c) and hierarchical follicle (d–f) granulosa cell apoptosis rate of laying hens. Granulosa cells stained with fluorescein isothiocyanate- (APC-) Annexin V/propidium iodide (PI). Flow cytometric analysis defined early stage apoptosis as APC-Annexin V⁺ and PI⁻. Late stage apoptosis was defined as APC-Annexin V⁺ and PI⁺ in cells. Flow cytometry profiles with APC-Annexin V (x-axis) and PI (y-axis) staining (a, d). Calculated proportion of cells undergoing apoptosis (b, c, e, f). Values are the means ± SEMs (n = 4). Means without a common letter are different; P < 0.05. CON: control; FP: fresh protein; OP: oxidized protein; FO: fresh oil; OO: oxidized oil; Combination: oxidized oil+oxidized protein.

Figure 3

Effect of dietary oxidized oil and oxidized protein on levels of apoptotic granulosa cells in prehierarchical follicles (a, b) and hierarchical follicles (c, d) granulosa cells of laying hens. Nuclei were blue (DAPI). White arrows indicated TUNEL-positive granulosa cells (green) and quantified (b, d) in at least ten images (bar = 100 μm). Values are the means ± SEMs (n = 4). CON: control; FP: fresh protein; OP: oxidized protein; FO: fresh oil; OO: oxidized oil; Combination: oxidized oil+oxidized protein.

Figure 4

Effect of dietary oxidized oil and oxidized protein on the expression of apoptosis-related genes in prehierarchical follicles (a–d) and hierarchical follicles (e–h) granulosa cells of laying hens. Values are the means ± SEMs (n = 4). All the data were acquired using real-time PCR. Means without a common letter are different; P < 0.05. FP: fresh protein; OP: oxidized protein; FO: fresh oil; OO: oxidized oil.

Figure 5

Effect of dietary oxidized oil and oxidized protein on the expression of FoxO1 (a, d), ERK (b, e), and JNK (c, f) in prehierarchical follicle and hierarchical follicle granulosa cells of laying hens. Values are the means ± SEMs (n = 4). All the data were acquired using real-time PCR. Means without a common letter are different; P < 0.05. FP: fresh protein; OP: oxidized protein; FO: fresh oil; OO: oxidized oil.

Figure 6

Effect of dietary oxidized oil and oxidized protein on ovary antioxidant enzyme activity of laying hens. Antioxidant enzyme activity assay was performed in the ovary in which small yellow follicles and hierarchical follicle were excluded. Values are the means ± SEMs (n = 6). Means without a common letter are different; P < 0.05. FP: fresh protein; OP: oxidized protein; FO: fresh oil; OO: oxidized oil; MDA: malondialdehyde; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; T-AOC: total antioxidant capacity.

Figure 7

Effect of dietary oxidized oil and oxidized protein on the expression of antioxidant-related genes in prehierarchical follicle (a) and hierarchical follicle (b) granulosa cells of laying hens. Values are the means ± SEMs (n = 4). All the data were acquired using real-time PCR. Means without a common letter are different; P < 0.05. FP: fresh protein; OP: oxidized protein; FO: fresh oil; OO: oxidized oil.