Tauroursodeoxycholic Acid Enhances the Quality of Postovulatory Aged Oocytes by Alleviating Oxidative Stress, Apoptosis, and Endoplasmic Reticulum Stress in Pigs

Abstract

One of the major factors causing reduced developmental capacity of aged porcine oocytes is the induction of oxidative stress during oocyte aging. Tauroursodeoxycholic acid (TUDCA) supports cellular function by acting as an antioxidant and free radical scavenger. The aim of this study is to evaluate whether exogenous supplementation of TUDCA to the porcine in vitro maturation system can ameliorate the compromised quality of aged oocytes by mitigating free radical production. We found that TUDCA was able to effectively maintain normal oocyte morphology, cortical granule distribution, and spindle structure during postovulatory aging. Additionally, the blastocyst rate and total cell number in blastocysts were significantly increased in aged porcine oocytes treated with TUDCA. Importantly, aged porcine oocytes treated with TUDCA reduced ROS levels, increased the expression levels of GSH and SOD1 genes, and improved the mitochondrial membrane potential ratio. Further study demonstrated that TUDCA significantly alleviated apoptosis in aged porcine oocytes, confirmed by the decreased Caspase 3 levels and ratio of BAX to BCL2. Interestingly, TUDCA could effectively alleviate the phenomenon of endoplasmic reticulum stress triggered during the oocyte aging process. Taking these findings together, our study demonstrates that TUDCA supplementation beneficially affects the quality of aged porcine oocytes by suppressing oxidative stress, apoptosis, and endoplasmic reticulum stress.

Keywords: ROS; apoptosis; endoplasmic reticulum stress; postovulatory aged oocyte; tauroursodeoxycholic acid.

Figure 1

Influences of TUDCA on morphology and developmental potential of aged oocytes. (A) Typical images of normal, fragmented, and dead oocytes derived from the Control (i.e., fresh oocyte group), Aging (i.e., aged oocyte group), and Aging + TUDCA (i.e., TUDCA-treated aged oocyte group) groups. (B) The percentage of oocytes with normal morphology (Control n = 487; Aging n = 473; Aging + 50 μM TUDCA n = 484; Aging + 200 μM TUDCA n = 474; Aging + 500 μM TUDCA n = 463). (C) Representative images of cleavage- and blastocyst-stage embryos derived from control, aged, and TUDCA-treated oocyte groups. (D) The rate of cleavage and blastocyst formation (Control n = 164; Aging n = 156; Aging + 50 μM TUDCA n = 159; Aging + 200 μM TUDCA n = 162; Aging + 500 μM TUDCA n = 157). (E) Representative images of blastocysts after DAPI staining. (F) Numbers of total cells (TCs) in blastocysts. Bars = 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Influences of TUDCA on CG distribution, spindle formation, and nuclear maturation in aged porcine oocytes. (A) Typical pictures of CG distribution types. Normal CGs are shown on the left (arrow), and abnormal CGs are indicated on the right (arrow). (B) Percentage of abnormal CG distributions (Control n = 39; Aging n = 41; Aging + TUDCA n = 41). (C) Typical images of spindle structures. The red color represents DNA, and the green color indicates the spindle. (D) The percentages of abnormal spindle morphologies in oocytes (Control n = 36; Aging n = 36; Aging + TUDCA n = 33). (E) The proportions of oocytes that showed MII stage (Control n = 151; Aging n = 152; Aging + TUDCA n = 149). Scale bars represent 30 μm. * p < 0.05, ** p < 0.01.

Figure 3

TUDCA enhanced the antioxidative ability in aged porcine oocytes. (A) Typical images of oocytes stained with H2DCFDA (green color) and CellTracker Blue CMF2HC (blue color). (B) Fluorescence intensity of ROS staining (Control n = 30; Aging n = 30; Aging + TUDCA n = 30). (C) Fluorescence intensity of GSH staining (Control n = 30; Aging n = 30; Aging + TUDCA n = 30). (D) Expression levels of antioxidative ability-related genes. Scale bars = 100 μm in (A). * p < 0.05. ** p < 0.01, *** p < 0.001.

Figure 4

Influences of TUDCA on mitochondrial membrane potential in aged porcine oocytes. (A) JC-1 staining. (B) JC-1 fluorescence intensity (red/green ratio) (Control n = 45; Aging n = 45; Aging + TUDCA n = 45). Scale bar = 100 μm. ** p < 0.01, *** p < 0.001.

Figure 5

TUDCA decreased apoptosis levels in aged porcine oocytes. (A) Typical images of oocytes stained with TUNEL, apoptotic cells stained in red (white arrows), and DNA stained in blue. (B) TUNEL-positive cell rate (Control n = 37; Aging n = 36; Aging + TUDCA n = 36). (C) Images of porcine oocytes stained with Caspase 3. (D) Fluorescence intensity of Caspase 3 (Control n = 23; Aging n = 21; Aging + TUDCA n = 21). (E) The expression levels of apoptosis-related genes. Scale bars = 100 μm. * p < 0.05. ** p < 0.01, *** p < 0.001.

Figure 6

TUDCA improved the quality of aged oocytes by alleviating ER stress. (A,B) Expression patterns (A) and levels (B) of XBP1-u and XBP1-s genes in aged oocytes treated with TUDCA. (C) Expression levels of ER stress-related genes. * p < 0.05. ** p < 0.01, *** p < 0.001 (Figure S1).

Figure 7

The levels of ROS, GSH, Caspase 3, and TUNEL-positive cell rate in oocytes following H₂O₂ and TUDCA treatment. (A) Fluorescence intensity of ROS (H₂O₂ n = 46; H₂O₂ + TUDCA n = 45) and GSH (H₂O₂ n = 45; H₂O₂ + TUDCA n = 45) staining. (B) Fluorescence intensity of Caspase 3 (H₂O₂ n = 35; H₂O₂ + TUDCA n = 35). (C) TUNEL-positive cell rate (H₂O₂ n = 43; H₂O₂ + TUDCA n = 44). * p < 0.05, ** p < 0.01, *** p < 0.001.