Status of autophagy, lysosome activity and apoptosis during corpus luteum regression in cattle

Abstract

Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.

Fig. 1.

Expression of autophagy-related genes during bovine CL regression. Relative mRNA levels of LC3α (A), LC3β (B), Atg3 (C), Atg7 (D) and mTOR (E). All data are normalized to GAPDH expression and shown as means ± SEM. *P < 0.05. **P < 0.01. n, total number of analyzed tissues.

Fig. 2.

Evaluation of LC3 proteins and autophagy activity during bovine CL regression. Expression of the active membrane-bound LC3-II protein and inactive LC3-I protein detected by western blotting analysis (A) and the relative level of active LC3-II protein to GAPDH in the mid and late CL (B). Fluorescence microscopy of the autophagic vacuole accumulation in mid (C) and late (D) CL; Strong and diffuse green signals were observed in the late CL (white arrows). Quantification of autophagy activity was performed by calculating the fluorescence intensity (E). The data are expressed as means ± SEM. *P < 0.05. n, total number of analyzed tissues.

Fig. 3.

Expression of cathepsin-encoding genes during bovine CL regression. Relative mRNA levels of CTSB (A), CTSZ (B) and CTSD (C). The data are normalized to GAPDH expression and are shown as means ± SEM. *P < 0.05. **P < 0.01. n, total number of analyzed tissues.

Fig. 4.

Detection of lysosomal cathepsin B protein, activity and active lysosomes in the regressing bovine CL. Immunohistochemical localization of cathepsin B protein in the mid (A) and late (B) CL. Green fluorescent dots of cathepsin B in the cytoplasm of late CL (white arrows) cells. Cathepsin B activity in the mid (C) and late (D) CL. The activity was indicated by diffuse red fluorescence in the cytoplasm of late luteal cells (white arrows). Relative fluorescence intensity of cathepsin B (E). Intracellular localization of lysosomes in the mid (F) and late (G) CL; Active lysosomes appeared as diffuse red fluorescence spots in the cytoplasm of luteal cells especially in the late CL (white arrows). Lysosomal size was performed (H). The data are expressed as means ± SEM. *P < 0.05. **P < 0.01. n, total number of analyzed tissues.