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. 2022 Jul 18:13:926795.
doi: 10.3389/fphys.2022.926795. eCollection 2022.

Coprophagy Prevention Decreases the Reproductive Performance and Granulosa Cell Apoptosis via Regulation of CTSB Gene in Rabbits

Guohua Song  1 , Yadong Wang  1 , Yaling Wang  1 , Yixuan Jiang  1 , Shuaijie Sun  1 , Hanfang Cai  1 , Guirong Sun  1 , Ming Li  1 , Massimo Bionaz  2 , Huifen Xu  1
Affiliations

Coprophagy Prevention Decreases the Reproductive Performance and Granulosa Cell Apoptosis via Regulation of CTSB Gene in Rabbits

Guohua Song et al. Front Physiol. .

Abstract

Coprophagy is an instinctive behavior in rabbit with important effects on growth and reproductive performance. The underlying mechanism of this effect in rabbit is unknown. Here, we used Elizabeth circle as a coprophagy preventing model in female rabbits and assess feed intake, growth, and reproductive performance. We found that preventing coprophagy did not affect feed intake but decreased body weight and weight of several organs and tissues and resulted in complete reproductive failure during the late pregnancy period, accompanied by reduced levels of plasma progesterone. RNA-seq analysis of rabbit ovarian tissues revealed that preventing coprophagy affected significantly 241 genes (DEGs), with the large majority being downregulated. Bioinformatic analyses revealed that those DEGs are mostly involved in apoptosis, immune response, and metabolic pathways. Among DEGs, the lysosomal cysteine protease cathepsin B (CTSB) was significantly downregulated in the coprophagy prevention group. Further studies using siRNA and adenovirus overexpression systems revealed that CTSB promotes the proliferation of rabbit granulosa cells (GCS) and prevents apoptosis. Measurement of transcripts coding for proteins related to apoptosis revealed a minor transcriptomic effect of CTSB, indicating that its effect is likely post-transcriptional. Overexpression of CTSB increased secretion of progesterone and estradiol, partly via upregulation of CYP19A1 while inhibition of CTSB decreased progesterone secretion partly via downregulation of the StAR gene. In conclusion, our study demonstrated the detrimental effect on reproduction by preventing coprophagy with a main role for this response played by CTSB on the granulosa cells of the ovary.

Keywords: CTSB; coprophagy; granulosa cells; rabbits; reproductive performance.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effects of coprophagy prevention on (A) feed intakes; (B) body weight; (C) weight of different tissues and internal organs; and (D) serum biochemical parameters in rabbits [progesterone (PROG), albumin (Alb), triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL), and LDL-cholesterol (LDL)]. Statistical differences between groups were indicated as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
FIGURE 2
FIGURE 2
Coprophagy prevention increased cell apoptosis in ovary of rabbits. (A) Representative images of ovarian stained by TUNEL assay, cell nuclei were stained by DAPI (blue), apoptotic cells were stained with TUNEL (green). (B) Quantification of the proportion of ovarian apoptotic cells as detected by TUNEL assay in each group of rabbits. Scale bar = 50 μm **p < 0.01.
FIGURE 3
FIGURE 3
Transcriptomic analysis of ovary tissues in rabbit where coprophagy was prevented (i.e., experimental group) and a control group (n = 3 per group). (A) Volcano plot of differentially expressed genes (expression ratio experimental/control group). (B) KEGG pathways enriched (p < 0.05) in DEGs by using KOBAS. (C) Summary of main categories of KEGG pathways generated by using the Dynamic Impact Approach. Blue horizontal bars denote impact (larger the bar, larger the impact) and squares besides the blue horizontal bars denote flux of the pathway that can be induced or inhibited as indicated in the bottom legend. (D) RNA-seq vs. RT-qPCR for transcripts selected among the ones related to metabolism, apoptosis, and signaling pathways. (E) Additional transcripts measured by RT-qPCR involved in apoptosis and related to cathepsin B activity.
FIGURE 4
FIGURE 4
Characterization of granulosa cells (GCS) isolate from ovaries of healthy female rabbits (5 months of age). (A) H&E staining (40×). (B) Expression of follicle-stimulating hormone receptor (FSHR) in GCS isolated from ovaries of rabbits as detected via immunofluorescence staining (red = FSHR; blue = nuclei) (200×). Pseudopodia between cells are indicated with white arrows.
FIGURE 5
FIGURE 5
Adenovirus-mediated overexpression and siRNA-mediated interference of CTSB in rabbit GCS. The transcription of mRNA via RT-qPCR (A,D) and the protein expression via Western blot (B,C,E,F) of CTSB were highly affected by overexpression and interference treatments. Each experiment was performed with three biological replicates, and the results are expressed as the mean ± SEM. *p < 0.05, **p < 0.01.
FIGURE 6
FIGURE 6
CTSB overexpression and interference on the proliferation and apoptosis of rabbit GCS. Transcription of genes associated with cell proliferation and apoptosis after CTSB overexpression (A) and knockdown (C). Rabbit GCS proliferation after CTSB overexpression (B) and knockdown (D). CTSB overexpression decreased the apoptosis in GCS as assessed by flow cytometer (E,F). CTSB knockdown increased apoptosis of GCS as assessed by using a flow cytometer (G,H). *p < 0.05; **p < 0.01.
FIGURE 7
FIGURE 7
Effects of CTSB overexpression and interference on progesterone and estradiol secretion in rabbit GCS. Transcription of key genes related to hormone secretion in GCS with CTSB overexpression (A) or interference (D). Level of progesterone and estradiol secretion in rabbit GCS after CTSB overexpression (B,C) and interference (E,F). Each experiment had three biological replicates and the results are expressed as the mean ± SE. * p < 0.05, ** p < 0.01.
FIGURE 8
FIGURE 8
Model summarizing findings from the experiment.
See this image and copyright information in PMC

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