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. 2025 Jun 2;21(1):396.
doi: 10.1186/s12917-025-04806-9.

Co-expression of LncRNA and mRNA in the cumulus-oocyte complex of rabbits exposed to ammonia

Jia Cui #  1   2 , Fengyang Wu #  2   3 , Xinyu Yang  2 , Yanhua Liu  2 , Shudong Liu  2 , Shuaijuan Han  2 , Jiawei Guo  4 , Baojiang Chen  5
Affiliations

Co-expression of LncRNA and mRNA in the cumulus-oocyte complex of rabbits exposed to ammonia

Jia Cui et al. BMC Vet Res. .

Abstract

Background: Ammonia (NH3) is an environmental pollutant and a potent reproductive stressor widely found in rabbit houses. Exposure to ammonia can result in follicle atresia, affect oocyte maturation and cause cumulus cell apoptosis. Long non-coding RNA (lncRNA) is an important factor in the regulation of cumulus cell development and oocyte maturation. The potential molecular mechanism of NH3 in the induction of cumulus-oocyte complex (COCs) toxicity and the regulatory role of lncRNA in COCs are currently unclear.

Methods: A total of 150 female IRA rabbits (35 days old) were randomly divided into three groups, and kept in environmental control rooms for four weeks. The rabbits in the control group (CG) were kept under an NH3 concentration of < 3 ppm. The two treatment groups were kept under NH3 concentrations of 10 ppm (low ammonia concentration, LAC) and 30 ppm (high ammonia concentration, HAC). We used a combination of RNA deep sequencing, quantitative real-time polymerase chain reaction (qRT-PCR), and bioinformatic analysis to explore the regulatory mechanism of lncRNA and messenger RNA (mRNA) in COCs.

Results: We found that primordial follicles and primary follicles were significantly decreased while atretic follicles were significantly increased in the NH3-treated groups. The results from Gene Ontology (GO) items showed that female meiosis sister chromatid cohesion and the regulation of follicle-stimulating hormone secretion were involved in the mechanism of rabbits exposed to NH3. The results demonstrated that the mammalian target of the rapamycin (mTOR) signaling pathway and the transforming growth factor-beta (TGF-beta) signaling pathway inhibits germ cell development and follicular growth in the LAC versus the CG group. LncRNAs were involved in the apoptosis of female germ cells via the hypoxia-inducible factor (HIF-1) signaling pathway in the HAC versus the CG group. Co-expression analysis found that lncRNA MAPK3 and lncRNA SHC1 were correlated with changes in cumulus cell and oocyte function after NH3 exposure.

Conclusions: These results indicate that NH3 affected the development and function of COCs by influencing lncRNA expression.

Keywords: Ammonia; Cumulus-oocyte complex; LncRNA; Rabbit; Reproductive dysfunction.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: All experiments were performed in accordance with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), approved by the Ethics Committee of Animal Experimentation of Hebei Agricultural University (Protocol 2021083) and obtained informed consent from Xingtai Kangming Breeding Co., LTD (Xingtai, China) to use the animals. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Effects of NH3 on ovarian structure and morphology (A-C) and the numbers of primordial, primary, secondary, and atretic follicles in the ovaries. (D) A: CG-< 3 ppm NH3, B: LAC-10 ppm NH3, C: HAC-30 ppm NH3; Key: Black arrows (↑) indicate primordial follicles. Red arrows (↑) indicate primary follicles. Green arrows (↑) indicate secondary follicles. Blue arrows (↑) indicate atretic follicles. Bars represent means ± SD, n = 4. * P < 0.05 and ** P < 0.01 indicate significantly different and highly significantly different values, respectively
Fig. 2
Fig. 2
Expression levels and genomic features of lncRNAs and mRNAs of rabbit cumulus-oocyte complexes under different levels of NH3 exposure. The length distribution of lncRNAs and mRNAs (A). Expression levels of mRNAs and lncRNAs in the CG, LAC and HAC groups were compared with the FPKM method (B). Boxplots of FPKM distributions were drawn using all the mRNAs and lncRNAs expression data, respectively. The lncRNAs from all samples generated exonic, intronic, and intergenic regions in the CG, LAC and HAC groups (C). CG: < 3 ppm NH3; LAC: 10 ppm NH3; HAC: 30 ppm NH3
Fig. 3
Fig. 3
Differentially expressed genes of rabbit cumulus-oocyte complexes under different levels of NH3 exposure (CG: < 3 ppm NH3; LAC: 10 ppm NH3; HAC: 30 ppm NH3). Differential expression levels of mRNAs (A) and lncRNAs (B). Hierarchical cluster analysis of significantly differentially expressed mRNAs (C) and lncRNAs (D). Red indicates up-regulated RNA and green indicates down-regulated RNA
Fig. 4
Fig. 4
Genes most enriched as determined by gene ontology (GO) and KEGG enrichment analysis of rabbit cumulus-oocyte complexes under different levels of NH3 exposure. The 20 most enriched differentially expressed genes (DEGs) by GO enrichment analysis in the LAC versus CG groups (A) and the HAC versus CG groups (B). The top 20 DEGs by KEGG enrichment analysis in the LAC versus CG groups (C) and the HAC versus CG groups (D). CG: < 3 ppm NH3; LAC: 10 ppm NH3; HAC: 30 ppm NH3
Fig. 5
Fig. 5
Co-expression network of representative lncRNAs and their partial target mRNAs in the CG vs. LAC (A) and the CG vs. HAC groups (B). The relationships between lncRNA-mRNA were reconstructed based on expression correlation coefficients (Pearson correlation > 0.99 or < − 0.99) using Cytoscape software (v2.8.3). Red indicates up-regulated RNA and green indicates down-regulated RNA. CG: < 3 ppm NH3; LAC: 10 ppm NH3; HAC: 30 ppm NH3
Fig. 6
Fig. 6
The lncRNA and mRNA expressions were validated using qRT-PCR. Differentially expressed lncRNAs of LAC (A) and HAC (B) groups and genes of LAC (C) and HAC (D) groups were validated using qRT-PCR. Data of Illumina Hiseq and qRT-PCR were analyzed using SPSS version 21. Bars represent means ± SD, n = 4. CG: < 3 ppm NH3; LAC: 10 ppm NH3; HAC: 30 ppm NH3. CYP11a: recombinant Cytochrome P450 11a; CYP19a: recombinant Cytochrome P450 19a; KITLG: KIT Ligand; SIRT1: sirtuin 1
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