CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes
MengQi Li
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China
Search for more papers by this authorChunYan Yang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorAnQin Duan
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorPeng Xiao
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China
College of Animal Science and Technology, Guangxi Vocational University of Agriculture, Nanning, China
Search for more papers by this authorXingRong Lu
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorXiaoYa Ma
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorYuanYuan Xu
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Search for more papers by this authorWei Zheng
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorChao Feng
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorXia Mo
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorChenQian Huang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorLiQing Huang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Search for more papers by this authorCorresponding Author
JiangHua Shang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Correspondence
HaiYing Zheng and JiangHua Shang, Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China.
Email: [email protected] and [email protected]
Search for more papers by this authorCorresponding Author
HaiYing Zheng
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Correspondence
HaiYing Zheng and JiangHua Shang, Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China.
Email: [email protected] and [email protected]
Search for more papers by this authorMengQi Li
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China
Search for more papers by this authorChunYan Yang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorAnQin Duan
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorPeng Xiao
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China
College of Animal Science and Technology, Guangxi Vocational University of Agriculture, Nanning, China
Search for more papers by this authorXingRong Lu
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorXiaoYa Ma
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorYuanYuan Xu
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Search for more papers by this authorWei Zheng
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorChao Feng
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorXia Mo
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorChenQian Huang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Search for more papers by this authorLiQing Huang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Search for more papers by this authorCorresponding Author
JiangHua Shang
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Correspondence
HaiYing Zheng and JiangHua Shang, Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China.
Email: [email protected] and [email protected]
Search for more papers by this authorCorresponding Author
HaiYing Zheng
Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning, China
Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning, China
Correspondence
HaiYing Zheng and JiangHua Shang, Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China.
Email: [email protected] and [email protected]
Search for more papers by this authorAbstract
This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB−) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB− group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB− group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB− group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB− group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.
CONFLICT OF INTEREST STATEMENT
None of the authors have any conflict of interest to declare.
Open Research
DATA AVAILABILITY STATEMENT
All data generated or analysed during this study are included in this published article.
REFERENCES
- Abazari-Kia, A. H., Mohammadi-Sangcheshmeh, A., Dehghani-Mohammadabadi, M., Jamshidi-Adegani, F., Veshkini, A., Zhandi, M., Cinar, M. U., & Salehi, M. (2014). Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte. Journal of Assisted Reproduction and Genetics, 31(3), 313–321.
- Aguila, L., Treulen, F., Therrien, J., Felmer, R., Valdivia, M., & Smith, L. C. (2020). Oocyte selection for in vitro embryo production in bovine species: Noninvasive approaches for new challenges of oocyte competence. Animals (Basel), 10(12), 2196.
- Ahuja, K., Batra, V., Kumar, R., & Datta, T. K. (2023). Transient suppression of Wnt signaling in poor-quality buffalo oocytes improves their developmental competence. Frontiers in Veterinary Science, 10, 1324647.
- Ashry, M., Lee, K., Mondal, M., Datta, T. K., Folger, J. K., Rajput, S. K., Zhang, K., Hemeida, N. A., & Smith, G. W. (2015). Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: Relevance to early embryonic development. Molecular Reproduction and Development, 82(3), 251–264.
- Azevedo, V. A. N., Barroso, P. A. A., Vasconcelos, E. M., Costa, F. C., Assis, E. I. T., Silva, B. R., Paulino, L. R. M., Silva, A. W. B., Donato, M. M. A., Peixoto, C. A., Silva, J. R. V., & Souza, A. L. P. (2022). Effects of Aloe vera extract on growth, viability, ultrastructure and expression of mRNA for antioxidant enzymes in bovine secondary follicles cultured in vitro. Animal Reproduction Science, 247, 107078.
- Bettegowda, A., Patel, O. V., Lee, K. B., Park, K. E., Salem, M., Yao, J., Ireland, J. J., & Smith, G. W. (2008). Identification of novel bovine cumulus cell molecular markers predictive of oocyte competence: Functional and diagnostic implications. Biology of Reproduction, 79(2), 301–309.
- Bhardwaj, R., Ansari, M. M., Pandey, S., Parmar, M. S., Chandra, V., Kumar, G. S., & Sharma, G. T. (2016). GREM1, EGFR, and HAS2; the oocyte competence markers for improved buffalo embryo production in vitro. Theriogenology, 86(8), 2004–2011.
- Chaubey, G. K., Kumar, S., Kumar, M., Sarwalia, P., Kumaresan, A., De, S., Kumar, R., & Datta, T. K. (2018). Induced cumulus expansion of poor quality buffalo cumulus oocyte complexes by interleukin-1beta improves their developmental ability. Journal of Cellular Biochemistry, 119(7), 5750–5760.
- Chen, L., Zhang, S., Gu, Y., Peng, Y., Huang, Z., Gong, F., & Lin, G. (2022). Vacuolization in embryos on days 3 and 4 of in vitro development: Association with stimulation protocols, embryo development, chromosomal status, pregnancy and neonatal outcomes. Frontiers in Endocrinology, 13, 985741.
- Dong, R., Lv, P., Han, Y., Jiang, L., Wang, Z., Peng, L., Ma, Z., Xia, T., Zhang, B., & Gu, X. (2022). Enhancement of astrocytic gap junctions Connexin43 coupling can improve long-term isoflurane anesthesia-mediated brain network abnormalities and cognitive impairment. CNS Neuroscience & Therapeutics, 28(12), 2281–2297.
- Duan, J., Chen, H., Li, Y., Xu, D., Li, X., Zhang, Z., Cheng, J., Yang, L., & Li, Q. (2021). 17β-estradiol enhances porcine meiosis resumption from autophagy-induced gap junction intercellular communications and Connexin 43 phosphorylation via the MEK/ERK signaling pathway. Journal of Agricultural and Food Chemistry, 69(40), 11847–11855.
- Dutta, R., Mandal, S., Lin, H. A., Raz, T., Kind, A., Schnieke, A., & Razansky, D. (2020). Brilliant cresyl blue enhanced optoacoustic imaging enables non-destructive imaging of mammalian ovarian follicles for artificial reproduction. Journal of the Royal Society Interface, 17(172), 20200776.
- Fathi, M., & Elkarmoty, A. F. (2021). Effect of adding growth factors during in vitro maturation on the developmental potentials of ewe oocytes selected by brilliant cresyl blue staining. Vet World, 14(2), 452–456.
- Ghanem, N., Ahmed, D. A. R., Dessouki, S. M., Faheem, M. S., Gad, A. Y., Peippo, J., & Barkawi, A. H. (2021). Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation. Zygote (Cambridge, England), 29(4), 314–324.
- Lee, S., Kang, H. G., Jeong, P. S., Nanjidsuren, T., Song, B. S., Jin, Y. B., Lee, S. R., Kim, S. U., & Sim, B. W. (2020). Effect of oocyte quality assessed by brilliant Cresyl blue (BCB) staining on cumulus cell expansion and sonic hedgehog signaling in porcine during in vitro maturation. International Journal of Molecular Sciences, 21(12), 4423.
- Memili, E., Peddinti, D., Shack, L. A., Nanduri, B., McCarthy, F., Sagirkaya, H., & Burgess, S. C. (2007). Bovine germinal vesicle oocyte and cumulus cell proteomics. Reproduction, 133(6), 1107–1120.
- Mohapatra, S. K., Sandhu, A., Neerukattu, V. S., Singh, K. P., Selokar, N. L., Singla, S. K., Chauhan, M. S., Manik, R. S., & Palta, P. (2015). Buffalo embryos produced by handmade cloning from oocytes selected using brilliant cresyl blue staining have better developmental competence and quality and are closer to embryos produced by in vitro fertilization in terms of their epigenetic status and gene expression pattern. Cellular Reprogramming, 17(2), 141–150.
- Mohr-Sasson, A., Orvieto, R., Blumenfeld, S., Axelrod, M., & Haas, J. (2020). The association between follicle size and oocyte development as a function of final follicular maturation triggering. Reproductive Biomedicine Online, 40, 887–893.
- Mourot, M., Dufort, I., Gravel, C., Algriany, O., Dieleman, S., & Sirard, M. A. (2006). The influence of follicle size, FSH-enriched maturation medium, and early cleavage on bovine oocyte maternal mRNA levels. Molecular Reproduction and Development, 73(11), 1367–1379.
- Mulchandani, V., Banerjee, A., Vadlamannati, A. V., Kumar, S., & Das, S. J. (2023). Connexin 43 trafficking and regulation of gap junctional intercellular communication alters ovarian cancer cell migration and tumorigenesis. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie, 159, 114296.
- Nivet, A. L., Vigneault, C., Blondin, P., & Sirard, M. A. (2013). Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine. Reproduction, 145(6), 555–565.
- Ochota, M., Pasieka, A., & Niżański, W. (2016). Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes. Theriogenology, 85(5), 922–927.
- Peddinti, D., Memili, E., & Burgess, S. C. (2010). Proteomics-based systems biology modeling of bovine germinal vesicle stage oocyte and cumulus cell interaction. PLoS One, 5(6), e11240.
- Piras, A. R., Ariu, F., Zedda, M. T., Paramio, M. T., & Bogliolo, L. (2020). Selection of immature cat oocytes with brilliant Cresyl blue stain improves in vitro embryo production during non-breeding season. Animals (Basel), 10(9), 1496.
- Salviano, M. B., Collares, F. J., Becker, B. S., Rodrigues, B. A., & Rodrigues, J. L. (2016). Bovine non-competent oocytes (BCB-) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development. Zygote (Cambridge, England), 24(2), 245–251.
- Shin, K. T., Nie, Z. W., Zhou, W., Zhou, D., Kim, J. Y., Ock, S. A., Niu, Y. J., & Cui, X. S. (2020). Connexin 43 knockdown induces mitochondrial dysfunction and affects early developmental competence in porcine embryos. Microscopy and Microanalysis, 26(2), 287–296.
- Sirait, B., Wiweko, B., Jusuf, A. A., Iftitah, D., & Muharam, R. (2021). Oocyte competence biomarkers associated with oocyte maturation: A review. Frontiers in Cell and Development Biology, 9, 710292.
- Sjöblom, P., Menezes, J., Cummins, L., Mathiyalagan, B., & Costello, M. F. (2006). Prediction of embryo developmental potential and pregnancy based on early stage morphological characteristics. Fertility and Sterility, 86(4), 848–861.
- Walker, B. N., Nix, J., Wilson, C., Marrella, M. A., Speckhart, S. L., Wooldridge, L., Yen, C. N., Bodmer, J. S., Kirkpatrick, L. T., Moorey, S. E., Gerrard, D. E., Ealy, A. D., & Biase, F. H. (2022). Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells. Reproductive Biology and Endocrinology, 20(1), 119.
- Walter, J., Monthoux, C., Fortes, C., Grossmann, J., Roschitzki, B., Meili, T., Riond, B., Hofmann-Lehmann, R., Naegeli, H., & Bleul, U. (2020). The bovine cumulus proteome is influenced by maturation condition and maturational competence of the oocyte. Scientific Reports, 10(1), 9880.
- Zhang, J., Riquelme, M. A., Hua, R., Acosta, F. M., Gu, S., & Jiang, J. X. (2022). Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress. Elife, 11, 11.
Citing Literature
