Aquaporin channels (AQPs) are known to play an important role in the development of ovarian follicles through their function in water transport pathways. Compared to other AQPs, research on the role of AQP4 in female reproductive physiology, particularly in cattle, remains limited. In our previous study, gene chip microarray data showed a downregulation of AQP4 in bovine cystic follicles. This study was performed to validate the AQP4 expression level at the protein level in bovine follicles using immunohistochemistry, Western blotting, and immunoprecipitation assays. Immunostaining data showed that AQP4 was expressed in granulosa and theca cells of bovine ovarian follicles. The ovarian follicles were classified according to size as small (< 10 mm) or large (> 25 mm) in diameter. Consistent with earlier microarray data, semi-quantitative PCR data showed a decrease in AQP4 mRNA expression in large follicles. Western blot analysis showed a downregulation of the AQP4 protein in large follicles. In addition, AQP4 was immunoprecipitated and blotted with anti-AQP4 antibody in small and large follicles. Accordingly, AQP4 exhibited a low expression in large follicles. These results show that AQP4 is downregulated in bovine ovarian large follicles, suggesting that the downregulation of AQP4 expression may interfere with follicular water transport, leading to bovine follicular cysts.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
CONFLICTS OF INTEREST
ACKNOWLEDGEMENTS
AUTHOR CONTRIBUTIONS
AUTHOR’S POSITION AND ORCID NO
REFERENCES

• Chang-Woon Kim(Department of Physiology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon 51353, Korea)
• Eun-Ju Choi(Department of Physiology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea)
• Eun-Jin Kim(Department of Physiology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea)
• Adrian S. Siregar(Department of Physiology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea)
• Jaehee Han(Department of Physiology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea)
• Dawon Kang(Department of Physiology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea) Corresponding author