(249) Bovine Oviductal Organoids Exhibit Rhythmic Expression of Core Clock and Reproductive Related Genes

Abstract Authors: Ahmed Gad¹; Brandi Dunn²; Riley E. Thompson¹; Mindy A. Meyers¹; Fiona K. Hollinshead¹

1. Animal Reproduction and Biotechnology Laboratory (ARBL), Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA

2. Animal Reproduction and Biotechnology Laboratory (ARBL), Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
 

Abstract Text:

Circadian rhythms, also known as the biological clock, are essential regulators of physiological processes, including reproductive function in the oviduct. These rhythms are driven by a central pacemaker located in the suprachiasmatic nucleus of the hypothalamus, which synchronizes internal biological clocks to external environmental cues, such as light and darkness. These internal clocks, known as peripheral clocks, are present in nearly all body cells, regulate tissue-specific functions, and are influenced by various physiological signals, including hormonal fluctuations, feeding cycles, and temperature changes.  Despite their importance, little is known about how these rhythms operate within oviductal cells and influence their role in reproductive fertility. This study aimed to investigate the expression, regulation, and synchronization of circadian rhythms in bovine oviductal organoids, a 3D in vitro model that closely replicates the in vivo oviduct environment. To assess circadian rhythmicity, organoids were cultured for 32 days with passaging every 14 days and then samples were collected every 6 hours over a 30-hour period (two biological replicates, each containing two technical replicates with four wells per technical replicate in a 48-well plate). The expressions of core circadian clock genes BMAL1, CLOCK, PER1, PER2, CRY1, CRY2, and functionally related genes ESR1, PGR, OVGP1 were quantified. Time-series normalized expression data were analyzed using the MetaCycle R package, incorporating three complementary methods: JTK_CYCLE, ARSER, and Lomb-Scargle to detect rhythmic patterns. The analysis revealed significant rhythmic patterns in six out of the nine examined genes, including BMAL1, CLOCK, ESR1, and OVGP1, with cycle periods ranging between 14 and 22 hours, shorter than the typical 24-hour circadian cycle. Interestingly, time-course visualization showed overlapping expression profiles for CLOCK, ESR1, and PGR genes, peaking at approximately 10 hours and cycling every ~17 hours, suggesting a potential functional connection between circadian regulation and reproductive processes. This study provides the first evidence of circadian regulation in oviductal organoids, revealing rhythmic expression of the core clock and reproductive genes. Future research will explore strategies to synchronize circadian rhythms in oviductal organoids using approaches such as serum shock, glucocorticoid treatment, and extracellular vesicles (EVs). In particular, EVs derived from synchronized oviductal fluids will be explored as a physiologically relevant strategy. These findings contribute to a deeper understanding of circadian rhythms in reproductive biology and their potential applications in improving animal and human reproductive health and fertility performance.