(234) Effects of Disrupted Circadian Rhythms on Clock and Mitochondrial-related Gene Profiles in Round and Elongated Spermatids and Spermatozoa

T. S. Kostic¹ , A.P. Becin¹, D. Z. Travicic¹, I. M. Keselj¹, S. A. Andric¹

¹University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Laboratory for Chronobiology and Aging, 21000 Novi Sad, Serbia

Abstract Text:

Our previous research linked circadian rhythm disruption to impaired sperm function, but its impact on male fertility is unclear. This study examines how circadian disruption affects germ cell maturation during spermiogenesis. Efficient sperm maturation requires precise coordination of critical biological processes, including energy balance. Therefore, the focus of this study is analyzing the expression of clock genes and mitochondria-related genes essential for maintaining the energy balance necessary for spermatozoa maturation. To assess the impact of circadian desynchrony on sperm maturation, round and elongated spermatids, along with spermatozoa, were isolated from rat testes and epididymis. Circadian desynchrony was induced by altering the light schedule for two months (two days of constant light, two days of darkness, and three days on a 14:10-hour light-dark cycle). The rats' activity patterns were monitored continuously, and gene expression related to temporal organization and cellular energetics during spermiogenesis was analyzed at active and inactive phases. Spermatozoa obtained from control rats exhibit decreased expression of clock genes compared to both round and elongated spermatids. Principal component analysis (PCA) revealed distinct expression patterns among these cell types: positive clock regulators such as Bmal1 and Rora, along with Reverba, displayed overlapping expression patterns in round and elongated spermatids, while negative clock regulators, including Per1, Per2, Cry1, Cry2, and Reverbb, formed a distinct cluster in spermatozoa. Additionally, PCA of mitochondria-related genes revealed distinct expression patterns across the three cell types. Genes involved in mitochondrial biogenesis and dynamics, such as Pprgc1a, Pparg, Cox4/2, Mfn1, and Fis1, exhibited more pronounced expression in elongated spermatids. In contrast, genes related to biogenesis, mitophagy, fusion, and fission showed similar expression levels across all three types of cells. These results align with the increased metabolic demands of elongated spermatids and suggest a shift towards glycolysis as the dominant ATP production pathway in spermatozoa, rather than relying on mitochondrial oxidative phosphorylation. Altogether, gene expression in spermatozoa from control rats differs from that in earlier stages of spermiogenesis, suggesting specialized regulatory roles for these mature male gametes. In response to circadian desynchrony, a third PCA analysis revealed significant variability in gene expression across the three cell types. While round and elongated spermatids exhibited overlapping expression patterns, spermatozoa demonstrated a distinct response characterized by clusters of genes involved in pro-biogenesis, pro-mitophagy, profission, and clock regulation. These findings indicate that spermatozoa have a specific and differentiated reaction to circadian disruptions, which may significantly impact their functionality. Overall, this research provides valuable insights into the molecular mechanisms underlying male fertility and highlights the need for further studies to explore how circadian disruptions affect reproductive health.

Acknowledgements. This study was supported by the Autonomic Province of Vojvodina (grant APV9418), Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grants No. 451-03-137/2025- 03/200125, 451-03-136/2025-03/200125 and CIV-CeRES-2024 grant), the and the ANDRONET action.