(241) Cannabinoid Exposure Alters Oviduct Fluid Composition in Vitro with Potential Implications for Embryo Epigenetic Reprogramming

Cannabinoid Exposure Alters Oviduct Fluid Composition in Vitro with Potential Implications for Embryo Epigenetic Reprogramming

Dailin M. Fuego¹; Iebu Devkota¹; Zachary L. Bonomo¹; Mahmoud Dondeti²; Anastasios Vourekas²; Constantine A. Simintiras¹

1. School of Animal Sciences, Agricultural Center, Louisiana State University, Baton Rouge, LA 70803, USA.

2. Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

Abstract Text:

The mammalian oviduct is the site of oocyte fertilization and early embryo development, including the activation of the embryonic genome. Therefore, a suboptimal oviductal microenvironment is likely to disrupt early embryo epigenetic programming, potentially leading to long-term health issues in adulthood, in line with the developmental origins of health and disease paradigm. Due to the ethical and technical challenges of in vivo oviductal research, we used bovine oviduct epithelial organoids (BOEO) as a model. Organoids are three-dimensional in vitro systems that closely replicate key in vivo characteristics, including the composition of intra-organoid fluid (IOF), which reflects the proteomic and metabolomic profile of the tissue of origin lumen. Cannabis is the most used drug among women of reproductive age in the United States, with increasing use during pregnancy. This is concerning as prenatal cannabinoid exposure is associated with negative offspring health. While the embryo expresses cannabinoid receptors (CB1 and CB2), this ongoing study investigates the overarching hypothesis that maternal cannabis exposure – specifically through its primary metabolites cannabidiol (CBD) and tetrahydrocannabinol (THC) – indirectly influences early embryo epigenetic programming, by modulating oviduct epithelial secretions. BOEO were established from synchronized crossbred beef cattle (n=4) post-mortem on Day 5 of the estrous cycle. Following a single sub-culture and a 14-day growth period, BOEO were supplemented with 50 nM 17β-estradiol (E2) for 72 hours. Subsequent treatments were either: (a) vehicle control, (b) 50 nM E2, (c) 1 µM medroxyprogesterone acetate (MPA), (d) E2+MPA, or (e) E2+MPA + 6 µM CBD + 6 µM THC for 6 days. BOEO were imaged every 24 h after media replenishment, followed by morphological, immunohistochemical (IHC), gene expression (RT-PCR), and IOF metabolomic analyses. All BOEO expressed the epithelial marker cytokeratin, oviduct specific glycoprotein 1 (OVGP1), CB1, and CB2. Hormonal responsiveness was confirmed through nuclear estrogen and progesterone receptor IHC. While there were no differences in BOEO count or circularity across treatments, the area of BOEO was significantly reduced (P< 0.05) in the THC-treated group compared to the others by Day 6, suggesting that CBD and THC affect BOEO proliferation. Furthermore, hormonal and cannabinoid treatments induced distinct changes in BOEO gene expression and IOF composition. Differentially abundant IOF metabolites included sucrose, glucose-1-phosphate, and propionylcarnitine, among others. The latter is of particular interest due to its potential role as an epigenetic modulator. In summary, this study demonstrates that BOEO respond to physiological and toxicological stimuli, with potential implications for embryo epigenetic reprogramming. Ongoing research aims to further investigate the impact of propionylcarnitine, among other metabolites, on bovine embryo epigenetic regulation and gene expression. This work was approved by the Louisiana State University (LSU) Agricultural Center Animal Care and Use Committee and is supported by the LSU Therapeutic Cannabis Research Committee (PG010126), the State of Louisiana Board of Regents [LEQSF(2023‐26)‐RD‐A‐03], the LSU Agricultural Center Collaborative Research Program (PG010315), and USDA Research Capacity (Hatch) funds (LAB-94578).