Transgenerational effects of in utero and nursing cannabis exposure on male and female reproductive functions in mice

Mingxin Shi¹, Yeongseok Oh¹, Debra A. Mitchell¹, James A. MacLean II¹, Ryan J. Mclaughlin², Kanako Hayashi¹

¹ School of Molecular Biosciences, Center for Reproductive Biology, Washington State University

² Department of Integrative Physiology and Neuroscience, Washington State University

Abstract Text:

Cannabis is considered the most widely used psychoactive drug in the US and worldwide. Nearly 50 million Americans have used cannabis. Unsurprisingly, adverse health outcomes associated with cannabis consumption have also been increasing. Although cannabis induces psychoactive effects, strong correlations exist between cannabis use by pregnant women and adverse obstetrical, pregnancy, and neonatal outcomes. Maternal cannabis use increases the risks of congenital malformation, psychosis, and delinquent behavior in utero-exposed offspring. However, the long-term consequences of cannabis use on reproductive functions and how it might impact the subsequent generations have not been examined. We thus assessed the transgenerational effects of in-utero and nursing cannabis vapor exposure on male and female reproductive functions. Pregnant female mice were exposed to cannabis vapor [(25, 100, or 200 mg/ml Δ9-tetrahydrocannabinol (THC) in the cannabis extract] from gestational day 1 to postnatal day 21 (twice/day). This window will cover the entire pregnancy through weaning. Based on plasma THC levels in F0 females, we chose 100 and 200 mg/ml THC in the cannabis extract for further analysis. The selected dose and exposure condition did not disrupt pregnancy and nursing in F0 females. Pregnancy and neonatal outcomes, including gestational length, litter size, and sexual ratio, were not affected by cannabis exposure. However, cannabis-exposed F1 males significantly reduced sperm count/motility and testosterone levels. Spermatogenesis was disrupted, observing abnormal testicular histology, such as increased apoptotic cells, the presence of vacuoles and multinucleated cells, and round spermatids sloughing off into the lumen. In the F1 neonatal testis, cannabis exposure increased apoptosis and DNA damage. Single-cell multiomics revealed disrupted transcriptomes and chromatin accessibilities in developing germ cells. While sperm count/motility was reduced by cannabis exposure in F2 males, it was no longer affected in F3 males. In F1 females, cannabis exposure delayed vaginal opening as a sign of puberty onset and disrupted estrous cyclicity. However, its effects were minor in F2 and F3 females. Furthermore, F1-F3 females did not show any abnormal ovarian and uterine histology and plasma estradiol-17β levels and could produce normal offspring without any pregnancy issues. These results suggest that in utero and nursing cannabis exposure strongly affects F1 male reproductive functions, disrupting spermatogenesis, probably due to spermatogenic defects in the developing testis. Developmental stages of F1 female reproduction were affected by cannabis exposure but did not compromise adult reproductive function. The present study indicates limited transgenerational effects of in utero and nursing cannabis exposure on male and female reproductive functions. Supported by NIH/NIDA R21DA057305 and the State of Washington Initiative Measure No. 171.