Prenatal exposure to cannabidiol affects spermatogonial homeostasis and the production of spermatozoa in adulthood

Pregnant women increasingly use cannabidiol (CBD) isolates to alleviate symptoms such as depression, anxiety, vomiting, or nausea. However, the effects of gestational CBD exposure on postnatal reproductive capacity remain poorly characterized. Since CBD interacts with the endocannabinoid system, which is critically involved in regulating male reproduction, we investigated the impact of prenatal CBD exposure on postnatal spermatogonial differentiation and adult reproductive parameters. Pregnant female mice were administered a daily oral dose of either vehicle (DMSO) or CBD (5 or 20 mg/kg/day) from embryonic day (E) 6.5 to E15.5. Male offspring gonads were analyzed at 5 days postpartum (DPP), when spermatogonia are in an undifferentiated state, and in adulthood, using morphological, cellular, and transcriptomic approaches. Prenatal CBD exposure did not affect litter parameters or overall testicular morphology in 5 DPP males but significantly altered spermatogonial homeostasis. CBD promoted the commitment of GFRA1-positive undifferentiated spermatogonia toward a differentiated state characterized by the expression of SOX3 and RARG. These effects were associated with alterations in CB1 expression and changes in histone marks, including an increase in H3K4me3 and a decrease in H3K27me3 in the prepubertal testis. Long-term analyses revealed that gestational CBD exposure impaired sperm production and modified the adult testicular transcriptome, notably through the downregulation of kallikrein genes encoding peptidases involved in semen quality. This study demonstrates that prenatal exposure to CBD can disrupt spermatogonia differentiation after birth and negatively affect spermatogenesis in adulthood. 1. Prenatal CBD exposure altered neonatal spermatogonial homeostasis, reducing stem-like (GFRA1 +) cells and increasing differentiation-committed (SOX3/RARG +) cells. 2. CBD exposure was associated with reduced CB1 protein and altered H3K4me3/H3K37me3 levels in testis. 3. Ex vivo PND5 testis cultures showed a shift toward differentiation commitment, supporting in vivo findings. Adult offspring displayed reduced sperm count, meiotic alterations, and testicular transcriptomic dysregulation.