Vapor Cannabis Exposure Generationally Affects Male Reproductive Functions in Mice

Shi, Mingxin; Langholt, Esther M; Harvey, M.; Wheeler, Emma C.; Zhao, Liang; MacLean, James A.; Oh, Yeongseok; Sabrowsky, Emily; Yu, Shiguang; Watson, Shane; Davis, Jon F.; Hayashi, Kanako

doi:10.1093/toxsci/kfab137

Cited by 4 publications

(1 citation statement)

References 65 publications

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“…And then, motile and non-motile sperm were counted under a dark-field microscope to determine sperm concentration and motility. For more sophisticated and automated analysis, sperm samples were incubated at 37 • C for 15 min and analyzed using computer-assisted sperm analysis (CASA) (SCA CASA system; Fertility Technology Resources, Inc.) following the manufacturer's instructions as previously described [14,15].…”

Section: Sperm Analysismentioning

confidence: 99%

Rhox8 homeobox gene ablation leads to rete testis abnormality and male subfertility in mice

Oh,

Kasu,

Bottoms

et al. 2023

Biology of Reproduction

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The reproductive homeobox X-linked (Rhox) genes encode transcription factors that are expressed selectively in reproductive tissues including the testis, epididymis, ovary, and placenta. While many Rhox genes are expressed in germ cells in the mouse testis, only Rhox8 is expressed exclusively in the Sertoli cells during embryonic and postnatal development, suggesting a possible role of Rhox8 in embryonic gonad development. Previously, Sertoli cell-specific knockdown of RHOX8 resulted in male subfertility due to germ cell defects. However, this knockdown model was limited in examining the functions of Rhox8 as RHOX8 knockdown occurred only postnatally, and there was still residual RHOX8 in the testis. In this study, we generated new Rhox8 knockout (KO) mice using the CRISPR/Cas9 system. Sex determination and fetal testis development were apparently normal in mutant mice. Fertility analysis showed a low fecundity in Rhox8 KO adult males, with disrupted spermatogenic cycles, increased germ cell apoptosis, and reduced sperm count and motility. Interestingly, Rhox8 KO testes showed an increase in testis size with dilated seminiferous tubules and rete testis, which might be affected by efferent duct Rhox8 ablation dysregulating the expression of metabolism and transport genes in the efferent ducts. Taken together, the data presented in this study suggest that Rhox8 in the Sertoli cells is not essential for sex determination and embryonic testis differentiation but has an important role in complete spermatogenesis and optimal male fertility.

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Section: Sperm Analysismentioning

confidence: 99%

Rhox8 homeobox gene ablation leads to rete testis abnormality and male subfertility in mice

Oh,

Kasu,

Bottoms

et al. 2023

Biology of Reproduction

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Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

Cournoyer³

et al. 2022

Toxicological Sciences

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Cannabidiol (CBD), one of the major cannabinoids in the plant Cannabis sativa L., is the active ingredient in a drug approved for the treatment of seizures associated with certain childhood-onset epileptic disorders. CBD has been shown to induce male reproductive toxicity in multiple animal models. We previously reported that CBD inhibits cellular proliferation in the mouse Sertoli cell line TM4 and in primary human Sertoli cells. In this study, using a transcriptomic approach with mRNA sequencing analysis, we identified molecular mechanisms underlying CBD-induced cytotoxicity in primary human Sertoli cells. Analysis of differentially expressed genes (DEGs) demonstrated that DNA replication, cell cycle, and DNA repair were the most significantly affected pathways. We confirmed the concentration-dependent changes in the expression of key genes in these pathways using real-time PCR. mRNA-sequencing showed upregulation of a group of genes tightly associated with senescence associated secretory phenotype (SASP) and with the activation of the p53 signaling pathway, a key upstream event in cellular senescence. Prolonged treatment of 10 μM CBD induced cellular senescence, as evidenced by the stable cessation of proliferation and the activation of senescence associated β-galactosidase (SA-β-gal), two hallmarks of senescence. Additionally, using real-time PCR and Western blotting assays, we observed that CBD treatment increased the expression of p16, an important marker of cellular senescence. Taken together, our results show that CBD exposure disturbs various interrelated signaling pathways and induces cellular senescence in primary human Sertoli cells.

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Environmentally-relevant doses of bisphenol A and S exposure in utero disrupt germ cell programming across generations resolved by single nucleus multi-omics

Zhao,

Shi,

Winuthayanon

et al. 2024

Preprint

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Background: Exposure to endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), disrupts reproduction across generations. Germ cell epigenetic alterations are proposed to bridge transgenerational reproductive defects resulting from EDCs. Previously, we have shown that prenatal exposure to environmentally relevant doses of BPA or its substitute, BPS, caused transgenerationally maintained reproductive impairments associated with neonatal spermatogonial epigenetic changes in male mice. While epigenetic alterations in germ cells can lead to transgenerational phenotypic variations, the mechanisms sustaining these changes across generations remain unclear. Objectives: This study aimed to systematically elucidate the mechanism of transgenerational inherence by prenatal BPA and BPS exposure in the murine germline from F1 to F3 generations at both transcriptomic and epigenetic levels. Methods: BPA or BPS with doses of 0 (vehicle control), 0.5, 50, or 1000 μg/kg/b.w./day was orally administered to pregnant CD-1 females (F0) from gestational day 7 to birth. Sperm counts and motility were examined in F1, F2, and F3 adult males. THY1+ germ cells on postnatal day 6 from F1, F2, and F3 males at a dose of 50 μg/kg/b.w./day were used for analysis by single-nucleus (sn) multi-omics (paired snRNA-seq and snATAC-seq on the same nucleus). Results: Prenatal exposure to BPA and BPS with 0.5, 50, and 1000 μg/kg/b.w./day reduced sperm counts in mice across F1 to F3 generations. In the F1 neonatal germ cells, ancestral BPA or BPS exposure with 50 μg/kg/b.w./day resulted in increased differentially expressed genes (DEGs) associated with spermatogonial differentiation. It also disrupted the balance between maintaining the undifferentiated and differentiating spermatogonial populations. Differentially accessible peaks (DAPs) by snATAC-seq were primarily located in the promoter regions, with elevated activity of key transcription factors, including SP1, SP4, and DMRT1. Throughout F1-F3 generations, biological processes related to mitosis/meiosis and metabolic pathways were substantially up-regulated in BPA- or BPS-exposed groups. While the quantities of DEGs and DAPs were similar in F1 and F2 spermatogonia, with both showing a significant reduction in F3. Notably, approximately 80% of DAPs in F1 and F2 spermatogonia overlapped with histone post-translational modifications linked to transcription activation, such as H3K4me1/2/3 and H3K27ac. Although BPA exerted more potent effects on gene expression in F1 spermatogonia, BPS induced longer-lasting effects on spermatogonial differentiation across F1 to F3 males. Interestingly, DMRT1 motif activity was persistently elevated across all three generations following ancestral BPA or BPS exposure. Discussion: Our work provides the first systematic analyses for understanding the transgenerational dynamics of gene expression and chromatin landscape following prenatal exposure to BPA or BPS in neonatal spermatogonia. These results suggest that prenatal exposure to environmentally relevant doses of BPA or BPS alters chromatin accessibility and transcription factor motif activities, consequently contributing to disrupted transcriptional levels in neonatal germ cells, and some are sustained to F3 generations, ultimately leading to the reduction of sperm counts in adults.

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Vapor Cannabis Exposure Generationally Affects Male Reproductive Functions in Mice

Cited by 4 publications

References 65 publications

Rhox8 homeobox gene ablation leads to rete testis abnormality and male subfertility in mice

Rhox8 homeobox gene ablation leads to rete testis abnormality and male subfertility in mice

Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

Environmentally-relevant doses of bisphenol A and S exposure in utero disrupt germ cell programming across generations resolved by single nucleus multi-omics

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