Chlorocholine chloride delays progenitor Leydig cell differentiation via PI3K/AKT pathway activation in rats.

Chlorocholine chloride (CCC) is a commonly used plant growth regulator. It was shown that CCC exposure could lead to decreased testosterone biosynthesis, delayed puberty onset, and damaged spermatogenesis in pubertal rodents, but the underlying mechanism is still not well understood. The testosterone level is determined not only by the functional activity of Leydig cell but also by its number and maturational status. To identify the critical developmental stage at which CCC impairs Leydig cell maturation and elucidate its underlying mechanisms, pubertal rats were exposed to 0, 75, 137.5, and 200 mg/kg bw/day CCC from PND14 (progenitor Leydig cells, PLCs) to PND28 (immature Leydig cells, ILCs) and from PND28 to PND56 (adult Leydig cells, ALCs) respectively. The results showed that, for exposure from PLCs stage, CCC exposure at 137.5 and 200 mg/kg bw/day led to histopathological changes of rat testes and impaired Leydig cell lineage specification in rats. CCC exposure at 200 mg/kg bw/day significantly decreased the mRNA expression of Star, Lhcgr, Cyp17a1, and Hsd17b3. Hormonal measurement and proteomic analysis revealed that both the disruption of the hypothalamic-pituitary-testis axis and the aberrant activation of the PI3K/AKT signaling pathway contribute to the impairment of Leydig cell lineage specification. For exposure from ILCs stage, Leydig cell development was not affected, but serum and testicular testosterone levels decreased resulting from the down-regulation of StAR, CYP11A1, and HSD3B1. In conclusion, our study found that CCC exposure could delay the development process of rat Leydig cells by disruption of Leydig cell differentiation during early developmental stage, which might lead to decreased testosterone level.