Morphological, histopathological, and transcriptomic analyses of gonads reveal reproductive impacts of levonorgestrel (LNG) exposure on Gambusia affinis.

Levonorgestrel (LNG), a synthetic progestin, is extensively utilized in contraceptives and hormone replacement therapies in daily medical practice. To date, LNG has been frequently detected in various water bodies worldwide, posing significant threats to aquatic organisms, particularly fish. Its presence disrupts the delicate hormonal balance and gonadal development pathways in fish, leading to adverse effects on their reproductive health and potentially altering population dynamics. In this study, adult female mosquitofish were chronically exposed to LNG at four distinct concentration levels (0 ng/L, 5 ng/L, 50 ng/L, and 500 ng/L) for a duration of 36 days. The masculinizing effects of LNG on female mosquitofish were evaluated through morphological, histological, and transcriptomic analyses. Results revealed that LNG exposure significantly reduces body weight and growth index while inducing masculinizing traits, including posterior orientation of the 14th, 15th, and 16th hemal spines and elongation of the third anal fin with an increased number of segments. Additionally, LNG suppresses ovarian development, particularly at concentrations of 50 ng/L and 500 ng/L, as evidenced by a reduction in the abundance of late vitellogenic oocytes. Transcriptomic analysis reveals substantial alterations in the expression of key genes associated with gonadal development, steroid metabolism, and reproductive function. Specifically, downregulation of genes such as cyp19a1 and cyp17a1, along with upregulation of dmrt1 and hsd17b3, highlights the disruptive impact of LNG on the hormonal balance and gonadal differentiation pathways in female mosquitofish. Within the context of this investigation, an excess of LNG might be directly transformed into testosterone, which suppressed the synthesis of both endogenous estrogens and androgens and induced masculinization.