Prepubertal exposure to 17β-estradiol downregulates motor protein genes, impairs sperm flagella formation, and reduces fertility in Nile tilapia (Oreochromis niloticus).

Studies have demonstrated that adult male fish exposed to estrogenic endocrine-disrupting compounds (EDCs) exhibit reduced sperm motility and fertility. However, the effects of prepubertal estrogenic EDC exposure on testicular development and subsequent reproductive capacity remain poorly understood. In this study, prepubertal male (XY) tilapia at 30 days after hatching (dah) were exposed to 17β-estradiol (E2) for 60 days, covering the prepubertal period, to investigate its impact on testicular development and fertility at sexual maturity. Morphological and histological analyses revealed that prolonged E2 exposure reduced number of spermatogenic cells, increased testicular cell apoptosis, and significantly decreased the gonadosomatic index in E2-treated XY (E2-XY) fish. Fertility analysis of sexually mature E2-XY fish at 180 dah showed a significant reduction in sperm fertilization capacity compared to Control-XY fish. Sperm quality analysis indicated that E2-XY fish had an increased proportion of immotile sperm and a decreased proportion of progressive sperm. Additionally, the straight-line velocity, curvilinear velocity, and beat frequency of sperm flagella were significantly reduced in E2-XY fish sperm. Scanning electron microscopy (SEM) and immunofluorescence analysis revealed a significant reduction in flagellum length in E2-XY sperm compared to Control-XY sperm. Transcriptome sequencing of 180 dah E2-XY testes demonstrated a significant downregulation of genes associated with motor activity, microtubule binding, microtubule motor activity, and dynein complex formation. Specifically, the expression of kinesin and dynein axonemal heavy chain gene family members was significantly reduced. qPCR and Western blot (WB) assays confirmed that the expression of kinesin family members (kifc1, kif2c, kif11, kif16b, kif17, kif20a, kif20ba, kif20bb, kif22) and dynein heavy chain domain-containing protein family members (dnah2, dnah5, dnah7, dnhd1), which are involved in flagellar movement and assembly, was significantly decreased in E2-XY testes compared to Control-XY testes. These findings demonstrate that prepubertal E2 exposure downregulates genes critical for sperm flagella formation and motility in tilapia, resulting in shortened flagella, reduced motility, and irreversible fertility impairment, in contrast to the recoverable fertility observed in adult fish following exposure to estrogenic EDCs. This study provides novel insights into the mechanisms underlying reduced fertility in male fish exposed to estrogenic EDCs.