Tfcp2l1 participates in paternal vitamin D deficiency–induced fetal lung maldevelopment in mice

Fetal lung development is crucial for neonatal respiratory function, with insufficient maturation contributing to neonatal respiratory distress syndrome. While maternal vitamin D deficiency (VDD) has been linked to adverse offspring outcomes, the impact of paternal VDD on fetal lung programming remains largely unexplored. This study investigates the effects of paternal VDD on fetal lung development and maturation, aiming to provide a theoretical basis for paternal preconception nutritional intervention and highlight the importance of paternal health in offspring respiratory outcomes. Male C57BL/6J mice were gavaged with diets containing vitamin D₃ (0 or 1000 IU kg⁻¹/d) for 90 days. Serum vitamin D concentrations were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Treated males were then mated with females to generate offspring. Mating outcomes were recorded to assess reproductive success. Fetal lungs were collected at embryonic days 15.5–17.5 for H&E staining, morphometric analysis. Interrogation of a publicly available single-cell RNA sequencing (scRNA-seq) dataset delineated cell-type-specific marker gene signatures and prioritized candidate transcription factors, whose expression patterns were subsequently validated in offspring under paternal VDD conditions by quantitative real-time PCR, Western blotting, and multiplexed fluorescence immunohistochemistry. In mice, paternal VDD did not significantly affect mating success or pregnancy establishment. However, paternal VDD led to fetal lung dysplasia, accompanied by reduced lung weight and distal alveolar expansion. Alveolar type I (AT1) and type II (AT2) markers were markedly downregulated, reflecting delayed distal epithelial maturation. Guided by analysis of a publicly available scRNA-seq dataset, Tfcp2l1 was prioritized as a candidate transcription factor. Subsequent experimental validation revealed its predominant localization to AT1 and AT2 cells, with downregulation associated with decreased AT2 proliferation, a concomitant reduction in AT1 abundance, and limited AT1 expansion. Paternal VDD is associated with altered fetal lung development, characterized by reduced lineage specification of distal alveolar epithelial cells (AT1 and AT2) and diminished alveolar sacculation. The transcription factor Tfcp2l1 may be involved in this process, with its downregulation under paternal VDD correlating with reduced AT2 cell proliferation and decreased AT2-to-AT1 differentiation, ultimately relating to insufficient pulmonary maturation.