1. Background
In male fetuses, high androgen levels are required for the bipotential genital tubercle (GT) to differentiate to a penis. Disrupted androgen signaling can cause hypospadias, a common genital malformation in newborn boys. Some human hypospadias cases are suspected of arising from fetal exposure to endocrine disruptors, but the mechanistic links remain elusive due to limited understanding of underlying developmental processes. Using the rat as a model, we mapped the cellular and transcriptional differentiation of the GT during normal and disrupted development.
2. Methods
We used a rat model of hypospadias involving in utero exposure to the androgen receptor (AR) antagonist flutamide. Rats were exposed to 0, 3, or 6 mg/kg/day from gestational day (GD) 7 until GD17, GD19, or GD21, on which days the dams were killed, and fetuses dissected. GTs from control males, control females, and hypospadiac males were isolated for single nuclei RNA-sequencing (n=2-4) and in situ hybridization analyses (n=2-3).
3. Results
The developing rat GT comprised 35 cell populations across sexes and ages. Hypospadias development was characterized by cellularity changes resembling a female developmental trajectory. Differential expression analysis within each cell type revealed time- and cell type-specific transcriptional changes, with the preputial and ventral mesenchymal cell populations particularly altered in hypospadiac males. Within these cell populations, enrichment analysis confirmed a significant overlap in differentially expressed genes between control females and hypospadiac males, when compared with control males. The ventral mesenchyme emerged as a central androgen-sensitive population, with effects on androgen, estrogen, and Wingless-type (Wnt) signaling in the hypospadiac penis.
4. Conclusion
Data supports a model in which androgen signaling coordinates multiple processes within distinct cell populations of the GT. This first single cell resolution map of hypospadias development lays the foundation for further mechanistic studies into the causal mechanisms underlying endocrine disruption-mediated hypospadias in humans.