Abstract
Hypospadias is a common disorder of sex development (DSD), resulting from the impaired formation of the penile urethra. In mammals, penile urethral development depends on androgens secreted during a limited time window immediately after the onset of testis development. In this study, we performed morphological and immunohistochemical analyses of a 15-day-old genetically XY Holstein calf with perineal hypospadias to characterize a unique phenotype providing critical insights into its pathogenic mechanisms. Although the penis and prostate were grossly developed, the penile urethra was not formed. Furthermore, the right testis presented as a streak gonad attached to the dorsal abdominal wall and was covered by a fibrous membrane. Extensive fatty infiltration and a lack of interstitial Leydig cells were observed throughout the testis; however, a few testicular cords containing GATA4/SOX9-positive Sertoli cells and VASA-positive germ cells were identified. In contrast, the left testis was morphologically normal, but exhibited increased expression of the androgen-biosynthetic enzyme P450C17, suggesting compensatory up-regulation of androgen production. These findings suggest that a transient androgen deficiency, caused by developmental arrest of the right testis after testicular cord formation, led to the failure of penile urethral formation. This case provides clinical evidence that the precise timing of unilateral testicular impairment is a critical determinant of the hypospadias phenotype in cattle.