Hypospadias is a congenital external genital malformation in which the urethral folds are incompletely fused, resulting in an abnormal opening of the ventral urethra of the penile [1]. Urethral seam fusion is formed proximally to distally along the GT. Any obstruction during seam fusion or remodeling along the tubercle may lead to an abnormal urethral orifice in hypospadias [2]. Hypospadias is a heterogeneous and multifactorial disease influenced by a combination of genetic, hormonal, and environmental factors [3]. Many studies have shown that bone morphogenetic protein (BMP), sonic hedgehog (Shh), fibroblast growth factor (FGF), transforming growth factor-β1 (TGF-β1), and other genes are involved in early urethral development [4,5]. A coordinated balance between androgens and estrogens in animals and humans is crucial to ensure normal penile development and function. Experiments with pregnant rats and mice treated with flutamide, an androgen receptor (AR) antagonist, resulted in hypospadias in the male offspring [6,7]. In addition, changes in estrogen levels might be a potential cause of hypospadias formation by mediating the action of androgens [8]. Animal studies have demonstrated a clear link between maternal exposure to endocrine-disrupting chemicals and male reproductive malformations [9,10]. Therefore, exploring the underlying mechanism of hypospadias is crucial to reducing its incidence.

In the RAS superfamily of small GTPases, Rho, Rab, and Arf subfamilies are involved in integrin trafficking, and an essential function of Rab and Arf members is to regulate various cellular processes such as membrane dynamics, cell differentiation, and receptor transport. Rab proteins regulate vesicle transport and intracellular signal transduction for normal cellular activities [11]. Changes in Rab proteins and their effectors are associated with many human diseases, including cancer. Rab25 (Rab11c) enhances the aggression of tumor cells by participating in various pathways related to cell proliferation, apoptosis, viability, and regulation of integrin circulation [12]. Rab25 regulates cell polarity, proliferation, and apoptosis during the development of multiple cell lines [11,13,14]. Rab25 can act as either a tumor suppressor or promoter, depending on the cancer context. There has been extensive research on Rab25 in various cancers, but few studies have evaluated the effect of Rab25 on external genitalia development and hypospadias formation. We previously reported a loss of Rab25 expression in the hypospadias group compared with that in the control group [15]. Determining the developmental defects of GTs is often challenging in the embryonic period. Rab25 expression was observed in GTs of male fetal mice at 18.5 days of gestation and the morphology of GTs tended to be complete at this time point, so we selected the GD 18.5 mouse model. Therefore, a better understanding of the function of Rab25 can contribute to determining the role of Rab25 in urethral development.