February 25, 2021

Cell ablation mouse model to study endocrine disrupting chemicals on hypospadias development

Each year over 20,000 boys are born with hypospadias in the United States (1). Hypospadias is a malformation of the penis where the urethra does not exit at the tip, but will occur somewhere along the shaft of the penis. Urethra defects are often accompanied with other defects of the penis, such as chordee, ventral curvature of the penis, or micropenis (2). Hypospadias and penis comorbidities often lead to difficulties with urination, intercourse, and can result in poor self-image and psychological issues. Boys with hypospadias often undergo some form surgical reconstruction to restore penis function and aesthetic appearance. However, lower urinary tract issues are twice as common in patients who underwent hypospadias surgery and patients often remain unsatisfied with the surgical outcome (3). Repair outcomes are largely correlated to the positioning of the urethra and the comorbidities in the penis.

Unfortunately, since the 1970’s there has been a consistent increase in hypospadias prevalence throughout the world (1). Genetic screens and heritability assays show that only 30% of hypospadias cases can be explained by genetic mutations (4, 5). However, exposure to environmental chemicals with endocrine disrupting properties are tightly linked to hypospadias presence (6). Mothers with high endocrine exposure risk occupation have a higher chance of developing hypospadias in their male fetuses (7, 8). Indeed, penis development is dependent on androgen signaling where anti-androgenic and estrogenic chemicals can consistently induce hypospadias (9). The endocrine properties of the chemical dictate the severity of hypospadias and the comorbidities within the penis. Although the scientific community has understood that antiandrogens and estrogens can cause malformed penis development for 60 years, we still have a very limited idea of how androgen, estrogen, and environmental chemicals influence penis development.

In our research we hypothesize that the cell populations within the penis have different responses to androgens and estrogens. We expect those differences in response dictate the severity of hypospadias and the types of comorbidities. We first aimed to classify the cell populations based on their transcriptome by using single cell RNA sequencing. With mice we collected penis samples from three critical time points during urethra formation. The data was then processed to investigate cell populations and RNA expression at each time. To our surprise the penis is made up of a diverse array of cell types. Each cell population had distinctive expression patterns of Androgen Receptor, Estrogen Receptors, androgen responsive, and estrogen responsive genes, supporting our hypothesis. After in depth analysis of genetic markers for each population and morphological mapping based on in-situ gene expression, we narrowed our investigation of cell populations to functionally investigate down to two.

Selection of these cell populations was based on anatomical positioning and the expression of androgen and estrogen related genes. At the start of penis development, the urethra is open at the base of the penis. As development progresses the urethra will start to close at the base and then continue to the tip of the penis. Concurrent with the tube forming, the cells that surround the urethra will migrate to the site of where the urethra closes to support the morphogenic process. We identified two cell populations that were positioned closely to the open urethra and migrated toward the site of urethra closure. The first cell population was uniquely enriched with Forkhead box L2 and other Forkhead box genes. Forkhead box proteins are transcription factors that function as coregulators of androgen and estrogen receptors to either enhance or suppress gene expression. The second population was marked by Steroidogenic Factor 1 expression, which is a master regulator of steroidogenic genes and is consistently involved in hormone signaling.

Based on gene expression these two cell types were ideal candidates to test. To test if these cells were involved in urethra closure we used a cell ablation mouse model. Basically, whenever a cell expresses Forkhead box L2 or Steroidogenic Factor 1 the cells in the penis will die. This technique allowed us to investigate how the penis will form in the absence of the cell populations of interest. Upon removal of the Forkhead box L2 population there was a urethra closure defect in the proximal most portion of the penis, but the urethra was closed along the rest of the penis. The rest of the penis look normal. However, when we removed the Steroidogenic Factor 1 cells from the penis, there were severe hypospadias defects. There was no urethra closure in many cases. In addition to no urethra closure, there was severe hypoplasia along the penis although the Steroidogenic Factor 1 cell population only composes <1% of the cells in the penis. These data display the unique functions of each of the two cell populations in the penis.

Truly the penis is composed of diverse populations of cells that play different roles in penis development. The unique role of each cell population is likely driven by the unique origins and differential responses to circulating hormones. Understanding the cell dynamics and cell responsiveness to hormones will help us identify the impacts of endocrine disrupting chemicals on penis development and help use better understand the penis defects and comorbidities we see in the human populations.

Contributed by Ciro Amato, NIEHS

References

1.         L. J. Paulozzi, J. D. Erickson, R. J. Jackson, Hypospadias Trends in Two US Surveillance Systems. Pediatr 100, 831-834 (1997).

2.         C. P. Nelson et al., The increasing incidence of congenital penile anomalies in the United States. J Urol 174, 1573-1576 (2005).

3.         S. P. Rynja, T. P. de Jong, J. L. Bosch, L. M. de Kort, Functional, cosmetic and psychosexual results in adult men who underwent hypospadias correction in childhood. J Pediatr Urol 7, 504-515 (2011).

4.         L. Ságodi, Á. Kiss, E. Kiss-Tóth, L. Barkai, Prevalence and possible causes of hypospadias. Orvosi Hetilap 155, 978–985 (2014).

5.         H. J. van der Horst, L. L. de Wall, Hypospadias, all there is to know. Eur J Pediatr 176, 435-441 (2017).

6.         N. Kalfa et al., Is Hypospadias Associated with Prenatal Exposure to Endocrine Disruptors? A French Collaborative Controlled Study of a Cohort of 300 Consecutive Children Without Genetic Defect. European urology 68, 1023-1030 (2015).

7.         E. Haraux et al., Maternal Exposure to Domestic Hair Cosmetics and Occupational Endocrine Disruptors Is Associated with a Higher Risk of Hypospadias in the Offspring. Int J Environ Res Public Health 14 (2016).

8.         C. M. Rocheleau, P. A. Romitti, L. K. Dennis, Pesticides and hypospadias: a meta-analysis. J Pediatr Urol 5, 17-24 (2009).

9.         M. H. Wang, L. S. Baskin, Endocrine disruptors, genital development, and hypospadias. J Androl 29, 499-505 (2008).