P-793 The effect of Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1) in human ovarian somatic cell development
Authors: Danti L , Lundin K , Sepponen K , Yohannes D.A , Kere J , Tuuri T , Tapanainen J 
Published: June 2022

Abstract

Study question
Can an inducible NR5A1 (encoding Steroidogenic Factor 1) cell line be used to study the functional role of NR5A1 in human female gonadal differentiation?

Summary answer
Conditional induction of NR5A1 in a human pluripotent stem cell (hPSC)-based model of gonadal differentiation allows us to study its effect on the differentiating cells.

What is known already
According to animal studies, NR5A1 is a critical factor in gonadal development. Previous research in our lab showed that CRISPR/Cas9 induced upregulation of NR5A1 during gonadal differentiation in male (XY) hPSCs increases expression of early Sertoli cell markers such as Inhibin A and Anti-Müllerian hormone (AMH) and thus pushes the cells into a more mature gonadal cell type. However, the role of NR5A1 in female gonadal differentiation has yet to be determined.

Study design, size, duration
The female (XX) human embryonic stem cell (hESC) line, H9, was edited using CRISPR/Cas9 to conditionally induce NR5A1. These cells were subjected to a 10/20-day gonadal differentiation protocol where NR5A1 was induced through addition of Doxycycline (DOX) and Trimethoprim (TMP) from day 4 until day 10/20. Starting from day 4 technical duplicates/triplicates from every second day were subjected to qRT-PCR. Technical triplicates from day 4, 6, 8 and 10 were subjected to bulk RNA-sequencing.

Participants/materials, setting, methods
The cell line that was used in this project is a female hESC line, H9 (WiCell). A previously established protocol for gonadal differentiation (Sepponen et al., 2017) was followed wherein different cell culture media and matrices were supplemented with small molecules, inhibitors and growth factors. The CRISPR/Cas9 genome-editing system was used to generate the NR5A1 inducible cell line. Cell characterisation was performed using qRT-PCR and bulk RNA sequencing (NovaSeq S4) methods.

Main results and the role of chance
qRT-PCR results showed that without NR5A1 induction there was almost no expression of this gene, but its expression increased drastically upon addition of DOX and TMP to the culture media. NR5A1 induction seemed to upregulate FOXL2 and WNT4, which are crucial genes for female gonadal differentiation, although the increase was not statistically significant. AMH showed a significant increase on day 8 of differentiation after NR5A1 induction. We also saw an increase in the expression of steroidogenic genes such as STAR, CYP11A1, CYP17A1 and CYP19A1 after NR5A1 induction. Subjecting the cells to NR5A1 induction for an additional 10 days resulted in the upregulation of bipotential gonadal markers GATA4, WT1 and EMX2 after NR5A1 induction. Similarly, the female gonadal markers RSPO1 and WNT4 were upregulated over time and WNT4 expression further increased upon NR5A1 induction.
NovaSeq S4 data showed that on day 8, after induction of NR5A1, genes related to reproductive system development and female pregnancy were upregulated. These included DHH, NR5A1, CYP19A1, AR, INHA and NR0B1. On day 10 genes related to reproductive system development such as FOXL2, NR5A1, INHA, LHCGR, and NR0B1 were also upregulated after NR5A1 induction.

Limitations, reasons for caution
The effect of NR5A1 induction on expression levels of bipotential gonadal markers GATA4, LHX9, WT1, EMX2 and female gonadal markers FOXL2, RSPO1, WNT4 was small in comparison to differentiation without additional NR5A1 induction. Moreover, we lack a protocol to generate bipotential gonadal cells expressing NR5A1 without inducing NR5A1 through CRISPR/Cas9.

Wider implications of the findings
Taken together the results will provide valuable insight and novel information on the function of NR5A1 in early female gonadal development and more in general on human ovarian development and sex determination. This knowledge will increase our understanding of pathological gonadal development, which often underlies disorders of sex development.

Trial registration number
Not applicable