Comparative Analysis of Microarray andBulkRNA-seq Data for Identifying Putative Interactionsof NR5A1 in Azoospermia

Abstract

Azoospermia is one of the major causes of male infertility and is described as theabsence of spermatozoa in the ejaculate. Azoospermia is the cause of infertilityinmore than one percent of males in the general population whereas 10\%–15\%of infertile men are affected by this problem. It is classified into two types i.e. obstructiveazoospermia (OA) and non-obstructive azoospermia (NOA). NOA is the most prevalent kind of azoospermia and affects approximately 60\% of azoospermic males. It is caused by spermatogenesis failure due to different factors. There is no proper treatment available for NOA, however, sperm can be retrieved in some cases for in- vitro fertilization. This process is very expensive and has a very low success rate. Treatment options are urgently needed to increase sperm production and for targetingunderlying causes. Multiple studies to understand the disease mechanismand improvethe sperm retrieval rate have been reported; however, they reveal a comparatively lowsuccess rate. Studies have identified a number of genes important for spermatogenesis. Among the most important genes, Nuclear receptor subfamily5, group A, member 1 (NR5A1, also known as steroidogenic factor 1[SF-1]) is important. NR5A1 is a nuclear hormone receptor, that plays a crucial role in regulating steroidhormone biosynthesis by targeting different genes in humans. Some transcriptionfactors, cofactors, and transcription co-activators participate with NR5A1 in regulatingNR5A1 target genes. Mutations identified in the NR5A gene have been acknowledgedas being causally associated with Non-Obstructive Azoospermia (NOA). Some studiessupport the association of NR5A1 mutations with NOA and some conflict itsassociation with NOA. Datasets from different platforms, one NGSdataset (GSE216907), and 2 micro-array datasets (GSE45885 and GSE 45887) were usedinthe current study for gene expression analysis of important genes associated withNOA. In the dataset, GSE45885, 839 genes demonstrated differential expression, whileGSE45887 displayed 772 differentially expressed genes, and GSE216907 exhibited1168 genes with differential expression. The number of common differentiallyexpressed genes in the three datasets was 16. The common DEGs were usedfor pathway enrichment analysis and the HedgeHog Signaling pathway was identifiedasimportant with P-value 0.04. However, the expression of NR5A1, target genesof NR5A1, and its regulating cofactors are normal in the used datasets. This study alsodemonstrates the interaction profile of NR5A1 with its target genes and with cofactors at the molecular level. The protein structures of NR5A1 and interacting partners weredocked using the High Ambiguity Driven protein–protein DOCKing (HADDOCK) server. The binding affinity and interaction profile of NR5A1 protein with all interacting partnerswere analyzed. The NR5A1 protein shows interaction with all its target proteins, cofactors, and coactivators. NR5A1 shows the strongest interaction with CTNNB1among all interacting proteins. The identified mutations of NR5A1 were searched intheinteracting residues of NR5A1 with their interacting partners. Fromthe identifiedmutations in different studies, only one mutation was present in the interacting residueand is present in only 0.4\% of the azoospermic cases used in that study. This studysuggests that the mutations identified are not in the interacting residues of NR5A1, andthe expression profile of NR5A1 and its interacting residues is also normal in theNGSdataset used in this study. The findings that negate the link between NR5A1 mutationsand NOA are supported by this study