Human male infertility associated with mutations in NR5A1 encoding steroidogenic factor 1

Abstract

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

Figure 1

Distribution of NR5A1 Mutations Associated with Spermatogenic Failure in Relation to the Protein The functional domains of the NR5A1 protein are shown. The DNA-binding domain containing two zinc-finger motifs is indicated. The FtzF1 box stabilizes protein binding to DNA. The hinge region is important for stabilizing the ligand-binding domain and interacts with other proteins that control NR5A1 transcriptional activity. The AF2 domain recruits cofactors necessary for NR5A1 transactivating activity. The position of the amino acid change and its evolutionary conservation are shown for each of the mutations identified.

Figure 2

Gonadal Histology of Subject 1 Photomicrographs demonstrating abnormal testicular histology showing areas of interstitial fibrosis (A–D) and hyalinization (C), with scattered residual abnormal seminiferous tubules containing occasional germ cells but no normal spermatogenesis. Within the fibrous areas, residual tubular structures are present (D). No normal testicular tissue is present. (ST, seminiferous tubules; ^∗, interstitial fibrosis; HT, hyalinised tubule; RT, residual tubular structures).

Figure 3

Cellular Localization of NR5A1 Mutants Cellular localization of GFP-SF1 fusion proteins (green), generated and expressed in tsa201 cells with the use of a pAcGFP-C1 vector. WT NR5A1 shows strong nuclear localization, with relative nucleolar exclusion and very occasional nuclear subfoci. An expression and localization pattern similar to that of the WT was seen for all the other mutant proteins.

Figure 4

Assays of NR5A1 Transcriptional Activity The transcriptional activity of WT NR5A1 and variants associated with male infertility was studied with the use of Cyp11a1 (A) and AMH (B) promoters in HEK293T cells. A previously described inactivating mutation of NR5A1, p.Gly35Glu, was included as a control in the transactivation studies. Results are expressed as a percentage of WT NR5A1 activity, which is considered to be 100%. Data represent the mean of three independent experiments, each performed in triplicate. The T bars represent the SEM.