The Nsun7 (A11337)-deletion mutation, causes reduction of its protein rate and associated with sperm motility defect in infertile men

Abstract

Purpose: Recent studies have shown that genetic abnormalities may be responsible for most unknown cases of male infertility. Human Nsun7 gene, which is located on chromosome4, has a role in sperm motility by encoding the putative methyltransferase Nsun7 protein. The aim of the present study was to investigate the mutations of exon4 in the Nsun7 gene, which is associated with sperm motility defect.

Methods: Semen samples including those of fertile normospermic (normal), infertile oligospermic (with normal sperm motility), and infertile asthenospermic (with reduced sperm motility) men were collected from the Omid and Fatemezahra IVF centres (Babol, Iran). These samples were then analysed on the basis of World Health Organization guidelines using the general phenol-chloroform DNA extraction method. Exon4 was amplified using Sun-F/Sun-R primers. Samples from asthenospermic men, which showed different patterns of movement on single-strand conformation polymorphism compared with normal and oligospermic samples, were identified and subjected to sequencing for further identification of possible mutations.

Results: Analysis of extracted sperm proteins showed that the rate of Nsun7 decreased. Likewise, direct sequencing of PCR products, along with their analysis, confirmed the deletion mutation of adenine in location 11337 of the Nsun7 gene in asthenospermic men. Comparison of normal and mutant protein structures of Nsun7 indicated that the A11337-deletion of the exon4 resulted in the valine residues-157 with GTA-codon in normospermic replaced with TAG-early stop codon in asthenospermic samples, causing an abortive protein product with amino acid sequence shorter than normal. The secondary structure of the protein, the protein folding, and ligand binding sites were changed, indicating the impairment of the protein function.

Conclusions: Because the Nsun7 gene products have a role in sperm motility, it will lead to impairment in the activity of the protein and motility of sperm flagella as well as male infertility if a mutation occurs in this gene.

Fig. 1

Total genomic DNA of spermatozoa and PCR products of the Nsun7 (exon 4): (a) Total genomic DNA of spermatozoa: Lane 1, total genomic DNA of normal spermatozoa; lane 2, total genomic DNA of spermtozoa from an oligospermic sample; lanes 3–6, total genomic DNA of spermatozoa from an astenospermic sample. (b) PCR products of the Nsun7 (exon 4) subjected to agarose gel electrophoresis: lane 1, exon 4 PCR product (121 bp) from total spermatozoa DNA of a normospermic sample; lane 2, exon 4 PCR product (121 bp) from total spermatozoa DNA of a oligospermic sample; lanes 3–6, exon 4 PCR product (121 bp) from total spermatozoa NA of an asthenospermic sample; lane 7, 1-kb DNA ladder (SM0311; Fermentas Company. (http://www.fermentas.com)

Fig. 2

Single-strand conformation polymorphism (SSCP) of exon 4 PCR products of the NOP2/Sun domain family, member 7 (Nsun7) gene; Lane 1, normospermic; lanes 2, oligospermic; Lanes 3–6, asthenospermic

Fig. 3

SDS-Poly acrylamide gel of extracted proteins of the total spermatozoa: Lane 1, Protein ladder; Lane 2, normospermic; lanes 3, oligospermic; Lanes 4–7, asthenospermic

Fig. 4

Direct sequencing of exon 4 of the NOP2/Sun domain family, member 7 (Nsun7) gene in asthenospermic men: (a) in the wild-type Nsun7 gene, ‘A’ is at nucleotide position 11337. (b) Sequence of a sample from an asthenospermic infertile man with poor sperm motility showing a Deletion ‘A’ which was observed in samples from asthenospermic infertile men

Fig. 5

Comparison of the primary structure of the amino acid sequences of Nsun7 proteins (b) and Blast analysis of exon 4 sequences in all asthenospermic (A) and normospermic (N) samples (a). The asterisks indicate similarity; different symbols indicate conversion of the valine (V) residue to stop-codon

Fig. 6

Comparison of the secondary structure of the Nsun7 protein using Protscale server. Alpha-helix in normospermic (a), Alpha-helix in asthenospermic (b), the secondary structure of the Nsun7 protein in asthenospermic (c), Polarity in normospermic (d), Polarity in asthenospermic (e). Revealed that there were changes in the secondary structure of the Nsun7 protein in asthenospermic sample and there is no beta strand in sample with deletion mutation in exon4

Fig. 7

Comparisons of the binding energy and three-dimensional structure of the Nsun7 protein and ligand (S-adenosyl-l-methionine) in (a, b) normospermic and (c, d) asthenospermic men. (a, c) Schematic diagram of protein folding of the Nsun7. (b, d) Binding energy and binding mode of the Nsun7 ligand (S-adenosyl-l-methionine) to the active site region of the Nsun7 protein