Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia

Abstract

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.

Figure  1.

Family with globozoospermia and a mutation in the SPATA16 gene. A, Sperm morphology. Fluorescent acrosin (green) staining (with fluorescein) of acrosomes and 4′,6-diamidino-2-phenylindole (blue) staining of nuclei. On the left is a sample from a fertile control, in which the most important content of the acrosome (acrosin) is clearly and abundantly present; on the right is a sample from a patient. Sperm morphology and acrosome structures are severely disrupted in patient cells. Remnants of acrosin staining were observed for some deformed sperm cells, but most signals represent nonspecific acrosin staining in the leukocytes. B, Chromatograms of the mutation. Shown are the sequences from a control sample, the heterozygous father, and one of the patients. C, The NciI recognition site (5′-CCCGG-3′) is lost because of the G→A mutation at the last nucleotide of exon 4. (The HpaII recognition site is not shown but overlaps at 5′-CCGG-3′). This mutation predicts a R283Q amino acid substitution, as well as the disruption of the 5′ splice site of intron 4. D, Pedigree of the Ashkenazi Jewish family in this study. The order of the 10 siblings is arbitrary. The segregation of the mutation was studied by NciI digestion of a PCR amplification of exon 4 and its flanking sequences. The first lane is the marker lane. As asterisk (*) indicates tested individuals. The two parents and two siblings are heterozygous for the mutation. The three affected males are homozygous, and one unaffected male and a control (C) are not carriers of the mutation.

Figure  2.

Sequence alignment of SPATA16

Figure  3.

Mutated donor splice site of SPATA16 intron 4 and U1 SnRNP binding to wild-type (WT) and mutant (MT) donor splice sites. A, Overview of the used prediction sites. B, Minigene constructs used to test the splicing of exon 4. T- = construct without exon; T-RT = construct without exon in RT-PCR. C, Gel showing that wild-type exon 4 is invariably included in the final mRNA. In sharp contrast, the mutant exon gives rise to two aberrant splicing forms. M = marker lane. D, U1 SnRNP binding analyzed by psoralen-mediated UV-crosslinking experiments, revealing that mutant exon 4 is not recognized by the splicing machinery, whereas wild-type exon 4 is clearly recognized. The identity of the U1 SnRNP was confirmed by RNAse H treatment with use of an oligodeoxynucleotide complementary to nucleotide positions 1–15 of U1 SnRNA.