KATNB1 in the human testis and its genetic variants in fertile and oligoasthenoteratozoospermic infertile men.

Oligoasthenoteratozoospermia (OAT) is a phenotype frequently observed in infertile men, and is defined by low spermatozoa number, abnormal spermatozoa morphology and poor motility. We previously showed that a mutation in the Katnb1 gene in mice causes infertility because of OAT. The KATNB1 gene encodes an accessory subunit of the katanin microtubule-severing enzyme complex; this accessory subunit is thought to modulate microtubule-severing location and activity. We hypothesized that KATNB1 may play a role in human spermatogenesis and that genetic variants in KATNB1 could be associated with OAT in humans. Using immunostaining, we defined the localization of the KATNB1 protein in human testes. KATNB1 was present during spermatid development, and in particular localized to the microtubules of the manchette, a structure required for sperm head shaping. To assess a potential association between genetic variants in the KATNB1 gene and infertile men with OAT, we performed direct sequencing of genomic DNA samples from 100 OAT infertile and 100 proven fertile men. Thirty-seven KATNB1 variants were observed, five of which had not previously been described. Ten variants were present only in OAT men, however, statistical analysis did not reveal a significant association with fertility status. Our results suggest that variants in the KATNB1 gene are not commonly associated with OAT infertility in Australian men.

Genetic variants in the human glucocorticoid-induced leucine zipper (GILZ) gene in fertile and infertile men.

Sertoli cell only (SCO) syndrome is the predominant histology for men with non-obstructive azoospermia (NOA) and is usually of unexplained aetiology. Studies in mouse models indicated that the X-linked gene glucocorticoid-induced leucine zipper (GILZ) is essential for survival and differentiation of spermatogonia, and meiosis. GILZ deficiency results in a rapid and progressive loss of germ cells with SCO tubules and sterility in adults. The role of GILZ in human fertility has not been examined. Here we show that GILZ is localized to spermatogonia and spermatocytes in the human testis in a pattern analogous to that seen in mice. To assess the potential for an association between GILZ variants and human infertility, we sequenced the entire protein-coding regions of the GILZ gene in 65 SCO and 87 fertile Australian men. We identified six genetic variants, three of which had not been reported previously. Three variants, 107018665 G>A, 107018485 C>G and 106959283 C>T, were found at a low frequency only in SCO men. Although none of the identified variants changed the protein code, sequence analysis indicated that two variants, 107018665 G>A and 107018485 C>G, would completely abolish the exonic splicing enhancer (ESE)-binding motifs for the splicing factors SF2/ASF and SC35 respectively. This result prompted an assessment of whether these two variants were associated with male infertility in a separate population of men. We used a PCR-based SNP detection approach to screen an additional 52 NOA and 153 fertile Australian men, and 86 SCO and 54 fertile American men. None of these men carried either of these two variants. The cumulative allelic frequency of these variants is less than 1% in SCO men and no association with fertility status was observed. Our study suggests that GILZ variants are not common causes of SCO and NOA in Australian or American men.

GGN1 in the testis and ovary and its variance within the Australian fertile and infertile male population.

Mouse gametogenetin (Ggn) is a testis-enriched gene that encodes multiple spliced transcripts giving rise to three predicted protein isoforms: GGN1, GGN2 and GGN3. Of these, GGN1 has been linked to germ cell development. Based on the spatial and temporal expression pattern of GGN1 during mouse spermatogenesis, it has been proposed as a candidate human infertility gene. Here, we report the localization of GGN1 in the human testis and ovary compared with the mouse orthologue. Within the testis, GGN1 was confined to pachytene spermatocytes and spermatids. During mid-prophase GGN1 redistributes from a solely cytoplasmic localization to both cytoplasmic and nuclear in late prophase spermatocytes and round spermatids, and is ultimately incorporated into the sperm tail. Within both mouse and human ovaries, GGN1 was localized within granulosa cells. Lower levels of expression were observed in mouse oocytes and the cumulus cells. Furthermore, to define the level of sequence variation in the fertile population and to assess the potential for an association with male infertility, we sequenced the coding region of human GGN in 100 idiopathic oligospermic infertile and 100 control men. Fifteen genetic variants were identified, of which 10 had not previously been reported. No significant associations with fertility status were observed, suggesting that variance in the GGN gene are not a common cause of oligospermic infertility in Australian men.

Polymorphisms in the human cysteine-rich secretory protein 2 (CRISP2) gene in Australian men.

BACKGROUND: Cysteine-rich secretory protein 2 (CRISP2) is localized to the human sperm acrosome and tail. It can regulate ryanodine receptors Ca(2+) gating and binds to mitogen-activated protein kinase kinase kinase 11 in the acrosome and gametogenetin 1 (GGN1) in the tail. METHODS AND RESULTS: In order to test the hypothesis that CRISP2 variations contribute to male infertility, we screened coding and flanking intronic regions in 92 infertile men with asthenozoo- and/or teratozoospermia and 176 control men using denaturing HPLC and sequencing. There were 21 polymorphisms identified, including 13 unreported variations. Three SNPs resulted in amino acid substitutions: L59V, M176I and C196R. All were only present in a heterozygous state and found in fertile men. However, the C196R polymorphism was of particular interest as it resulted in the loss of a strictly conserved cysteine involved in intramolecular disulphide bonding. Screening of an additional 637 infertile men identified 23 heterozygous C196R men to give an overall frequency of 3.6%, compared with 3.4% in control men. The functional significance of the C196R polymorphism was defined using a yeast two-hybrid assay. The C196R substitution resulted in the loss of CRISP2-GGN1 binding. CONCLUSIONS: Although none of the many polymorphisms identified herein showed a significant association with male infertility, functional studies suggested that the C196R polymorphism may compromise CRISP2 function.