MOLECULAR REPRODUCTION AND DEVELOPMENT 74:795–799 (2007) Polymorphisms of the Human PUMILIO2 Gene and Male Sterility K. KUSZ, 1 B. GINTER-MATUSZEWSKA, 1 K. ZIOLKOWSKA, 1 A. SPIK, 1 J. BIERLA, 2 P. JEDRZEJCZAK, 3 A. LATOS-BIELENSKA, 4 L. PAWELCZYK, 3 AND J. JARUZELSKA 1 * 1 Institute of Human Genetics Polish Academy of Sciences, Poznan, Poland 2 Department of Histology, University of Medical Sciences, Poznan, Poland 3 Division of Infertility and Reproductive Endocrinology, University of Medical Sciences, Poznan, Poland 4 Department and Chair of Medical Genetics University of Medical Sciences, Poznan, Poland ABSTRACT The highly conserved Pumilio protein plays crucial roles in fertility of many organisms acting as a repressor of translation, and causing infertility when mutated. Although one of two human Pumilio homologs, PUMILIO2 is expressed mainly in the germ line, its role in mammalian germ cell development has not been reported yet. To shed light on the role of PUMILIO2 in development of the human male germ line, we screened this gene for mutations in 137 patients presenting a variety of phenotypes with spermatogenic failure. The first variant, we identified was a single base substitution within intron 15 (IVS15 þ 6G > A). This variant was found in three azoospermic males, the second allele being the wild type. However, this variant was also present among fertile males, as frequently as in the patients. Although location of IVS15 þ 6G > A substitution in close proximity to the canonical donor splice site GT, indicates that its influence on splicing cannot be excluded, our preliminary cDNA analysis has not revealed evidence of a splicing abnormality of PUMI- LIO2 pre-mRNA carrying this variant. Nevertheless, this study provides new interesting variant containing a donor splice site variant, which can be relevant for understanding of splicing mechanism of mammalian genes. The second variant, c.774 C > T transversion (Y258Y) in exon 6 was found only in one patient, but an influence on PUMILIO2 function is not obvious. Altogether, this study shows that variation in the PUMILIO2 gene is very low and it seems improbable that mutations of this gene significantly contribute to male infertility in humans. Mol. Reprod. Dev. 74: 795–799, 2007. ß 2006 Wiley-Liss, Inc. Key Words: germ cells; male infertility; mRNA splicing; PUMILIO2; spermatogenesis INTRODUCTION Pumilio protein is evolutionary conserved from yeast to humans and plays multiple roles in development of model organisms. It is a key regulator of specific mRNAs acting as a translational repressor during morphogen- esis and in germ cell development. In D melanogaster, Pumilio is essential for mitotic and transcriptional quiescence of primordial germ cells during their migra- tion to primary gonads (Asaoka-Taguchi et al., 1999). Later in development, Pumilio is involved in mainte- nance of germ-line stem cells (Forbes and Lehmann, 1998). A homologous role in germ-line stem cell main- tenance was shown for fbf-1 (fem-3 binding factor) and fbf-2 respectively, the two Pumilio homologs in C elegans (Crittenden et al., 2002). A third Pumilio homolog in the same organism, puf-8, was found to be responsible for meiotic completion (Subramaniam and Seydoux, 2003). Pumilio plays a role in the analogous process in Xenopus, namely in meiotic oocyte reactivation (Naka- hata et al., 2003). Moreover, it was recently demon- strated that Pumilio function in the Xenopus oocyte is to repress translation of a specific mRNA involved in the translational regulatory cascade (Padmanabhan and Richter, 2006, Vasudevan et al., 2006). However, the role of Pumilio homologs in mammalian fertility is unsolved as a mouse model of Pumilio gene dysfunction has not been established yet. There are two Pumilio paralogs in humans, PUMI- LIO1 and PUMILIO2 (Spassov and Jurecic, 2002). Although, similarity in the amino acid sequence is very high, these proteins significantly diverge in tissue expression patterns. While PUMILIO1 mRNA was detected in all the human tissues tested (Jaruzelska, unpublished), PUMILIO2 is expressed mainly in ovary and testis (Moore et al., 2003). This indicates that in the course of mammalian evolution, one of the Pumilio homologs, PUMILIO2 might have become specialized in germ cell-specific regulatory processes. Namely, it was demonstrated that PUMILIO2 protein is present in the human spermatogonia stem cells indicating a potential role in stem cell maintenance as in model organisms. PUMILIO2 was previously shown to form a complex ß 2006 WILEY-LISS, INC. Grant sponsor: State Committee for Scientific Research; Grant number: 3PO5E 013 25. *Correspondence to: J. Jaruzelska, PhD, Institute of Human Genetics Polish Academy of Sciences, 32, Strzeszynska, 60-479 Poznan, Poland. E-mail: jaruzjad@man.poznan.pl Received 13 September 2006; Accepted 13 October 2006 Published online 8 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20683