The SNARE-associated component SNAPIN binds PUMILIO2 and NANOS1 proteins in human male germ cells.

It has been reported that a highly conserved human protein PUMILIO2 forms a complex with NANOS1 in human male germ cells, as does the Drosophila ancestor Pumilio, which binds Nanos to regulate translation of specific mRNAs. Here, we found that PUMILIO2 interacts also with SNAPIN, a modulator of SNARE complex assembly, which is involved in vesicle trafficking. We demonstrated that SNAPIN interacts additionally with NANOS1 protein. This is the first report demonstrating that the N-terminal region of NANOS1 is necessary for protein binding. In human testis, SNAPIN co-localizes with PUMILIO2 and NANOS1 in prenatal and also in spermatogenic germ cells of the adult. We describe for the first time the expression of SNAPIN in germ cells which raises possibility that SNAPIN plays an extra role in mammals which is germ cell specific. The presence of a coiled-coil domain responsible for protein-protein interaction could enable SNAPIN to be an adaptor of PUMILIO2 and NANOS1, binding other factors to regulate translation in the development of the human germ cells.

The highly conserved NANOS2 protein: testis-specific expression and significance for the human male reproduction.

The highly conserved Nanos gene was found to encode a translational repressor necessary for germ-cell development in lower organisms. The mammalian homologue, Nanos2, was recently found to be expressed in the mouse germ cells. Since its disruption caused infertility exclusively in males, we sought to study the significance of this gene in human male reproduction. Here, we describe for the first time the expression pattern of the NANOS2 gene in human tissues and show that it is testis specific. We found that NANOS2 protein is present in prenatal germ cells and at later stages in spermatogenesis. To elucidate the role of NANOS2 in human germ-line development, we screened this gene for mutations in 214 males with isolated sterility and spermatogenic abnormalities. We identified two heterozygous variants, each in a different oligospermic patient, the second allele being the wild-type. The influence of the first variant, a missense mutation H68Q on the sterility phenotype, was not obvious since it was accompanied by a microdeletion within the AZF region of the Y chromosome. The second variant contained a silent mutation, H109H. Although both mutations were situated within the most conserved RNA-binding domain and were absent in 400 fertile males, it is not obvious that they cause male infertility.

Spatial and temporal distribution of the neutral polymorphisms in the last ZFX intron: analysis of the haplotype structure and genealogy.

With 10 segregating sites (simple nucleotide polymorphisms) in the last intron (1089 bp) of the ZFX gene we have observed 11 haplotypes in 336 chromosomes representing a worldwide array of 15 human populations. Two haplotypes representing 77% of all chromosomes were distributed almost evenly among four continents. Five of the remaining haplotypes were detected in Africa and 4 others were restricted to Eurasia and the Americas. Using the information about the ancestral state of the segregating positions (inferred from human-great ape comparisons), we applied coalescent analysis to estimate the age of the polymorphisms and the resulting haplotypes. The oldest haplotype, with the ancestral alleles at all the sites, was observed at low frequency only in two groups of African origin. Its estimated age of 740 to 1100 kyr corresponded to the time to the most recent common ancestor. The two most frequent worldwide distributed haplotypes were estimated at 550 to 840 and 260 to 400 kyr, respectively, while the age of the continentally restricted polymorphisms was 120 to 180 kyr and smaller. Comparison of spatial and temporal distribution of the ZFX haplotypes suggests that modern humans diverged from the common ancestral stock in the Middle Paleolithic era. Subsequent range expansion prevented substantial gene flow among continents, separating African groups from populations that colonized Eurasia and the New World.

Human Y-chromosome variation in the western Mediterranean area: implications for the peopling of the region.

Y-chromosome variation was analyzed in a sample of 1127 males from the Western Mediterranean area by surveying 16 biallelic and 4 multiallelic sites. Some populations from Northeastern Europe and the Middle East were also studied for comparison. All Y-chromosome haplotypes were included in a parsimonious genealogic tree consisting of 17 haplogroups, several of which displayed distinct geographic specificities. One of the haplogroups, HG9.2, has some features that are compatible with a spread into Europe from the Near East during the Neolithic period. However, the current distribution of this haplogroup would suggest that the Neolithic gene pool had a major impact in the eastern and central part of the Mediterranean basin, but very limited consequences in Iberia and Northwestern Europe. Two other haplogroups, HG25.2 and HG2.2, were found to have much more restricted geographic distributions. The first most likely originated in the Berbers within the last few thousand years, and allows the detection of gene flow to Iberia and Southern Europe. The latter haplogroup is common only in Sardinia, which confirms the genetic peculiarity and isolation of the Sardinians. Overall, this study demonstrates that the dissection of Y-chromosome variation into haplogroups with a more restricted geographic distribution can reveal important differences even between populations that live at short distances, and provides new clues to their past interactions.

Haplotype analysis of phenylalanine hydroxylase alleles in polish families with phenylketonuria.

Eight polymorphic restriction enzyme sites at phenylalanine hydroxylase locus from the parental chromosomes in Polish families with phenylketonuria were analyzed. Among 28 chromosomes studied, we identified haplotypes found within the Danish population. Haplotype 2 has been found in 25% of affected alleles. One of the patients studied is homozygous for this haplotype.

A familial X/Y translocation: cytogenetic and molecular study.

In this study we describe a 3-generation family carrying a (X;Y)(p22.3;q11.2) translocation in seven individuals of both sexes. Molecular analysis of the aberrant (X;Y)(p22.3;q11.2) chromosome was performed by FISH using X and Y-specific painting probes and also PCR amplification of the Y-specific sequences. Using these approaches it was demonstrated that the translocation resulted in a deletion of both X and Y pseudoautosomal regions. Moreover, using RBG banding it was shown that in all females the X-derivative chromosome was inactive in over 90% of mitoses. From the preliminary results obtained in this study we assumed that in this particular family the observed phenotype of the patients was caused by a deletion of the cluster of pseudoaotosomal genes responsible for the stature. More proximal loci, like STS or MRX49, were probably not deleted, since neither ichtyosis nor mental retardation was observed in this family.

[Molecular analysis of protein 4.1 gene in teratozoospermic and azoospermic patients]. / Analiza molekularna genu kodujacego bialko 4.1 u pacjentów z teratozoospermia i azoospermia.

In normal sperm, the 135 kD isoform of protein 4.1 is replaced by the 80 kD variant. Transcript for protein 4.1 loses the 'upstream' initiation codon by a stage-dependent alternative splicing and in mature sperm only 'downstream' initiation codon is active. A mutation in the 'downstream' initiation codon may be a reason for sperm differentiation arrest (azoospermia) or can be associated with the presence of amorphous spermatozoa in ejaculate (teratozoospermia). The aim of the study was the molecular analysis of gene coding for the protein 4.1, carrying a 'downstream' translation initiation codon. We have screened DNA samples obtained from azoospermic (blood) and teratozoospermic (spermatozoa) patients using PCR amplification of gene fragment, AUG containing exon with subsequent digestion (NlaIII) of CATG sequence. The absence of a cleavage site for this restriction enzyme would suggest the presence of mutation in the AUG codon. Analysis of DNA samples obtained from both azoospermic and teratozoospermic patients did not reveal any changes in the 'downstream' translation initiation codon. We concluded therefore that observed by others a defective expression of protein 4.1 in amorphous sperm cells is probably due to the other factor(s) than mutation in the 'downstream' translation initiation codon.

[A case of true hermaphroditism]. / Przypadek obojniactwa prawdziwego.

In the paper we present the case of true hermaphroditism in young girl. The examinations revealed the presence of an ovary with left corn of the uterus at the left side and immature testis with seminal duct the right side.

[Importance of cytogenetic analysis in patients with azoospermia or severe oligozoospermia undergoing in vitro fertilization]. / Znaczenie badan cytogenetycznych u pacjentów z azoospermia lub ciezka postacia oligozoospermii, korzystajacych z zaplodnienia in vitro.

The karyotypic analysis was performed to assess the importance of genetic factor in male infertility. For that purpose, chromosomal analysis in blood lymphocytes was performed in 28 males, candidates for ICSI with azoospermia or severe oligozoospermia and in their spouses. Although chromosomal aberrations were identified in as many as 11 couples, (in 6 couples aberrations were identified in male, in 4 other couples in female partner, whereas in 1 one couple they were detected in both partners) their risk for potential offspring is unequal. Balanced autosomal aberrations detected in two males (7%) constitute a high risk since they can cause not only infertility but also severe somatic abnormalities if transferred as the unbalanced ones to the next generation. The remaining 9 chromosomal aberrations identified in this study were present in mosaic additional cell lines with low representation. In 8 of them sex chromosomes and in 1 an autosom were involved. Although these mosaic chromosomal aberrations can lower efficiency of in vitro fertilisation, the probability that they can be transferred to the next generation causing somatic abnormalities is not high. This study indicates that in case of azoospermia or severe oligozoospermia, the karyotypic analysis should be performed in both partners prior to in vitro fertilisation.

Polymorphisms of the human PUMILIO2 gene and male sterility.

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 + 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 + 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 PUMILIO2 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.

Is selection responsible for the low level of variation in the last intron of the ZFY locus?

DNA variability was investigated in the last intron of the Y-chromosome-specific zinc finger gene, ZFY, and its X homolog on Xp21.3, ZFX. No polymorphisms were found in the 676-bp ZFY segment in a sample of 205 world-wide-distributed Y chromosomes, other than a solitary nucleotide variant in one individual (nucleotide diversity pi = 0.0014%). In contrast, 10 segregating sites (pi = 0.082%) were identified within 1,089 bp of the ZFX sequence in a sample of 336 X chromosomes. Four of these polymorphisms, which contributed most of the diversity, were located within an Alu insert disrupting the ZFY-ZFX homology (pi Alu = 0.24%). The diversity in the homologous portion of the ZFX intron, although higher than that in ZFY, was lower than that found in genomic segments believed to evolve neutrally; interspecies divergence in both segments was also reduced. Although this suggests that the evolution of both ZFY and ZFX homologs may not be entirely neutral, both Tajima and HKA tests did not reject neutrality. The lack of statistical significance may be attributed to a lack of power in these tests (the low divergence and variability values reduce the power of the HKA and Tajima tests, respectively); furthermore, Homo sapiens has recently undergone a rapid population growth, and selection is more difficult to detect in an expanding population. Therefore, the failure to reject neutrality does not necessarily indicate the absence of selection. In this context, the phylogenetic argument was given more weight in out interpretations. The high level of sequence identity in ZFY and ZFX segments, in spite of their separation 80-130 MYA, reflects a lower mutation rate as compared with other segments believed to undergo unconstrained evolution. Thus, the possibility of weak selection contributing to the low level of nucleotide diversity in the last ZFY intron cannot be excluded and should be kept in mind in the population genetics studies based on Y chromosome variability.

Deletion mapping of interval 6 of the human Y chromosome.

DNAs of four individuals demonstrating abnormalities in sexual development and mosaic 45,XO/46,XY karyotypes with terminal deletions of Yq were studied using a number of Y-specific probes. The results of these analyses allowed us to map several known DNA fragments within deletion interval 6 in the following order: Ycen-pDP105B/52dA, 50f2E, Fr25-II/Fr15-II, 50f2C, 49f-Yqter (groups of fragments in undetermined order separated by diagonal lines).

Small nuclear RNAs synthesis in PHA-stimulated and nonstimulated human peripheral blood lymphocytes.

Five fractions of small nuclear RNAs (snRNAs) were identified in nuclei of human peripheral blood lymphocytes. They have been designated as: H, G', D, C and A (nomenclature according to Weinberg) in order to decreasing electrophoretic mobility. Their synthesis was observed from the first day of treatment with mitogen and was continued with different intensity during four days of culture. In nonstimulated lymphocytes exclusively A and G' fractions were detected but no (14C)-uridine incorporation was observed.