An azoospermic man with a double-strand DNA break-processing deficiency in the spermatocyte nuclei: case report.

BACKGROUND: The mechanisms of meiotic arrest in human spermatogenesis are poorly known. METHODS AND RESULTS: A testicular biopsy from an azoospermic male showed complete spermatogenesis arrest at the spermatocyte stage, asynapsis, lack of formation of the XY body, partial reversion to a mitotic-like division and cell degeneration both at the prophase and at the abnormal cell divisions. Synaptonemal complex analysis showed minor segments of synapsis and mainly single axes. Fluorescent immunolocalization of meiotic proteins showed normal SYCP3, scarcity of SYCP1, null MLH1 foci, about 10 patches of gamma-H2AX, abnormal presence of BRCA1 among autosomal axes, absence of RAD51 in early and advanced spermatocytes and permanence of gamma-H2AX labelling up to the abnormal spermatocyte divisions that are the most advanced stage reached. There are at least six dominions of evenly packed chromatin resembling that of the normal XY body, but no true XY body. CONCLUSIONS: The protein phenotype and the fine structure of the nuclei are compatible with a deficiency of the processing of double-strand DNA breaks in the zygotene-like spermatocytes, but the features of this defect do not agree with Spo11, Sycp1, Atm and Dmc1 null mutations, which give absence of XY body, synapsis disturbances and spermatocyte apoptosis in mice.

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells.

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.

Fine structure and meiotic behaviour of the male multiple sex chromosomes in the genus Alouatta.

The meiotic cytology and fine structure of the sex multiples in males from two species of the genus Alouatta are presented and compared with descriptions from other species of this genus. As shown in pachytene by synaptonemal complex analysis and in metaphase I by spreading, there is a quadrivalent in male meiosis in A. caraya, which is formed by an X(1)X(2)Y(1)Y(2) complex, while in A. palliata there is a trivalent formed by an X(1)X(2)Y(1) complex. Chromosome painting with human probes shows that A. caraya sex multiples share the same components as those of A. seniculus sara and A. seniculus arctoidea. However, as shown here for A. palliata and by others in A. fusca, there are differences among the multiples of some species. It is shown that in this genus there are several varieties of sex multiples that share some features, and that the origin of these multiples is most probably a primitive development in the genus Alouatta.

Ultrastructural and immunocytochemical analysis of the XY body in rat and Guinea pig.

The formation of the XY body involves the compaction of the extended chromatin to form a mesh of fibrogranular structures. During this process the ribonucleoprotein particles (RNP), which were associated with the chromatin filaments progressively disappear. High resolution immunolocalization indicates that the mature XY body does not contain RNA polymerase II, hnRNPs, or snURNPs. Occasionally chromatin fibrils extend outside of the XY body. These fibrils are frequently associated with nascent RNP fibrils and granules indicating that not all the DNA of the sex chromosomes is transcriptionally inactive. However, transcription is located outside the sex body. The recombination protein Dmc1 is present in nodules associated with the unpaired chromosomal axes of the sex chromosomes located in the XY body. Cytochemical staining methods and in situ hybridization at electron microscopic level show that RNA is present in the unpaired chromosomal axes suggesting that the presence of RNA in the chromosomal axes and in forming synaptonemal complexes is related with the process of final pairing. The sex body and the nucleoli associated with it do not interweave and do not exchange RNA or DNA-containing filaments. These observations indicate that the spatial relation between these structures is just a close proximity, which is, however, very frequent.

Spermatocytes and round spermatids of rat testis: the difference between in vivo and in vitro protein patterns.

During mammalian spermatogenesis meiotic cell division and spermiogenesis occurs. Gene expression during this process is temporally regulated at the transcriptional and translational levels but the mechanisms are not well understood. In this publication we have investigated the synthesis of proteins in vitro to detect the proteins with a high metabolic turnover and to compare them with the in vivo protein map. RNA of spermatocytes and round spermatid cell populations, purified by centrifugal elutriation, and total testis was isolated. The poly A+ mRNA fraction was translated using a rabbit reticulocyte lysate. The translation products were separated by two-dimensional (2-D) gel electrophoresis using nonlinear 3.5-10 immobilized pH gradients for the first-dimensional separation. The gels with 35S-translated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and scanned using a phosphorimager. A highly reproducible and complex protein pattern was obtained using this methodology. Only rat testis messages were translated. Using Melanie 2 software we could compare and detect more than 1000 proteins on 2-D radioactive images. Some changes could be observed in protein expression between the different cell types but they were not statistically significant. The comparison between the 2-D rat testis map and the in vitro translated patterns show no matching between any spots. This result suggests that the post-transcriptional modifications occurring in the reticulocyte system are not the same as those that occur in vivo in the testis. Rabbit reticulocyte proteins were detected by staining PVDF membranes with colloidal gold. Rat testis and reticulocyte patterns were completely different.