J. del Mazo

Regulated expression of p14 (cofactor A) during spermatogenesis

The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself. Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance. As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation.

Multistranded organization of the lateral elements of the synaptonemal complex in the rat and mouse

Using electron microscopy, we have analyzed the structure of the lateral elements (LEs) of the synaptonemal complex (SC) in rat and mouse spermatocytes under different spreading conditions. Our observations indicate that in nearly 50% of the cells it is possible to detect more than one structural component of the LEs in the XY pair. When the same spreading conditions are used, the structure of the LEs is not the same in the autosomes and the XY pair. Moreover, the correlation between different spreading conditions and the detection of more than one sublateral element (subLE) was different in pachytene and diplotene spreads; it also varied depending on the species and meiotic stage. Our study supports the notion that the LEs of the SC are multistranded.

Ilf2 is regulated during meiosis and associated to transcriptionally active chromatin.

Analysis of gene expression during testis development demonstrated accumulation of Ilf2 mRNA in pachytene spermatocytes. In these cells, the protein was localized in the nucleus, but it was absent from chromatin of the XY pachytene bivalent, in which there is no transcriptional activity. Nucleolar signal is inmmunolocalized in spermatogonia, Sertoli cells and oocytes. By in situ hybridisation, Ilf2 expression is detected in proliferative cells of adult ovary and a defined pattern is also exhibited in different tissues of embryos. The presence of ILF2 in active chromatin is corroborated in NIH3T3 cultured cells after transfection with Ilf2-EGFP constructs.

Trisomy 21: Origin of non-disjunction

SummaryThe Q-band heteromorphisms of chromosome 21 were used in a sample of 48 families with a Downs syndrome child to evaluate the origin of non-disjunction.The parental origin and the meiotic error were determined in 27 families, and in eight families only partial information was obtained. Paternal and maternal origin of non-disjunction was in a 1:3 ratio. Failures were five times more frequent in first than in second meiotic division in both sexes.The mean parental age and environmental factors in relation to the origin of the anomaly are discussed.Our results are compared with those obtained previously in similar studies by other authors.

A new contribution to the study of 22 trisomy

SummaryThe case of a 21/2-month-old male child with intrauterine distrophy features and multiple congenital malformations is presented. Cytogenetic studies of the child and his parents, completed with Q- and G-banding techniques led us to conclude that it is a case of 22 trisomy inherited from his mother.

Partial deletion of 4p16 band in a ring chromosome and Wolf Syndrome.

SummaryA new case of ring chromosome 4 in a 2-day-old female child with multiple malformations is described. By means of the GTG-banding technique, a karyotype 46,XX,r(4), (p16→q35) was determined. The characteristics of the childs karyotype and the relationship with the structure of the chromosome, especially the location of the deletion that produces the syndrome, are compared with previous reports.

Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells

Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.

Centromere pattern in different mouse seminiferous tubule cells

Centromere arrangement in different mouse seminiferous tubule cells was analyzed using an anticentromere antiserum from a patient with the CREST syndrome of scleroderma. A peptide of 18 kd was recognized by this serum on immunoblotting of mouse nuclear proteins from seminiferous tubule cells. In the cells studied by immunofluorescence, different patterns of centromere arrangement were observed. A speckled arrangement of centromeres was found in spermatogonia, double spots corresponding to meiotic bivalents were found in pachytene cells, and clusters of a haploid numer of centromeres were found in early and acrosome phase spermatids. In Sertoli cells, only three centromeric spots were detected, corresponding to the nucleolar organizer chromosome pairs. A relationship between the functional stage of the cell and the arrangement of and conformational changes in the centromeres is considered.

Centromeric proteins recognized by CREST sera and meiotic chromosome segregation

Analysis of the peptides recognized by CREST sera was carried out in different mouse tissues and cells, including spermatozoa. In all cases, a polypeptide of Mr = 18000 was recognized by the sera and occasionally two other proteins of Mr = 80000 and Mr = 140000 were observed after immunoblotting of nuclear proteins. In both early and late spermatids, centromeric staining was observed after incubation and immunofluorescence with CREST sera. After detergent treatment, it was even possible to detect centromeric staining in mature spermatozoa. In spermatid cells, the immunofluorescent pattern presented a binomial distribution of the number of fluorescent spots, with a mean value around half of the haploid number of chromosomes. Since this pattern is the result of chromosome segregation after meiosis II, our data suggest that this centromeric peptide is not directly implicated in the chromosome segregation process. On the other hand, the distribution of spots after immunofluorescence suggests a different organization of centromeric components in meiosis I and meiosis II.

Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15(-/-)mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.