Rafael Díaz de la Guardia

Cytogenetic peculiarities in the Algerian hedgehog: silver stains not only NORs but also heterochromatic blocks

Hedgehogs belong to one of the several mammalian taxa in which karyotype differences are based on variations in heterochromatin content. Furthermore, the number and location of nucleolar organizer regions (NORs) can also vary widely. In the present study these cytogenetic features were investigated in the Algerian hedgehog, Erinaceus (Aethechinus) algirus. The heterochromatin and NOR distribution patterns in the karyotype of this species are new among hedgehogs, whereas the euchromatic regions, including their G-band pattern, are similar to those reported by others. In addition, silver staining revealed a cytogenetic feature exclusive to the heterochromatic blocks of E. algirus: their silver staining with standard cytogenetic procedures. Because no similar phenomenon has been described previously in a mammalian species, several hypotheses about the significance and specificity of silver staining to NOR sites are discussed. Finally, the existence of different types of heterochromatin in the species analysed here, lead us to propose that what hedgehogs have inherited from their common ancestor is a mechanism which permits the accumulation of heterochromatin on specific chromosomes, rather than the heterochromatin itself.

Females of four mole species of genus Talpa (Insectivora, mammalia) are true hermaphrodites with ovotestes

We studied the anatomical, histological, and genetic features of the sexual tract in four European mole species of the genus Talpa (Insectivora, mammalia): T. occidentalis, T. europaea, T. romana, and T. stankovici. All XY individuals had a normal male phenotype, whereas all XX individuals in all four species had features that identified them as intersexes. These individuals were nonetheless presumed to be functionally fertile females. Intersexuality was manifested mainly as gonadal hermaphroditism, with all females possessing bilateral ovotestes. The gonads were composed of a small portion of histologically normal ovarian tissue and a variably sized, generally large mass of disgenetic testicular tissue, accompanied by a small, rudimentary epididymis. The rest of the sexual tract was typically female, including oviducts, uterus, and vagina of normal appearance. Polymerase chain reaction (PCR) and Southern blotting analyses showed that the mammalian testis‐determining gene SRY is present in males but not in females. Part of the conserved sequence of the mole SRY gene was cloned and sequenced after PCR amplification in two of the four mole species (T. occidentalis from Spain and T. romana from Italy). Sequences were identical in these two species and were very similar to those of the human and mouse SRY gene. Our findings constitute the first evidence of the existence of a genus‐specific case of true hermaphroditism, probably due to a very ancient mutation that fixed in populations of the ancestral species from which contemporary moles evolved. The possible nature of this mutation is discussed with regard to the cytologic, histologic, and genetic features of the gonads in Talpa females.

A Rapid, Simple and Reliable Combined Method for G-Banding Mammalian and Human Chromosomes

A simple and reliable method for G-banding chromosomes from human and mammalian cells is described. This rapid method combines hot saline and trypsin treatments and yields high quality G-bands in both bone marrow and cultured cells.

Molecular and cytogenetic characterization of highly repeated DNA sequences in the vole Microtus cabrerae.

The genus Microtus presents several species with extremely large sex chromosomes that contain large blocks of constitutive heterochromatin. Several cytogenetic and molecular studies of the repetitive sequences in species of the genus Microtus have demonstrated that the heterochromatin is highly heterogeneous. We have cloned and characterized a family of repetitive DNA sequences from M. cabrerae, a species with large heterochromatic blocks on the giant sex chromosomes. These repetitive sequences are 65.84% A–T rich, organized in tandem, with a 161-bp unit and are located on the centromeric region of autosomes and the X chromosome. In addition, this repetitive DNA is located throughout the entire heterochromatic block of the X chromosome and on three interstitial bands in the heterochromatic block of the Y chromosome. Comparative analysis of this family of repetitive sequences from three Microtus species revealed that the development of these sequences has occurred by concerted evolution. Our results support the hypothesis that the heterochromatic blocks from the sex chromosomes of different species are evolving independently and they probably have the genetic capacity to amplify and retain different satellite DNAs. For a topic related to the location of these repetitive DNA sequences on the Y chromosome of M. cabrerae, we propose a model to explain the origin of a length polymorphism previously described for this chromosome.

Interchromosomal, intercellular and interindividual variability of NORs studied with silver staining and in situ hybridization.

We investigated the relationship between transcriptional activity and the quantity of ribosomal cistrons of the nucleolar organizing regions (NORs) in chromosomal pair 3 of the Spanish mole Talpa occidentalis. Transcriptional activity was estimated by the size of conventional silver-staining signals. The number of ribosomal cistrons was estimated by the size of the in situ hybridization signals obtained with a ribosomal DNA probe. A sample of cells was studied with each method in four different individuals, and the results obtained with the two techniques were compared. Furthermore, sequential analysis on the same cells was carried out to study the association of interstitial nucleolar constrictions with silver staining and in situ hybridization. Interchromosomal, intercellular and interindividual variability were found with both silver staining and in situ hybridization methods. Our results show that transcriptional activity of NORs does not depend exclusively on the number of ribosomal cistrons they have as a high percentage of cells had two NORs with abundant ribosomal cistrons, only one of which was active. Additional factors, probably responsible for the activation of transcription, may be involved in this variability.

Ovotestis variability in young and adult females of the mole Talpa occidentalis (Insectivora, Mammalia)

The age-related evolution and ontogenic origin of the ovotestes in fertile females of the Spanish mole (Talpa occidentalis) were studied. Volume of the ovotestis and its ovarian and testicular components, size of the epididymis and testicular cords, number of ovarian follicles and testicular cords, uterus weight, and age index were analyzed statistically in a large sample of young and adult individuals of this species. Comparison of means and linear correlation analyses were done. Most variables were shown to be age dependent, with a period of rapid change during puberty. In adult animals, volume of the ovarian portion and uterus weight followed a seasonal cycle of sexual activity. Interindividual variability was evident in most of the variables investigated except for the number of testicular cords per ovotestis, which remained unchanged throughout the animals life and hence was not inversely correlated with the number of ovarian follicles. This finding ruled out an ovary-testis transdifferentiation hypothesis for the ontogenic origin of the testicular tissue in the ovotestes of female moles. An alternative hypothesis based in the absence of oocytes in a portion of the undifferentiated fetal gonad is proposed in accordance with a new general model for mammalian sex determination.

Expression profile of telomere-associated genes in multiple myeloma.

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14‐3‐3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM).

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2☆

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2.