M. Burgos

A study of the Ag-staining significance in mitotic NOR's

The biological significance of nucleoli and mitotic Ag-staining NORs has been the cause of controversy for several years. Whereas several authors state or assume a direct correlation between transcriptional activity and Ag-staining, others deny this hypothesis or claim a correlation between Ag-staining and decondensation of the NOR chromatin. Our results show that a decondensed state of the NOR chromatin is necessary but not sufficient to permit silver impregnation, allowing us to conclude that transcriptional NOR activity in the previous interphase is additionally required for silver staining.

Inactive ribosomal cistrons are spread throughout the B chromosomes of Rattus rattus (Rodentia, Muridae). Implications for their origin and evolution.

In-situ hybridization with a rDNA probe has demonstrated the presence of non-transcribed ribosomal genes in the B chromosomes of the black rat Rattus rattus. To test whether methylation of ribosomal DNA present in the B chromosomes could account for their inactivation, we performed in-situ digestions and Southern analyses of DNA digested with the isoschizomers MspI and HpaII. Our results suggest that the accessory chromosomes of this species have originated from one of the smaller NOR-carrying chromosome pairs. In the course of evolution, repetitive sequences invaded this supernumerary element and its ribosomal DNA content was dispersed throughout the chromosome and inactivated by heterochromatinization and methylation.

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.

Multiple, polymorphic copies of SRY in both males and females of the vole Microtus cabrerae

In mammals, sex determination is controlled by the Y-linked gene SRY. Although SRY is male-specific in most eutherian and marsupial species, with a single copy on the Y chromosome, several rodent species have multiple Y-linked copies of SRY, and two mole-vole species of the genus Ellobius determine sex without the Y chromosome or the SRY gene. We searched for homologs of SRY in three vole species of the genus Microtus and concluded that this gene is not male-specific in M. cabrerae, as it is present in multiple, polymorphic copies in both males and females. In contrast, SRY is male-specific in the related species M. agrestis and M. nivalis. Up to 15 different partial sequences of the SRY gene were found in M. cabrerae. Southern blots suggest that most of the extra copies of SRY are X-linked. One of the copies observed only in males has a sequence identical to that of the SRY gene in M. agrestis and may represent a functional copy of the gene in this species. The rest are probably nonfunctional pseudogenes.

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.

Heterogeneous heterochromatin and size variation in the sex chromosomes of Microtus cabrerae.

The sex chromosomes of Microtus cabrerae are characterized by large segments of constitutive heterochromatin in both the X and Y. Alkali treatment, enzymatic digestion (trypsin), and fluorochrome staining reveal considerable heterogeneity within these heterochromatic blocks and allow us to distinguish several regions. In addition, variation in the length of these segments is also present, probably as a result of deletions. A possible explanation for the origin of these heterochromatic blocks is discussed.

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.

A MicroRNA (mmu-miR-124) Prevents Sox9 Expression in Developing Mouse Ovarian Cells

ABSTRACT In mammals, sex differentiation depends on gonad development, which is controlled by two groups of sex-determining genes that promote one gonadal sex and antagonize the opposite one. SOX9 plays a key role during testis development in all studied vertebrates, whereas it is kept inactive in the XX gonad at the critical time of sex determination, otherwise, ovary-to-testis gonadal sex reversal occurs. However, molecular mechanisms underlying repression of Sox9 at the beginning of ovarian development, as well as other important aspects of gonad organogenesis, remain largely unknown. Because there is indirect evidence that micro-RNAs (miRNA) are necessary for testicular function, the possible involvement of miRNAs in mammalian sex determination deserved further research. Using microarray technology, we have identified 22 miRNAs showing sex-specific expression in the developing gonads during the critical period of sex determination. Bioinformatics analyses led to the identification of miR-124 as the candidate gene for ovarian development. We knocked down or overexpressed miR-124 in primary gonadal cell cultures and observed that miR-124 is sufficient to induce the repression of both SOX9 translation and transcription in ovarian cells. Our results provide the first evidence of the involvement of a miRNA in the regulation of the gene controlling gonad development and sex determination. The miRNA microarray data reported here will help promote further research in this field, to unravel the role of other miRNAs in the genetic control of mammalian sex determination.

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.

Role of Apoptosis and Cell Proliferation in the Testicular Dynamics of Seasonal Breeding Mammals: A Study in the Iberian Mole, Talpa occidentalis

Apoptosis and cell proliferation are two important cellular processes known to be involved in the normal functioning of the testis in nonseasonally breeding mammals, but there is some controversy concerning their roles in the gonads of males from seasonally breeding species. We have studied the processes of apoptosis and cell proliferation in the testes of males of the Iberian mole (Talpa occidentalis), a species showing a strict seasonal reproduction pattern. Both males and females are sexually active during the winter and completely inactive in the summer, with two transitional periods, in the autumn and the spring. Adult males from these four reproductive stages were captured, and their testes were immunohistochemically studied for the presence of apoptotic and proliferation molecular markers as well for other testicular and meiotic cell-specific markers. We found that apoptosis varies in a season-dependent manner in the testes of male moles, affecting mainly late zygotene and pachytene cells during the period of sexual inactivity, but it does not differentially affect the number of Sertoli cells. More interestingly, apoptosis is not responsible for the massive germ-cell depletion occurring during mole testis regression. In addition, a wave of spermatogonial cell proliferation appears to restore the number of spermatogonia lost during the period of testis inactivity. According to current knowledge, data from moles indicate that mammals do not form a homogeneous group regarding the mechanisms by which the cell-content dynamics are regulated in the testes of males from seasonally breeding species.