A. Sánchez

Achiasmatic giant sex chromosomes in the vole Microtus cabrerae(Rodentia, Microtidae)

Conventional and microspread preparations of Microtus cabrerae spermatocytes were made to investigate the chromosomes of this species. Three different types of Y chromosomes, varying in size of the heterochromatic block, were observed; they were alike, however, in regard to synapsis, which was consistently absent. Our results suggest that the heterochromatic blocks are not involved in the lack of synapsis and that asynapsis is a cytological feature common to all species of the family Microtidae. In addition, the co-aligned configuration of the ends of the sex-chromosome axes of this species and the lack of silver-stainable threads or filaments connecting them suggest the existence of two mechanisms for association of the sex chromosomes during prophase I and metaphase I: attachment of the ends of both sex chromosome axes to the nuclear envelope and heterochromatin stickiness.

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.

Multiple mono- and polymorphic Y-linked copies of the SRY HMG-box in Microtidae

Sex determination in mammals is controlled by SRY (sex-determining region of the Y chromosome), a single-copy gene located on the Y-specific region. Several exceptions to this rule have been described: some rodent species present Y-specific multiple copies (either mono- or polymorphic) of this gene, and two Ellobius species and one Tokudaia species determine sex without a Y chromosome or the SRY gene. Recently, we have described multiple polymorphic copies of the SRY gene in both males and females of the vole species Microtus cabrerae. The female location and the presence of stop codons in some copies from males and females also suggest that they are nonfunctional copies of this gene (pseudogenes). We have investigated the SRY HMG-box in nine species of the family Microtidae; we report here the presence, in eight of these species, of multiple mono- or polymorphic copies of the SRY gene located on the Y chromosome.

Achiasmatic sex chromosomes in Pitymys duodecimcostatus: mechanisms of association and segregation

The meiotic behavior of the sex chromosomes of Pitymys duodecimcostatus was studied by electron microscopy of whole-mount synaptonemal complex preparations. The results established that the sex chromosomes of this species are achiasmatic and remain unassociated throughout meiotic prophase I in most spermatocytes. In other cells, nonspecific association of the X and Y occurred by means of filamentous bridges. Pitymys duodecimcostatus represents an additional example of a mammalian species lacking a homologous pairing segment in its sex chromosomes and extends current knowledge about this controversial subject. In this regard, we suggest that sex-chromosome association is a characteristic that probably followed different evolutionary paths in different mammals, leading to loss of the homologous segment in some species and its conservation in others. It is also suggested that in P. duodecimcostatus, and probably in many other species as well, three mechanisms may act in concert to permit joining of the X and Y chromosomes during meiotic prophase, and, consequently, to ensure proper segregation during anaphase I: (1) joining of the sex-chromosome axes at their ends to the nuclear membrane, (2) formation of fibrillar structures to hold the sex chromosomes together, and (3) cohesiveness due to sex-vesicle formation.

Sex chromosomes, sex determination, and sex-linked sequences in Microtidae

The Arvicolidae is a widely distributed rodent group with several interesting characteristics in their sex chromosomes. Here, we summarize the actual knowledge of some of these characteristics. This mammalian group has species with abnormal sex determination systems. In fact, some species present the same karyotype in both males and females, with total absence of a Y chromosome, and hence of SRY and ZFY genes. Other species present fertile, sex-reversed XY females, generally due to mutations affecting X chromosomes. Furthermore, in Microtus oregoni males and females are gonosomic mosaic (the females are XO in the soma and XX in the germ cells, while the males are XY in the soma and OY in the germ cells). Regarding sex chromosomes, some species present enlarged (giant) sex chromosomes because of the presence of large blocks of constitutive heterochromatin, which have been demonstrated to be highly heterogeneous. Furthermore, we also consider the alterations affecting composition and localization of sex-linked genes or repeated sequences. Finally, this rodent group includes species with synaptic and asynaptic sex chromosomes. In fact, several species with asynaptic sex chromosomes have been described. It is interesting to note that within the genus Microtus both types of sex chromosomes are present.

Recent evolution of NOR-bearing and sex chromosomes of the North African rodent Lemniscomys barbarus

The karyotype and meiotic phases of Lemniscomys barbarus from Morocco were extensively studied with G- and C-banding, Ag-NOR and fluorochrome staining, in situ hybridization with an rDNA probe, and synaptonemal complex analysis. Comparison of the data with those previously published for an Algerian specimen revealed in the Moroccan specimens the presence of large heterochromatic segments in the sex chromosomes, a new nucleolar organizer at chromosome pair 1, and silent NORs on both sex chromosomes — features that are not present in the Algerian specimen. These findings demonstrate that during the very recent evolution of the karyotype of this species a new NOR was acquired by pair 1, possibly by amplification of ribosomal genes after a translocation event. This new NOR changed the preference of activatio n of the NORs in these individuals and became the preferentially activated NOR. Another autosome—sex chromosome translocation led to the presence of NORs on the sex chromosomes, which were then inactivated by the invasion of repetitive sequences. These silent NORs may be involved in the pairing of the two sex chromosomes.

Fluorescence banding in four species of Microtidae: an analysis of the evolutive changes of the constitutive heterochromatin

Three different fluorochrome and specific counterstain combination (DAPI/AMD, DA/DAPI and CMA/DA) treatments were applied to the chromosomes of four Microtidae (Rodentia) species. The results complete the data obtained in our previous paper (Burgos, M., Jiménez, R., & Dìaz de la Guardia, R., Genome 30:540–546, 1988) and prove that the changes in the constitutive heterochromatin in the evolution of the karyotypes of these species are not only due to gain or loss of heterochromatin, but are qualitative with respect to their nucleotide composition, repeated base pair organization or DNA-protein complex modification. These variations lead to the differential response to the fluorescence dye combinations used.

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.

Restriction enzyme banding and in situ nick-translation on different types of hetero- and euchromatin

We studied the role of chromatin accessibility and methylation in the banding patterns produced by means of in situ nick-translation (NT) and restriction enzyme (RE) banding techniques. For these studies we used the X chromosomes of Microtus cabrerae because of their large segment with four different types of constitutive heterochromatin and because in these chromosomes we can also compare active and inactive euchromatin. The results demonstrate that constitutive heterochromatin in the X chromosomes of M. cabrerae is methylated at specific sequences in both active and inactive Xs. They also show that NT-based techniques are suitable for detecting weak differences in chromatin accessibility, such as differences between active and inactive euchromatin, and are able to distinguish methylation only at the accessible sites. Thus, when methylation has to be mapped in situ, additional experiments have to be performed in order to distinguish findings due to differential accessibility. RE banding seems less sensitive to slight differences in chromatin accessibility, and might thus be more suitable than in situ NT-based techniques for methylation mapping. In harmony with these results, HpaII-based RE banding is able to distinguish between active and inactive euchromatin, possibly depending on its methylation status.

Synaptonemal complex analysis of spermatocytes of Talpa occidentalis(Insectivora, Mammalia): autosomal synapsis and substaging of zygonema and pachynema

Spermatocytes from the mole, Talpa occidentalis, a species that includes both XX males and intersexes, were surface-spread and silver-stained to substage meiotic prophase from early zygonema through pachynema. In zygonema, only the Z2 and Z3 substages were found. This stage differed in comparison with such species as the Chinese hamster, laboratory mouse, and deer mouse, which belong to orders other than Insectivora. Pachynema, in which five substages were established (P1-P5), seems to be a more homogeneous stage, and remarkable differences with respect to the above-mentioned species were not found. Synaptic adjustment was demonstrated in X-Y pairing. Nonhomologous pairing was evident at the Y-centromeric region and considered likely in the proximal arm of this chromosome. In addition to sequencing the events taking place during zygonema and pachynema in males from a wild population in which some members show sex reversal, our finding represents the first attempt to substage zygonema and pachynema in an Insectivore species, thus contributing to current knowledge of the nature and degree of variability in the mammalian synaptic process.