Julio Sanchez Rufas

Sex determination in mammals is usually provided by a pair of chromosomes, XX in females and XY in males. Mole voles of the genus Ellobius are exceptions to this rule. In Ellobius tancrei, both males and females have a pair of XX chromosomes that are indistinguishable from each other in somatic cells. Nevertheless, several studies on Ellobius have reported that the two X chromosomes may have a differential organization and behavior during male meiosis. It has not yet been demonstrated if these differences also appear in female meiosis. To test this hypothesis, we have performed a comparative study of chromosome synapsis, recombination, and histone modifications during male and female meiosis in E. tancrei. We observed that synapsis between the two X chromosomes is limited to the short distal (telomeric) regions of the chromosomes in males, leaving the central region completely unsynapsed. This uneven behavior of sex chromosomes during male meiosis is accompanied by structural modifications of one of the X chromosomes, whose axial element tends to appear fragmented, accumulates the heterochromatin mark H3K9me3, and is associated with a specific nuclear body that accumulates epigenetic marks and proteins such as SUMO-1 and centromeric proteins but excludes others such as H3K4me, ubiH2A, and γH2AX. Unexpectedly, sex chromosome synapsis is delayed in female meiosis, leaving the central region unsynapsed during early pachytene. This region accumulates γH2AX up to the stage in which synapsis is completed. However, there are no structural or epigenetic differences similar to those found in males in either of the two X chromosomes. Finally, we observed that recombination in the sex chromosomes is restricted in both sexes. In males, crossover-associated MLH1 foci are located exclusively in the distal regions, indicating incipient differentiation of one of the sex chromosomes into a neo-Y. Notably, in female meiosis, the central region of the X chromosome is also devoid of MLH1 foci, revealing a lack of recombination, possibly due to insufficient homology. Overall, these results reveal new clues about the origin and evolution of sex chromosomes.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouseMus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

The histone variant H2AX phosphorylated on serine 139, named γ-H2AX, is a canonical DNA double-strand breaks marker. During mammalian meiotic prophase I, γ-H2AX participates in meiotic recombination, meiotic sex chromosome inactivation and meiotic silencing of unsynapsed chromatin. In this study, we have analyzed the distribution of γ-H2AX during male mouse meiosis by immunofluorescence on spread and squashed spermatocytes. We have found that γ-H2AX locates at the inner kinetochore plate of meiotic kinetochores in both meiotic divisions. Therefore our results, for the first time, uncover a novel role for γ-H2AX at mammalian meiotic kinetochores.

Natural populations of the house mouse Mus musculus domesticus show great diversity in chromosomal number due to the presence of chromosomal rearrangements, mainly Robertsonian translocations. Breeding between two populations with different chromosomal configurations generates subfertile or sterile hybrid individuals due to impaired meiotic development. In this study, we have analyzed prophase-I spermatocytes of hybrids formed by crossing mice from Vulcano and Lipari island populations. Both populations have a 2n = 26 karyotype but different combinations of Robertsonian translocations. We studied the progress of synapsis, recombination, and meiotic silencing of unsynapsed chromosomes during prophase-I through the immunolocalization of the proteins SYCP3, SYCP1, γH2AX, RAD51, and MLH1. In these hybrids, a hexavalent is formed that, depending on the degree of synapsis between chromosomes, can adopt an open chain, a ring, or a closed configuration. The frequency of these configurations varies throughout meiosis, with the maximum degree of synapsis occurring at mid pachytene. In addition, we observed the appearance of heterologous synapsis between telocentric and metacentric chromosomes; however, this synapsis seems to be transient and unstable and unsynapsed regions are frequently observed in mid-late pachytene. Interestingly, we found that chiasmata are frequently located at the boundaries of unsynapsed chromosomal regions in the hexavalent during late pachytene. These results provide new clues about synapsis dynamics during meiosis. We propose that mechanical forces generated along chromosomes may induce premature desynapsis, which, in turn, might be counteracted by the location of chiasmata. Despite these and additional meiotic features, such as the accumulation of γH2AX on unsynapsed chromosome regions, we observed a large number of cells that progressed to late stages of prophase-I, indicating that synapsis defects may not trigger a meiotic crisis in these hybrids.

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early “meiotic” repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a “somatic-like” repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase. DNA repair is critical for both somatic and meiotic cells. During meiosis, hundreds of DNA double strand breaks (DSBs) are introduced endogenously. To repair this damage, meiotic cells use a specialized version of the homologous recombination (HR) pathway that uses specific meiotic recombinases, such as DMC1, to promote repair with the homologous chromosome instead of the sister chromatid. This process is important to ensure chromosome segregation during meiosis and, as a side consequence, increases the genetic diversity of offspring. Nevertheless, under specific circumstances, meiotic cells can use other DNA repair mechanisms such as non-homologous end joining (NHEJ), which is error-prone. We investigated the response of mouse spermatocytes to increased DNA damage caused by gamma radiation, which is commonly used in cancer therapy. We found that the excess of DSBs produced by irradiation is processed by the meiotic HR recombination pathway in spermatocytes at the early stages of first meiotic prophase. However, this response is not inducible from the mid-pachytene stage onwards. From this point on, spermatocytes rely on a response that shares many features with that of somatic cells. In this response, the NHEJ pathway is first used to repair DNA damage but is subsequently replaced by a HR mechanism that does not use DMC1. Instead, it relies only on RAD51, which is known to function in both somatic and meiosis cells and, contrary to DMC1, has a preference for the sister chromatid. This switch from a meiotic to a somatic-like response is accompanied by a conspicuous change in the epigenetic response to DNA damage, reinforcing the idea that a functional transition occurs in meiotic cells during the mid-pachytene stage.

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated first by NHEJ, involving Ku70/80 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early “meiotic” repair pathway that acts by default at the beginning of meiosis is replaced from mid pachytene onwards by a “somatic-like” repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, expression of a new gene profile and acquisition of competence to proceed to metaphase.

Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species.

During meiosis, transcription is precisely regulated in relation to the process of chromosome synapsis. In mammals, transcription is very low until the completion of synapsis in early pachytene, and then reactivates during mid pachytene, up to the end of diplotene. Moreover, chromosomes or chromosomal regions that do not achieve synapsis undergo a specific process of inactivation called meiotic silencing of unpaired chromatin (MSUC). Sex chromosomes, which are mostly unsynapsed, present a special case of inactivation named meiotic sex chromosome inactivation (MSCI). Although processes that are similar to MSUC/MSCI have been described in other species like Sordaria and Caenorhabditis elegans, very few studies have been developed in insects. We present a study on the relationships between synapsis and transcription in two hemipteran species (Graphosoma italicum and Carpocoris fuscispinus) that possess holocentric chromosomes but develop different synaptic patterns. We have found that transcription, revealed by the presence of RNA polymerase II, is very low at the beginning of meiosis, but robustly increases during zygotene, long before the completion of synapsis, excepting in the sex chromosomes. In fact, we show that histone H3 methylation at lysine 9 (H3K9me3) may be present in the sex chromosomes at leptotene, thus acting as a likely epigenetic mark for this inactive state. Our results suggest that the meiotic transcription in these two species is differently regulated from that of mammals and, therefore, offer new opportunities to understand the relationship between synapsis and transcription and the mechanisms that govern MSUC/MSCI processes.

We analyzed the distribution of 2 repetitive DNAs, i.e. ribosomal DNA (rDNA) and a satellite DNA (satDNA), on the B chromosomes found in 17 natural populations of the grasshopper Eyprepocnemis ploransplorans sampled around the western Mediterranean region, including the Iberian Peninsula, Balearic Islands, Sicily, and Tunisia. Based on the amount of these repetitive DNAs, 4 types of B variants were found: B1, showing an equal or higher amount of rDNA than satDNA, and 3 other variants, B2, B24 and B5, bearing a higher amount of satDNA than rDNA. The variants B1 and B2 varied in size among populations: B1 was about half the size of the X chromosome in Balearic Islands, but two-thirds of the X in Iberian populations at Alicante, Murcia and Albacete provinces. Likewise, B2 was about one-third the size of the X chromosome in populations from the Granada province but half the size of the X in the populations collected at Málaga province. The widespread geographical distribution of the B1 variant makes it the best candidate for being the ancestor B chromosome in the whole western Mediterranean region.