SCP3 is a meiosis-specific structural protein appearing at axial elements and lateral elements of the synaptonemal complex. We have analysed the behaviour of SCP3 and the cohesin subunit Rad21 in mouse spermatocytes by means of a squashing technique. Our results demonstrate that both proteins colocalize and are partially released from chromosome arms during late prophase I stages, although they persist at the interchromatid domain of metaphase I bivalents. Thus, Rad21 cannot be considered a `mitotic'-specific variant, but coexists with Rec8. During late prophase I SCP3 and Rad21 accumulate at centromeres, and together with the chromosomal passenger proteins INCENP and aurora-B kinase, show a complex `double cornet'-like distribution at the inner domain of metaphase I centromeres beneath the associated sister kinetochores. We have observed that Rad21 and SCP3 are displaced from centromeres during telophase I when sister kinetochores separate, and are not present at metaphase II centromeres. Thus, we hypothesise that Rad21, and the superimposed SCP3 and SCP2, are involved in the monopolar attachment of sister kinetochores during meiosis I, and are not responsible for the maintenance of sister-chromatid centromere cohesion during meiosis II as previously suggested.

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.

Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse. Cells have different mechanisms to assess the proper occurrence of cellular events. These mechanisms are called checkpoints and are involved in the surveillance of processes such as DNA replication and cell division. A checkpoint at the pachytene stage arrests meiosis when defects in the process of homologous chromosome synapsis and recombination are detected. In mammals, both transcriptional inactivation of chromosomal regions that are not correctly synapsed at pachytene and activation of sex chromosome genes that are normally silent during this stage could contribute to meiotic arrest. We found that when Robertsonian translocations appear in heterozygosis, many synapsis defects occur, and mechanisms that trigger transcriptional silencing of the unsynapsed chromatin are activated. However, meiotic prophase-I progression is not greatly compromised. This questions the ability of the meiotic checkpoints to halt meiosis progression when synapsis is not completed, allowing cells with synapsis defects to reach the first meiotic division. The fertility reduction of Robertsonian heterozygous mice seems to be mainly caused by errors detected by the metaphase-I checkpoint, when most of the spermatocytes die, rather than by synapsis defects. In an evolutionary context, a permissive pachytene checkpoint could contribute to increasing the chances of Robertsonian translocations to spread into natural populations.