José Angel Suja

Background/Objectives: We analyzed the relationship between synapsis, recombination, and transcription during the spermatogenesis of the grasshopper Eyprepocnemis plorans carrying B chromosomes (type B1). Methods: The progression of synapsis was interpreted according to the dynamics of the cohesin subunit SMC3 axes. DNA double-strand breaks were revealed by RAD51 immunolabeling, while transcriptional activity was determined by the presence of RNA polymerase II phosphorylated at serine 2 (pRNApol II) immunolabeling. The two repressive epigenetic modifications, histone H3 methylated at lysine 9 (H3K9me3) and histone H2AX phosphorylated at serine 139 (γ-H2AX), were employed to reveal transcriptional inactivity. Results: During prophase I, spermatocytes with one B1 chromosome showed overall transcription except in the regions occupied by both the X and the B1 chromosomes. This transcriptional inactivity was accompanied by the accumulation of repressive epigenetic modifications. When two B1 chromosomes were present, they could appear as a fully synapsed monochiasmatic bivalent, showing intense H3K9me3 labeling and absence of pRNApol II, while γ-H2AX labeling was similar to that shown by the autosomes. Conclusions: According to our results, B1 transcriptional inactivation was triggered in spermatogonia, long before the beginning of meiosis, and was accompanied by H3K9me3 heterochromatinization that was maintained throughout spermatogenesis. Moreover, when two B1 were present, the transcriptional inactivation did not preclude synapsis and recombination achievement by these chromosomes.

PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. One of the main centromeric regulatory signaling pathways is the haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin−/− mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1 (Δ/Δ) indicates a central role of Polo-like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo-like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.

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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.

Cohesin cofactors regulate the loading, maintenance and release of cohesin complexes from chromosomes during the mitotic cell cycle but little is known on their role during vertebrate meiosis. One such cofactor is PDS5, which exists in two versions in somatic and germline cells, PDS5A and PDS5B, with unclear functional specificity. Here we have analyzed their distribution and functions in mouse spermatocytes. We show that simultaneous elimination of PDS5A and PDS5B results in severe defects during prophase I while their individual depletion does not, suggesting a functional redundancy of the two factors. Shortened axial/lateral elements and a reduction of early recombination nodules are observed in the absence of both PDS5 proteins. Moreover, telomere integrity and their association to the nuclear envelope are severely compromised. As these defects occur without detectable reduction in chromosome-bound cohesin, we propose that the dynamic behavior of the complex, mediated by PDS5 proteins, is key for successful completion of meiotic prophase I.

A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.

Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins.

The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis‐specific cohesin subunits REC8 and SMC1β. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II. The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2‐PP2A. Synopsis This study analyses the distribution of Sororin, a regulatory factor of the cohesin complex, during male mouse meiosis. The results suggest different functions of Sororin at the synaptonemal complex and at the centromere. Sororin loads to the central region of the synaptonemal complex in a SYCP1‐dependent manner, but independently of cohesin complexes containing meiosis‐specific REC8 and SMC1β subunits. Sororin accumulates at the centromere by late prophase I and remains there until anaphase II. Sororin could participate in the regulation of centromeric cohesion during meiosis in collaboration with the SGO2‐PP2A complex.