CNTD1 coordinates the maturation and designation of meiotic crossover sites from an excess pool of double-strand break intermediates by regulating dynamic changes in key protein complexes associated with these sites.

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM.

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse ( Mlh3 DN/DN ) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3 DN/DN males, like fully null Mlh3 -/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3 -/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3 DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3 DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3 -/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3 DN/DN males. Double mutant mice homozygous for the Mlh3 DN/DN and Mus81 -/- mutations show losses in chiasmata close to those observed in Mlh3 -/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3 DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3 DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3 DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.

The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.

Fancj, the gene associated with Fanconi anemia (FA) Complementation Group J, encodes a DNA helicase involved in homologous recombination repair and the cellular response to replication stress. FANCJ functions in part through its interaction with key DNA repair proteins, including MutL homolog-1 (MLH1), Breast Cancer Associated gene-1 (BRCA1), and Bloom syndrome helicase (BLM). All three of these proteins are involved in a variety of events that ensure genome stability, including the events of DNA double strand break (DSB) repair during prophase I of meiosis. Meiotic DSBs are repaired through homologous recombination resulting in non-crossovers (NCO) or crossovers (CO). The frequency and placement of COs are stringently regulated to ensure that each chromosome receives at least one CO event, and that longer chromosomes receive at least one additional CO, thus facilitating the accurate segregation of homologous chromosomes at the first meiotic division. In the present study, we investigated the role of Fancj during prophase I using a gene trap mutant allele. FancjGT/GT mutants are fertile, but their testes are very much smaller than wild-type litter-mates, predominantly as a result of impeded spermatogonial proliferation and mildly increased apoptosis during testis development in the fetus. This defect in spermatogonial proliferation is consistent with mutations in other FA genes. During prophase I, early events of synapsis and DSB induction/repair appear mostly normal in FancjGT/GT males, and the FANCJ-interacting protein BRCA1 assembles normally on meiotic chromosome cores. However, MLH1 focus frequency is increased in FancjGT/GT males, indicative of increased DSB repair via CO, and is concomitant with increased chiasmata at diakinesis. This increase in COs in the absence of FANCJ is associated with increased localization of BLM helicase protein, indicating that BLM may facilitate the increased rate of crossing over in FancjGT/GT males. Taken together, these results demonstrate a critical role for FANCJ in spermatogenesis at two stages: firstly in the proliferative activity that gives rise to the full complement of testicular spermatogonia and secondly in the establishment of appropriate CO numbers during prophase I.

words)The MLH1-MLH3 complex is essential for crossing over in mammalian meiosis. We generated a mutation in mouse MLH3 that alters its conserved endonuclease domain and show that it disrupts crossing over in a manner distinct from the full null Mlh3 mouse, but also results in male infertility. 5 3 ABSTRACT During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are 10 designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3 DN/DN ) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3 DN/DN males, like fully null Mlh3 -/males, have no spermatozoa and are infertile, yet spermatocytes have normal 15 DSBs and undergo normal synapsis events in early prophase I. Unlike Mlh3 -/males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3 DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be 20 selected. While Mlh3 DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3 -/males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3 DN/DN males. Double mutant mice homozygous for the Mlh3 DN/DN and Mus81 -/mutations show losses in chiasmata that approach levels observed 25 in Mlh3 -/males, indicating that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in Mlh3 DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3 DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3 DN/DN allele show defects in the repair of meiotic 30

Meiosis is the specialized cell division in sexually reproducing organisms in which haploid gametes are produced. Meiotic prophase I is the defining stage of meiosis, when pairing and synapsis occur between homologous chromosomes, concurrent with reciprocal recombination (or crossing over) events that arise between them. Any disruption of these events during prophase I can lead to improper segregation of homologous chromosomes which can cause severe birth defects in the resulting progeny, and this occurs with alarming frequency in human oocytes. Thus, while the pathways that regulate these events in prophase I are highly conserved in both males and females, the stringency with which these events are monitored and/or controlled appears to be dramatically lower in females. These observations underscore the need to examine and compare meiotic mechanisms across the sexes. However, the study of female meiosis is impeded by the early start of meiosis during fetal development and the very limited amount of ovarian tissue available for meiotic analyses. Here we describe three different techniques which are useful for meiotic prophase I analysis in mouse/human oocytes, ranging from early prophase I events through until the resolution of crossing over at the first and second meiotic divisions.

Inspired by gecko’s adhesive feet, a wheeled wall climbing robot is designed in this paper with the synchronized gears and belt system acting as the wheels by considering both motion efficiency and adhesive capability. Adhesion of wheels is obtained by the bio-inspired adhesive material wrapping on the outer surface of wheels. A ducted fan mounted on the back of the robot supplies thrust force for the adhesive material to generate normal and shear adhesion force whilemoving on vertical surfaces. Experimental verification of robot climbing on vertical flat surface was carried out. The stability and the effect of structure design parameters were analyzed.