Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination

The Ubiquitin Ligase Ubr2, a Recognition E3 Component of the N-End Rule Pathway, Stabilizes Tex19.1 during Spermatogenesis

Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19) has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway

High-Speed Mouse Backcrossing Through the Female Germ Line

<div><p>Transferring mouse mutations into specific mouse strain backgrounds can be critical for appropriate analysis of phenotypic effects of targeted genomic alterations and quantitative trait loci. Speed congenic breeding strategies incorporating marker-assisted selection of progeny with the highest percentage target background as breeders for the next generation can produce congenic strains within approximately 5 generations. When mating selected donor males to target strain females, this may require more than 1 year, with each generation lasting 10 to 11 weeks including 3 weeks of gestation and 7 to 8 weeks until the males reach sexual maturity. Because ovulation can be induced in female mice as early as 3 weeks of age, superovulation-aided backcrossing of marker-selected females could accelerate the production of congenic animals by approximately 4 weeks per generation, reducing time and cost. Using this approach, we transferred a transgenic strain of undefined genetic background to >99% C57BL/6J within 10 months, with most generations lasting 7 weeks. This involved less than 60 mice in total, with 9 to 18 animals per generation. Our data demonstrate that high-speed backcrossing through the female germline is feasible and practical with small mouse numbers.</p></div

Oct4 distribution and level in mouse clones: consequences for pluripotency

Somatic cell clones often fail at a developmental stage coincident with commencement of differentiation. The transcription factor Oct4 is expressed during cleavage stages and is essential for the differentiation of the blastocyst. Oct4 expression becomes restricted to the inner cell mass and epiblast. After gastrulation Oct4 is active only in germ cells and is silent in somatic cells. Here, Oct4 and an Oct4–GFP transgene were used as markers for which gene reprogramming could be directly related to the developmental potential of somatic cell clones. Cumulus cell clones initiated Oct4 expression at the correct stage but showed an incorrect spatial expression in the majority of blastocysts. The ability of clones to form outgrowths was reduced, and the outgrowths had low or even undetectable levels of Oct4 RNA or GFP. The quality of GFP signals in blastocysts correlated with the ability to generate outgrowths that maintain GFP expression and the frequency of embryonic stem (ES) cell derivation. Abnormal Oct4 expression in clones is either directly or indirectly caused by reprogramming errors and is indicative of a general failure to reset the genetic program. The abnormal Oct4 expression may be associated with aberrant expression of other crucial developmental genes, leading to abnormalities at various embryonic stages. Regardless of other genes, the variations observed in Oct4 levels alone account for the majority of failures currently observed for somatic cell cloning

Pluripotency deficit in clones overcome by clone–clone aggregation: epigenetic complementation?

Abnormal gene expression patterns in somatic cell clones and their attrition in utero are commonly considered a consequence of errors in nuclear reprogramming. We observe that mouse clone blastocysts have less than half the normal cell number, and that higher cell number correlates with correct expression of Oct4, a gene essential for peri-implantation development and embryonic pluripotency. To increase the cell number, we aggregated genetically identical clones at the 4-cell stage. Clone–clone aggregates did not form more blastocysts, but the majority expressed Oct4 normally and had higher rates of fetal and postnatal development. Fertilized blastocysts with low cell numbers, induced by removal of two blastomeres at the 4-cell stage, did not exhibit abnormal Oct4 expression, indicating that improved gene expression and post-implantation development of clone–clone aggregates is not a consequence of increased cell number. Rather, we propose that complementation of non-cell-autonomous defects of genetically identical, but epigenetically different, embryos results in improved gene expression in clone–clone aggregates

Backcrossing Outcomes.

<p>Backcrossing Outcomes.</p

High-speed backcrossing through the female germline.

<p>(A) Approximate duration of approaches to obtain congenic strains, based on 5 generations for marker-assisted strategies (high-speed, speed) and 9 generations for traditional backcrossing (normal). Assumed durations: gestation, 20 days; weaning/genotyping, 21 days; breeding, 32 days for speed and normal (assuming maturity of males at 7.5 weeks), and 5 days for high-speed (superovulation at pre-pubertal age). (B) Diagram illustrating the experimental approach for high-speed backcrossing through the female germline. Star, mutation of interest.</p

NMDA receptor channels: subtype specific potentiation by reducing agents

Sulfhydryl redox agents affect NMDA receptor activity. We investigated a putative redox site in four recombinant NMDA receptors. In 293 cells expressing NR1-NR2A channels dithiothreitol (DTT) rapidly potentiated L- glutamate-activated whole-cell currents and decreased the time course of desensitization and deactivation. Part of the current potentiation (reversible component) and all kinetic changes reversed upon washout. The remaining potentiation (persistent component) was abolished by an oxidizing agent. The N-terminal 370 residues of NR2A mediate the reversible component in chimeric NR2 subunits. In cells expressing the NR1-NR2B, -NR2C, and -NR2D channels DTT elicited only a slowly developing, persistent potentiation and increased the deactivation time course. In these, but not in NR1-NR2A, the DTT effect was rendered insensitive to reoxidation by alkylation. Reduced glutathione mimicked the DTT effects only in the NR1-NR2A receptor. Hence, molecularly distinct NMDA receptors differ profoundly in their responses to sulfhydryl redox agents