Disruption of Chtf18 Causes Defective Meiotic Recombination in Male Mice

Abstract

CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.

Genetic Etiology of Chromosome 21 Nondisjunction and Down syndrome Birth: Aberrant Recombination and Beyond

“…In a different study mutant allele of chtf 18 causing impairment in spermatogenesis and defective meiotic recombination with premature chromatid separation in prophase I in male mouse has been documented. Additionally reduction in MLH1 foci has been found in this mouse strain [15]. These findings are suggestive of probable existence of similar etiology in Ch 21 nondisjunction in human, though such study is yet to be conducted.…”

Evidence for Paternal Age-Related Alterations in Meiotic Chromosome Dynamics in the Mouse

“…Specifically, our data suggest that the presence of "univalents" at the diplotene stage, which has been used in recent studies as a measure of recombination failure (e.g., Berkowitz et al 2012), may not provide an accurate assessment. The lack of obvious connections between homologs at diplotene may be misleading.…”

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

“…Each clump yields 2 slides and the number of slides yielded depends on the size of testes. We obtain 8 slides from wild type and 6 slides from subfertile Chtf18 -null P16 testes (which are smaller due to decreased numbers of meiotic and mitotic germ cells 8 ). One adult testis weighing 100 mg or more would be expected to yield 16 slides.…”

“…However, optimal results are best obtained for some proteins if immunostaining is performed immediately or within 2 weeks of preparing surface-spread nuclei. Slides can be immunostained with a standard protocol using antibodies to proteins of interest, followed by incubation with secondary antibodies conjugated to fluorescent proteins or dyes, then imaged with fluorescence microscopy 8 . At postnatal day 15 – 21 there is sufficient tissue per pair of wild type testes to yield 6 – 10 slides, and older mice (including adults) will yield more slides.…”