117. Meiosis arrest in a new animal model – a mutation on chromosome 5

Abstract

Many causes of male infertility are currently unknown and there is a great need for the identification of new genes involved in spermatogenesis. The chemical mutagen, N-ethyl-N-nitrosourea (ENU) was utilized to induce random point mutations in the germline of C57Bl-6J mice in order to generate models of male infertility. Three generation breeding programs produced mice homozygous for ~3–4 point mutations. Following a phenotype-based screen of 1650 G3 males from 155 pedigrees, 15 lines were identified with abnormal male fertility parameters consistent with a recessive mutation. One infertile line, ENU23, exhibited a complete meiosis arrest with no germ cells ever proceeding to become spermatozoa. No infertile females have been observed. Light microscopic examination of Bouin’s fixed paraffin embedded testis sections showed that the arrest occurred post-prophase and prior to the completion of metaphase I, since normal metaphase was not observed. In cells beyond the prophase stage of development, spermatocytes appeared condensed, became enlarged and subsequently underwent cell death by apoptosis confirmed by TUNEL analysis. Electron microscopy visualized normal synaptonemal complexes. The degenerating primary spermatocytes exhibited condensed chromosomes irregularly arranged on a poorly formed microtubular spindle, indicating an abortive attempt to complete metaphase. The percentage of tubules with diplotene/metaphase-like cells was calculated for 3 abnormal and 3 normal ENU23 mice and showed 10.8% compared to 1.37%, respectively, indicating that prior to cell death the cells undergo a lag period. To identify the ENU23 causal mutation, the mutation was bred onto a mixed C57Bl-6J/CBA background and linkage analysis was performed, identifying a region on chromosome 5 between microsatellite markers D5Mit7 and D5Mit32. Candidate genes within this region are currently being sequenced in the search for the ENU-induced mutation causing the ENU23 phenotype. We believe this model of metaphase arrest is unique and will provide insights into the male specific events of meiosis.