Search bioRxiv⌕ Search

Biology subjects

Bradley, R. A.

Publications and source records attributed to Bradley, R. A..

2 recordsLinked to original sources

Cyclin N-terminal domain-containing 1 (CNTD1) is critical for crossover designation during meiotic prophase I and for maintenance of ovarian reserve in mammalian oocytes

In meiotic prophase I, hundreds of double-strand breaks (DSBs) are formed throughout the genome. A majority of these breaks are repaired as non-crossovers (NCOs), while a minor subset are repaired as crossovers (CO). COs are essential for the faithful segregation of homologous chromsomes at the end of prophase I and errors in CO designation can result in aneuploidy, germ cell death, birth defects, or infertility. These errors are more evident in female meiosis compared to males and suggests that the events of meiotic prophase I are sexually dimorphic with respect to CO formation, placement, resolution, and/or surveillance. Here, we demonstrate a critical role for Cyclin N-Terminal Domain Containing 1 (CNTD1) protein in ensuring appropriate CO frequency and distribution across the genome during meiosis in females. We find that CNTD1 localizes with the heterodimer, MutL{gamma}, which marks the majority of CO that emerge in pachynema of prophase I, implicating CNTD1 in late-stage CO designation and/or maturation. Accordingly, loss of Cntd1 in oocytes results in failure to load MutL{gamma} and thus results in a catastrophic loss of chiasmata and sterility. Further investigation yielded a distinct phenotype in which the primordial follicles that form upon dictyate arrest are steadily lost from birth onwards, a temporal loss of follicles that is different to that seen in other CO mutants. We find that this follicle loss in Cntd1 mutants is dependent on the checkpoint kinase CHK2. Thus, in females, loss of Cntd1 appears to result in phenotypes that are temporally disconnected from early and late CO mutants such as MutS{gamma} and MutL{gamma}, which show early prophase I disruption and ablation of ovary structure, and no prophase I disruption and an appearance of wildtype ovaries, respectively. These data suggest novel dual roles for CNTD1 in CO designation and faithful progression of oocytes into dictyate arrest at late pachynema, the latter being critical for establishing the ovarian reserve in female mice.

genetics↗

Dynamic regulatory phosphorylation of mouse CDK2 occurs during meiotic prophase I

During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of homologous chromosomes during the first meiotic division. The mechanism by which DSBs become selected to repair as crossovers is unknown, although the crossover positioning and levels in each cell indicate it is a highly regulated process. One of the proteins that localises to crossover sites is the serine/threonine cyclin-dependent kinase CDK2. Regulation of CDK2 occurs via phosphorylation at tyrosine 15 (Y15) and threonine 160 (T160) inhibiting and activating the kinase, respectively. In this study we use a combination of immunofluorescence staining on spread spermatocytes and fixed testis sections, and STA-PUT gravitational sedimentation to isolate cells at different developmental stages to further investigate the temporal phospho regulation of CDK2 during prophase I. Western blotting reveals differential levels of the two CDK2 isoforms (CDK233kDa and CDK239kDa) throughout prophase I, with inhibitory phosphorylation of CDK2 at Y15 occurring early in prophase I, localising to telomeres and diminishing as cells enter pachynema. Conversely, the activatory phosphorylation on T160 occurs later, specifically the CDK233kDa isoform, and T160 signal is detected in spermatogonia and pachytene spermatocytes, where it co-localises with the Class I crossover protein MLH3. Taken together, our data reveals intricate control of CDK2 both with regards to levels of the two CDK2 isoforms, and differential regulation via inhibitory and activatory phosphorylation.

cell biology↗