shRNA was injected to mice through intra-ovarian injection.

BACKGROUND/AIMS: Circadian locomotor output cycles protein kaput (CLOCK) plays a key role in maintaining circadian rhythms and activation of downstream elements. However, its function on human female reproductive system remains unknown. METHODS: To investigate the potential role of CLOCK, CLOCK-shRNAs were transfected into mouse 129 ES cells or injected into the ovaries of adult female mice. Western blotting was utilized to analyze the protein interactions and flow cytometry was used to assess apoptosis. RESULTS: The expression of CLOCK peaked at the 6th week in the healthy fetuses. However, an abnormal expression of CLOCK was detected in fetuses from spontaneous miscarriage. To determine the effect of CLOCK on female fertility, a small hairpin RNA (shRNA) strategy was used to specifically knockdown the CLOCK gene expression in vitro and in vivo. Knockdown of CLOCK induced apoptosis in mouse embryonic stem (mES) cells and inhibited the proliferation in mES cells in vitro. CLOCK knockdown also led to decreased release of oocytes and smaller litter size compared with control in vivo. CONCLUSIONS: Collectively, theses findings indicate that CLOCK plays an important role in fertility and that the CLOCK knockdown leads to reduction in reproduction and increased miscarriage risk.