RNAi-mediated knockdown of inhibin α subunit increased apoptosis in granulosa cells and decreased fertility in mice

Inhibin α (INHα), a member of TGFβ superfamily, is an important modulator of reproductive function that plays a vital role in follicular changes, cell differentiation, oocyte development, and ultimately in mammalian reproduction. However, the role of inhibin α in female fertility and ovarian function remains largely unknown. To define its role in reproduction, transgenic mice of RNAi-INHα that knock down the INHα expression by shRNAi were used. Inhibin α subunit gene was knocked down successfully at both transcriptional and translational levels by RNAi PiggyBac transposon (Pbi) mediated recombinant pshRNA vectors and purified DNA fragments were microinjected into mouse zygotes. Results showed that transgenic female mice were sub-fertile and exhibited 35.28% reduction in litter size in F1 generation relative to wild type. The decreased litter size associated with the reduction in the number of oocytes ovulated after puberty. Serum INHα level was significantly decreased in both 3 and 6 weeks; whereas, FSH was significantly increased in 3 weeks but not in 6 weeks. Furthermore, suppression of INHα expression significantly promoted apoptosis by up-regulating Caspase-3, bcl2, INHβB and GDF9 and down regulated Kitl and TGFβRIII genes both at transcriptional and translational levels. Moreover, it also dramatically reduced the progression of G1 phase of cell cycle and the number of cells in S phase as determined by flow cytometer. These results indicate that suppression of INHα expression in RNAi-transgenic mice leads to disruption of normal ovarian regulatory mechanism and causes reproductive deficiencies by promoting cellular apoptosis, arresting cellular progression and altering hormonal signaling.