Functional evaluation of miR-212-132 and miR-183-96-182 clusters during follicle-luteal transition in the monovular ovary

Abstract

Low fertility is a major cause of lost productivity in the cattle industry. In addition, cattle provide a convenient model to study ovarian physiology in monovular species including humans. Our previous microarray studies in the bovine ovary showed the upregulation of two clusters, miR-212-132 and miR-183-96-182, in luteal relative to follicular tissues. The studies in this thesis were aimed at establishing the functional involvement of these miRNAs during the follicle-luteal transition using a bovine model as well as human tissues. The aim of the first study was to characterise the expression of miR-132 and miR-96 within luteal tissue using ISH and FACS. The expression of miR-132 was detected in most luteal compartments while miR-96 was not detectable using ISH. Further examination using FACS showed that miR-212-132 expression remained unchanged in sorted endothelia (CD144+) and steroidogenic (CD144-Nile Red (NR) +) cell fractions. In contrast, expression of miR-183 and miR-96 was significantly increased in CD144+ compared to CD144-NR+ fractions. To elucidate potential roles of these miRNAs in the CL, I used existing online databases to identify putative miRNA targets. I identified 3042 predicted bovine gene targets of these miRNAs as well as 174 miRNA targets that had been experimentally validated in human, mouse and/or rat. I also identified putatively targeted signalling pathways primarily involved in cell survival, proliferation and differentiation. For further investigation, I narrowed my list of targets to FOXO1 and ADCY6, the expression of which was naturally down- regulated during luteinisation. The second study used an in vitro model of bovine granulosa cell luteinisation. Levels of miR-183-96-182 and miR-212-132 increased significantly (P<0.05) during the first 4 days of luteinisation in vitro. The function of miR-132 and miR-96 during luteinisation in vitro was studied. Transfection of bovine granulosa cells with specific miRNA inhibitors or mimics of miR-132 and miR-96 led, respectively, to abolished expression and a significant increase in the levels of these miRNAs (P<0.01) within 4 days. These changes in miRNA levels did not have any effect on transcript levels of the predicted mRNA targets, FOXO1 and ADCY6, during luteinisation. However, progesterone production by luteinising granulosa cells decreased (P<0.05) on day 2 after transfection with miR-132 inhibitor. The results demonstrated that putative miRNA target genes remained unchanged during in vitro luteinisation which was not consistent with in vivo results. The third study aimed to elucidate the effect of miRNA inhibition in bovine luteal cells in culture. The loss of miR-132 led to an increase (P<0.05) in FOXO1 transcript but not protein levels. In contrast, inhibition of miR-96 increased protein but not transcript levels of FOXO1. Moreover, miR-96 inhibition induced an increase in the caspase 3/7 response of luteal cells to serum deprivation indicating an anti-apoptotic effect of this miRNA on these cells. In the fourth study, I investigated the role of miR-132 and miR-96 in human luteinised granulosa cells obtained from IVF patients. The levels of FOXO1 protein were significantly increased following depletion of miR-132 and miR-96, whereas caspase3/7 increased in response to miR-96 inhibition, regardless of whether cells had been serum deprived or not. Similarly, using Annexin V and Trypan blue staining an increase in numbers of apoptotic cells was observed in response to miR- 96 inhibition. In addition, reduction of FOXO1 with the siRNA inhibited the apoptotic effect of miR-96 inhibition. Interestingly, inhibition of pooled miR-132 and miR-96 reduced progesterone secretion. However, this effect was prevented by transfecting cells with FOXO1 siRNA. These results suggest that the effects of these miRNAs on cell survival and progesterone production are mediated through targeting FOXO1. In summary, my results identify miR-96 and miR-132 as potentially critical factors in ensuring luteal cell survival and steroidogenesis in both cattle and human.