Functional evaluation of miR-212-132 and miR-183-96-182 clusters during follicle-luteal transition in the monovular ovary
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Date
08/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Mohammed, Bushra Taher
Metadata
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.