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Title : Flow cytometric quantification of receptors and apoptosis 1 in granulosa cells and ovulation rate

22 The aim of the study was to determine the direct cause of the mutation-induced, increased 23 ovulation rate in Booroola merino sheep (BB). Granulosa cells were removed from antral 24 follicles before ovulation and post-ovulation from BB (n=5) and wild type (Wt) (n=12) 25 merino ewes. Direct immunofluorescent measurement of mature cell surface receptors using 26 flow cytometry demonstrated a significant up-regulation of follicle stimulating hormone 27 receptor (FSHR), the transforming growth factor beta (TGFβ) type 1, bone morphogenetic 28 protein receptor (BMPR1B), and luteinizing hormone receptor (LHR) in the BB. The 29 increased density of FSHR and LHR provide novel evidence of a mechanism for increasing 30 the number of follicles recruited during dominant follicle selection. The compounding 31 increase of receptors with increasing follicle size maintained the multiple follicles and 32 reduced the apoptosis, contributing to the high ovulation rate in the BB. In addition, we report 33 a mutation-independent mechanism of down-regulation to reduce receptor density of the 34 leading dominant follicle in each sheep. The suppression of receptor density coincides with 35 the cessation of mitogenic growth and steroidogenic differentiation as part of luteinisation of 36 the follicle. The BB mutation-induced attenuation of BMPR1B signalling led to an increased 37 density of the FSH and LH receptors with a concurrent reduction in apoptosis to increase the 38 ovulation rate. The role of BMPs in receptor modulation is implicated in the development of 39 multiple ovulations. 40