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Studying the molecular principles of selection and maturation of ovarian dominant follicles - Liat Alyagor

The ovary consists of follicles, each of which contains a single oocyte surrounded by a variable number of somatic cells. In each reproductive cycle, a cohort of follicles respond to the pituitary derived gonadotropin, follicle stimulating hormone (FSH) and grows. Only a small portion of these follicles, known as the dominant follicles (DF), will further respond to the pituitary derived luteinizing hormone (LH) and ovulate. One mechanism for DF selection is the expansion of the vasculature, thereby amplifying the exposure to gonadotropins. The molecular mechanisms that drive the differential angiogenic capacity between DF and subordinate follicles (SF) are unclear. This is probably due to the fact that the detection of DF in mice is challenging, and the characteristics of their vasculature are poorly studied. In this study we aim at investigating the regulating mechanisms of angiogenesis involved in DF selection. We also aim at utilizing characteristics of DF vasculature for their identification. We propose that upon their selection, DF may secrete soluble vascular endothelial growth factor receptor 1 (sVEGFR1/sFlt) to their surroundings, arresting SF angiogenesis and development. We further suggest that the DF is protected from this sFlt effect by maintaining a tolerance mechanism based on VEGF or membrane bound VEGF receptors. In addition, we wish to establish an imaging method for in vivo detection of mouse DF based on vasculature parameters. Our preliminary results show that sFlt mRNA is upregulated at 5 hours after PMSG (a FSH analog) and remains high for at least 48 hours  and is further upregulated after hCG (a LH analog).  Moreover, large follicles isolated from ovaries of PMSG-treated mice secreted more sFlt than small follicles. Although other time points need to be analyzed, we could not find an elevation in VEGFA mRNA in large follicles as compared to small follicles 48 hours after PMSG administration. Dynamic contrast enhanced (DCE)-MRI studies raised a possible approach for DF detection using the differential patterns of changes in permeability surface area (PS) and fractional blood volume (fBV). These parameters showed different average values and distribution between mice 36-48 hours after PMSG injection and non injected mice. Furthermore, a difference in PS and fBV values distribution was measured for the same ovary before and after ovulation, suggesting that these parameters can serve as indicators for the DF population. In summary, our findings show that sFlt mRNA up-regulation coincides with the timing of DF selection, and that this protein originates from large follicles. This suggests a potential role for sFlt in inter follicular relationships between DF and SF. The localization of sFlt expression to DF, its effect on SF and the mechanism by which DF remain unresponsive to it will be studied. In addition, PS and fBV average values and their distribution are presented here as possible parameters for the in vivo detection of DF. Further validation and calibration of this method will be done in order to study, for the first time, the in vivo dynamics of the DF selection process and vasculature remodeling.

Figure 1 | Permeability and fractional blood volume changes during the DF selection process. Representative PS and fBV maps of PMSG injected and non injected ovaries(A). The ovary is marked in yellow circle. Blue represents pixels with low PS/fBV and red represents pixels with high PS/fBV. Anatomical position is demonstrated by a T1 weighted image. A difference in values and distribution of high value pixels is visible both in the fBV and the PS maps. Representative fluorescent staining of biotin- BSA-GdDTPA (MRI contrast agent, green), BSA-ROX (red) and hoechst (blue) in sections of ovaries 36-48 hours after PMSG administration and of non injected ovaries, taken after MRI imaging.