Imaging the ovary Reproductive BioMedicine Online Yi Feng, Amin Tamadon, Aaron J.W. Hsueh  Reproductive BioMedicine Online  Volume 36, Issue 5, Pages 584-593 (May 2018) DOI: 10.1016/j.rbmo.2018.02.006 Copyright © 2018 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Different techniques for real-time live imaging of the ovary. (A) B-mode ultrasound imaging showing the fuzzy outline of an ovary in an adult C57/BL6 mouse. (B) Positron emission tomography/dual-modality computed tomography (PET/CT) imaging of an ovary for tracing a tumour in a C57BL/6 mouse (with permission from publisher (Ocak et al., 2015)). (C) Magnetic resonance imaging (MRI) of a shadow image of the right ovary in an adult mouse (with permission from the publisher (Ocak et al., 2015)). (D) Optical coherence tomography (OCT) imaging of the mouse ovary in vivo (with permission from the publisher and author; Burton et al., 2015). (E) Intravital microscopic image of an adult mouse ovary showing blood vessels and largest follicles (with permission from the publisher and author; Bochner et al., 2015). (F) Fluorescence molecular tomography (FMT) of ovarian tissue in an adult mouse (with permission from the publisher (Ocak et al., 2015)). (G) NIR-II live imaging of an ovary from an adult mouse using single-wall carbon nanotube (SWCNT). (H) NIR-II live imaging of an ovary using FSH conjugated to a near-infrared dye (with permission from the publisher and authors (Feng et al., 2017b)). Reproductive BioMedicine Online 2018 36, 584-593DOI: (10.1016/j.rbmo.2018.02.006) Copyright © 2018 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Imaging of ex-vivo ovarian tissues. (A) Haematoxylin and eosin staining of an ovarian section of a day-10 mouse ovary. (B) Electron microscopic image of a primordial follicle in a mouse (photo by Professor Fakhrodin Mesbah, School of Medicine, Shiraz University of Medical Sciences, with permission). (C) Monochromatic synchrotron X-ray of an ovary from an adult mouse (with permission from the publisher (Kim et al., 2012)). (D) Ovarian vasculature from an adult mouse labelled with BriteVu contrast agent (with permission from ScarletImaging.com). Images were acquired at Cornell's BRC imaging facility on a Zeiss XRM-520 3D X-ray microscope. (E) In-vitro confocal scanning of an ovary from adult Balb/c mice by using fluorophore CH1055 conjugated to follicle stimulating hormone (FSH-CH) under the NIR-II window (Feng et al., 2017b). (F) CLARITY 3D images of an ovary from a day-10 C57/BL mouse. Primordial and primary follicles were stained using a non-specific tyrosine hydroxylase (TH) antibody, whereas the secondary follicles were located in the centre of the ovary showing strong staining to the anti-Müllerian hormone (AMH) antibody. (G) Processing of an ovary using CLARITY, an ovary from an adult proestrus mouse was processed using the CLARITY method, followed by incubation in the clearing buffer for 4 weeks before immunostaining. After staining, samples were incubated for 1 h at 37°C in the FocusClear medium for refractive index (RI) homogenization. Although initial clearing led to tissue shrinkage, subsequent RI homogenization restored the original size (Feng et al., 2017a). (H) Three-dimensional (3D) and 2D images of CLARITY allowed investigators to analyse the follicle number, size, type, location and interrelationship. A light-sheet or two-photon microscope could enhance the scanning depth and increase the resolution, allowing study of the human ovary and ovaries in diseased states (Feng et al., 2017a). Reproductive BioMedicine Online 2018 36, 584-593DOI: (10.1016/j.rbmo.2018.02.006) Copyright © 2018 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Development of murine follicles from the primordial to the pre-ovulatory stage is accompanied by major increases in oocyte and follicle diameters. Using the CLARITY approach and marker immunostaining, individual follicles and corpora lutea in intact ovaries were identified. Follicle and oocyte volume were calculated using Imaris software (Bitplane 8.0, Switzerland) with the Spot automatic algorithm and 3D measurement. The pictures are drawn to scale. Reproductive BioMedicine Online 2018 36, 584-593DOI: (10.1016/j.rbmo.2018.02.006) Copyright © 2018 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Interactions between ovarian local vasculature and follicles. Growing follicles secrete local angiogenic factors (vascular endothelial growth factor 1 [VEGF1], VEGF2 and VEGF3, basic fibroblast growth factor [bFGF], angiopoietin1, prokineticin, etc.) to promote increases in local blood vessel growth, whereas increases in vascular supply to neighbouring follicles provide increased concentration of local gonadotrophin hormones [GnHs], nutrients and oxygen to promote follicle growth. Reproductive BioMedicine Online 2018 36, 584-593DOI: (10.1016/j.rbmo.2018.02.006) Copyright © 2018 Reproductive Healthcare Ltd. Terms and Conditions