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Imaging the ovary

  • Author Footnotes
    1 These authors contributed equally to this work.
    Yi Feng
    Correspondence
    Corresponding authors.
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Institutes of Brain Science, Brain Science Collaborative Innovation Centre, State Key Laboratory of Medical Neurobiology, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, Shanghai 200032, China
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  • Amin Tamadon
    Affiliations
    Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Institutes of Brain Science, Brain Science Collaborative Innovation Centre, State Key Laboratory of Medical Neurobiology, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, Shanghai 200032, China
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to this work.
    Aaron J.W. Hsueh
    Correspondence
    Corresponding authors.
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Program of Reproductive and Stem Cell Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA
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  • Author Footnotes
    1 These authors contributed equally to this work.
Published:March 06, 2018DOI:https://doi.org/10.1016/j.rbmo.2018.02.006

      Highlights

      • Near infrared fluorophores can be used to image ovarian follicles in live animals to detect preantral follicles.
      • Digital 3D visualization of ovarian structures using CLARITY in fixed whole ovary can be used to elucidate three-dimensional interrelationships among follicles, corpora lutea, and ovarian vasculature.
      • Advances in ovarian imaging techniques provide new understanding of ovarian physiology and allow for the development of better tools to diagnose ovarian pathophysiology.

      Abstract

      During each reproductive cycle, the ovary exhibits tissue remodelling and cyclic vasculature changes associated with hormonally regulated folliculogenesis, follicle rupture, luteal formation and regression. However, the relationships among different types of follicles and corpora lutea are unclear, and the role of ovarian vasculature in folliculogenesis and luteal dynamics has not been extensively investigated. Understanding of ovarian physiology and pathophysiology relies upon elucidation of ovarian morphology and architecture. This paper summarizes the literature on traditional approaches to the imaging of ovarian structures and discusses recent advances in ovarian imaging. Traditional in-vivo ultrasound, together with histological and electron microscopic approaches provide detailed views of the ovary at organ, tissue and molecular levels. However, in-vivo imaging is limited to antral and larger follicles whereas histological imaging is mainly two-dimensional in nature. Also discussed are emerging approaches in the use of near-infrared fluorophores to image follicles in live animals to detect preantral follicles as well as visualizing ovarian structures using CLARITY in fixed whole ovaries to elucidate three-dimensional interrelationships among follicles, corpora lutea and ovarian vasculature. Advances in ovarian imaging techniques provide new understanding of ovarian physiology and allow for the development of better tools to diagnose ovarian pathophysiology.

      Graphical Abstract

      Keywords

      Introduction

      Using mainly histological analyses of fixed ovarian tissues and ultrasound imaging of ovaries in vivo, earlier imaging studies have established the basic framework of ovarian folliculogenesis as well as luteal formation and regression. The ovary contains individual follicles as functional structures, together with corpora lutea, interstitial tissues, innermost medulla and the outmost layer of the surface epithelium. Most of the 800,000 primordial follicles found at birth in human females remain at the dormant stage. After birth, some of these follicles gradually initiate growth (~1000 per month) and progress into primary, secondary and antral stages (
      • Macklon N.S.
      • Fauser B.C.
      Aspects of ovarian follicle development throughout life.
      ,
      • McGee E.A.
      • Hsueh A.J.W.
      Initial and cyclic recruitment of ovarian follicles.
      ). Under the regulation of pituitary gonadotrophins, follicles start growing, with most of them becoming atretic at the early antral stage. Most of the ~20 early antral follicles degenerate during the early follicular phase in women and only one reaches the pre-ovulatory stage. Regulating female reproductive organs by secreting ovarian steroids, the pre-ovulatory follicle eventually ruptures to release the mature egg for fertilization and propagation of the species (
      • Macklon N.S.
      • Fauser B.C.
      Aspects of ovarian follicle development throughout life.
      ,
      • McGee E.A.
      • Hsueh A.J.W.
      Initial and cyclic recruitment of ovarian follicles.
      ). After rupture, the pre-ovulatory follicle undergoes luteinization and secretes progesterone to maintain pregnancy, followed by luteal regression if no pregnancy is established. In adult mammals, the ovary and uterus are the only two organs undergoing hormonally regulated neo-angiogenesis, which is otherwise only found during tumorigenesis (
      • Hazzard T.M.
      • Stouffer R.L.
      Angiogenesis in ovarian follicular and luteal development.
      ). Cyclic remodelling of ovarian structures and vasculature takes place during each reproductive cycle. This review summarizes traditional approaches to in-vivo imaging of antral/pre-ovulatory follicles, together with two-dimensional histological analyses of relationships among follicles and corpora lutea, followed by recent advances in monitoring preantral follicles using near-infrared imaging in live animals and three-dimensional analyses of relationships among follicles and corpora lutea using a CLARITY approach.

      Real-time live imaging of the ovary: from ultrasound to near-infrared imaging

      Different approaches have been used to perform real-time ovary imaging in patients and animals. These technologies, including ultrasound, magnetic resonance imaging (MRI), computed tomography (CT), optical coherence tomography (OCT), fluorescence molecular tomography (FMT) and near-infrared imaging, have been developed in mouse models to enhance the ability of clinicians to diagnose ovarian diseases. Each real-time imaging modality has its own limitations and strengths in imaging the ovary, as summarized in Table 1.
      Table 1In-vivo real-time ovary imaging approaches in research and the clinic.
      Applications/speciesUSMRICTOCTFMTIVMIR
       Rodents+ (
      • Jaiswal R.S.
      • Singh J.
      • Adams G.P.
      High-resolution ultrasound biomicroscopy for monitoring ovarian structures in mice.
      )
      + (
      • Israely T.
      • Dafni H.
      • Nevo N.
      • Tsafriri A.
      • Neeman M.
      Angiogenesis in ectopic ovarian xenotransplantation: multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI.
      )
      + (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      )
      + (
      • Burton J.C.
      • Wang S.
      • Stewart C.A.
      • Behringer R.R.
      • Larina I.V.
      High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography.
      )
      + (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      )
      + (
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      ,
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      )
      + (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      )
       Non-human primate+ (
      • Bishop C.V.
      • Sparman M.L.
      • Stanley J.E.
      • Bahar A.
      • Zelinski M.B.
      • Stouffer R.L.
      Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high-resolution ultrasonography.
      )
      + (
      • Zeleznik A.J.
      • Wolf G.L.
      Preliminary studies on the use of magnetic resonance imaging with Gd-DTPA to monitor ovarian function in subhuman primates.
      )
      + (
      • Jones J.C.
      • Appt S.E.
      • Werre S.R.
      • Tan J.C.
      • Kaplan J.R.
      Validation of multi-detector computed tomography as a non-invasive method for measuring ovarian volume in macaques (Macaca fascicularis).
      )
      NDNDNDND
       Human+ (
      • Deb S.
      • Campbell B.K.
      • Clewes J.S.
      • Raine-Fenning N.J.
      Quantitative analysis of antral follicle number and size: a comparison of two-dimensional and automated three-dimensional ultrasound techniques.
      )
      + (
      • Leonhardt H.
      • Hellström M.
      • Gull B.
      • Lind A.-K.
      • Nilsson L.
      • Janson P.O.
      • Stener-Victorin E.
      Ovarian morphology assessed by magnetic resonance imaging in women with and without polycystic ovary syndrome and associations with antimullerian hormone, free testosterone, and glucose disposal rate.
      )
      + (
      • Occhipinti K.A.
      • Frankel S.D.
      • Hricak H.
      The ovary. Computed tomography and magnetic resonance imaging.
      )
      NDNDNDND
      Pre-ovulatory/large antral follicles
       Rodents+ (
      • Jaiswal R.S.
      • Singh J.
      • Adams G.P.
      High-resolution ultrasound biomicroscopy for monitoring ovarian structures in mice.
      )
      + (
      • Burton J.C.
      • Wang S.
      • Stewart C.A.
      • Behringer R.R.
      • Larina I.V.
      High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography.
      )
      + (
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      )
      + (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      )
       Non-human primate+ (
      • Bishop C.V.
      • Sparman M.L.
      • Stanley J.E.
      • Bahar A.
      • Zelinski M.B.
      • Stouffer R.L.
      Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high-resolution ultrasonography.
      )
      NDND
       Human+ (
      • Deb S.
      • Campbell B.K.
      • Clewes J.S.
      • Raine-Fenning N.J.
      Quantitative analysis of antral follicle number and size: a comparison of two-dimensional and automated three-dimensional ultrasound techniques.
      )
      + (
      • Leonhardt H.
      • Hellström M.
      • Gull B.
      • Lind A.-K.
      • Nilsson L.
      • Janson P.O.
      • Stener-Victorin E.
      Ovarian morphology assessed by magnetic resonance imaging in women with and without polycystic ovary syndrome and associations with antimullerian hormone, free testosterone, and glucose disposal rate.
      )
      + (
      • Occhipinti K.A.
      • Frankel S.D.
      • Hricak H.
      The ovary. Computed tomography and magnetic resonance imaging.
      )
      NDND
      Antral follicles
       Rodents+ (
      • Jaiswal R.S.
      • Singh J.
      • Adams G.P.
      High-resolution ultrasound biomicroscopy for monitoring ovarian structures in mice.
      )
      + (
      • Burton J.C.
      • Wang S.
      • Stewart C.A.
      • Behringer R.R.
      • Larina I.V.
      High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography.
      )
      + (
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      )
      + (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      )
       Non-human primate+ (
      • Bishop C.V.
      • Sparman M.L.
      • Stanley J.E.
      • Bahar A.
      • Zelinski M.B.
      • Stouffer R.L.
      Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high-resolution ultrasonography.
      )
      NDND
       Human+ (
      • Haadsma M.L.
      • Bukman A.
      • Groen H.
      • Roeloffzen E.M.A.
      • Groenewoud E.R.
      • Heineman M.J.
      • Hoek A.
      The number of small antral follicles (2–6 mm) determines the outcome of endocrine ovarian reserve tests in a subfertile population.
      )
      + (
      • Leonhardt H.
      • Hellström M.
      • Gull B.
      • Lind A.-K.
      • Nilsson L.
      • Janson P.O.
      • Stener-Victorin E.
      Ovarian morphology assessed by magnetic resonance imaging in women with and without polycystic ovary syndrome and associations with antimullerian hormone, free testosterone, and glucose disposal rate.
      )
      NDND
      Preantral follicles
       Rodents+ (
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      )
      + (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      )
       Non-human primateNDND
       HumanNDND
      Vasculature/blood flow
       Rodents+
      • Bar-Joseph H.
      • Ben-Aharon I.
      • Tzabari M.
      • Tsarfaty G.
      • Stemmer S.M.
      • Shalgi R.
      In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels.
      )
      + (
      • Israely T.
      • Dafni H.
      • Nevo N.
      • Tsafriri A.
      • Neeman M.
      Angiogenesis in ectopic ovarian xenotransplantation: multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI.
      )
      + (
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      ,
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      )
      + (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      )
       Non-human primate+ (
      • Bishop C.V.
      • Sparman M.L.
      • Stanley J.E.
      • Bahar A.
      • Zelinski M.B.
      • Stouffer R.L.
      Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high-resolution ultrasonography.
      )
      NDNDND
       Human+ (
      • Mercé L.T.
      • Gómez B.
      • Engels V.
      • Bau S.
      • Bajo J.M.
      Intraobserver and interobserver reproducibility of ovarian volume, antral follicle count, and vascularity indices obtained with transvaginal 3-dimensional ultrasonography, power Doppler angiography, and the virtual organ computer-aided analysis imaging program.
      )
      NDNDND
      + = in use; – = not in use; CT = computed tomography; FMT = fluorescence molecular tomography; IVM = intravital multiphoton microscopy; IR = infrared imaging; MRI = magnetic resonance imaging; ND = no data; OCT = optical coherence tomography; PET = positron emission tomography; US = ultrasound.
      Ultrasound is the most widely used in-vivo approach to ovarian imaging; it uses sound waves to create an image on a video screen. Sound waves are emitted from a small probe placed transvaginally in the woman or on the surface of her abdomen to create echoes from the ovaries. The same probe detects the echoes that bounce back and a computer translates the pattern of echoes into a picture. Because of the unique position of the ovary inside the body, vaginal ultrasound provides close imaging of ovarian structures for detecting large antral follicles, ovarian tumours and fluid-filled cysts. With the transvaginal route, high-frequency ultrasound probes (>6 MHz), which have a better spatial resolution but less examination depth, are useful because the presence of fatty tissue does not interfere with imaging (
      • Balen A.H.
      • Laven J.S.E.
      • Tan S.L.
      • Dewailly D.
      Ultrasound assessment of the polycystic ovary: international consensus definitions.
      ). However, it is difficult to image preantral (secondary, primary and primordial) follicles in women using transvaginal ultrasound, making this approach inadequate for checking residual follicles in patients with diminished ovarian reserve (
      • Levi A.J.
      • Raynault M.F.
      • Bergh P.A.
      • Drews M.R.
      • Miller B.T.
      • Scott Jr., R.T.
      Reproductive outcome in patients with diminished ovarian reserve.
      ). As shown in Figure 1A, B-mode ultrasonography for imaging of the ovary allowed visualization of large follicles with an antral cavity in an adult mouse but smaller follicles without an antrum were not detectable. Furthermore, the power Doppler ultrasound imaging can be applied to evaluate mouse ovarian vasculature with comparable resolution (
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      ).
      Figure 1
      Figure 1Different 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 M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      )). (C) Magnetic resonance imaging (MRI) of a shadow image of the right ovary in an adult mouse (with permission from the publisher (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      )). (D) Optical coherence tomography (OCT) imaging of the mouse ovary in vivo (with permission from the publisher and author;
      • Burton J.C.
      • Wang S.
      • Stewart C.A.
      • Behringer R.R.
      • Larina I.V.
      High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography.
      ). (E) Intravital microscopic image of an adult mouse ovary showing blood vessels and largest follicles (with permission from the publisher and author;
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      ). (F) Fluorescence molecular tomography (FMT) of ovarian tissue in an adult mouse (with permission from the publisher (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      )). (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 Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      )).
      In addition to ultrasonography, CT and MRI scans represent advances in the live imaging field. The CT scan uses an X-ray procedure to produce cross-sectional images of the body. A CT scanner takes many pictures as it rotates around the body, before digitally transforming images into multiple slices of body images. CT scans do not show small ovarian follicles, but they can detect large follicles and tumours (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      ). Using vaginal ultrasound provides similar resolution but at a lower cost, so the CT approach is not routinely used for follicle imaging. As shown in Figure 1B, a CT scan of an adult mouse ovary showed poor differentiation of the ovary from surrounding organs.
      MRI scans use radio waves and strong magnets instead of X-rays. The energy from the radio waves is absorbed and a computer translates the pattern of radio waves given off by tissues into a detailed image of parts of the body. In addition, a contrast material might be injected to refine scanned images. As shown in Figure 1C, MRI was used for imaging of an adult mouse ovary in vivo (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      ). Although MRI provides clear imaging of large follicles in the ovary in vivo, this approach has not been routinely applied to ovarian follicle detection in the clinic due to the difficulties involved in accessing the imaging probes to the ovary.
      Optical coherence tomography (OCT) imaging in vivo, based on near-infrared light (Figure 1D), has been adopted for evaluation of mouse ovaries (
      • Burton J.C.
      • Wang S.
      • Stewart C.A.
      • Behringer R.R.
      • Larina I.V.
      High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography.
      ). The use of relatively long wavelength light provides cross-sectional views of the subsurface microstructure of biological tissues (
      • Schmitt J.M.
      Optical coherence tomography (OCT): a review.
      ). To decrease the effect of tissue motion (breathing or muscle contraction) during live imaging using OCT, an OCT system with higher imaging speed has been recommended (
      • Burton J.C.
      • Wang S.
      • Stewart C.A.
      • Behringer R.R.
      • Larina I.V.
      High-resolution three-dimensional in vivo imaging of mouse oviduct using optical coherence tomography.
      ). The OCT approach, however, suffers from the design of practical scanning systems for clinical application.
      As an experimental tool, intravital multiphoton microscopy, a fluorescence imaging technique that allows imaging of living tissue up to about 1 mm in depth (Figure 1E), was recently used to monitor blood flow of individual vessels and the thickness of the apical follicle wall during ovulation in murine models in vivo (
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      ). Constriction of apical vessels was found to occur within hours preceding follicle rupture (
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      ). Furthermore, preventing the peri-ovulatory rise in the expression of the vasoconstrictor endothelin 2 inhibited ovulation whereas infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored ovulation (
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      ).
      A similar intravital window approach for live imaging in situ was used to enable longitudinal microscopic analyses of ovarian morphology and orthotopic tumour invasion (
      • Bochner F.
      • Fellus-Alyagor L.
      • Kalchenko V.
      • Shinar S.
      • Neeman M.
      A novel intravital imaging window for longitudinal microscopy of the mouse ovary.
      ). The morphological structures of the murine ovarian cortex were examined together with cellular dynamics after gonadotrophin treatment in vivo. Of interest is the longitudinal imaging of the orthotopic tumour formation in vivo by following infiltration of tumour cells through the mesothelial layer and formation of new collagen fibres. As shown in Figure 1E, intravital multiphoton microscopy can be applied for imaging of the ovarian surface epithelium. However, this approach is restricted by the depth of the observable field and has limited clinical application.
      FMT (fluorescence molecular tomography) of ovarian tissue is another approach to live imaging of ovarian tissues in mice (Figure 1F). However, the resolution of FMT images is inferior to MRI imaging (
      • Ocak M.
      • Gillman A.G.
      • Bresee J.
      • Zhang L.
      • Vlad A.M.
      • Müller C.
      • Schibli R.
      • Edwards W.B.
      • Anderson C.J.
      • Gach H.M.
      Folate receptor-targeted multimodality imaging of ovarian cancer in a novel syngeneic mouse model.
      ). Potential clinical applications await further improvements.
      Recent advances in live imaging in vivo with quantum dot fluorescent agents in the long near-infrared (NIR) region (1.0–1.7 µm, called the NIR-II region) allowed reduction of photon scattering and autofluorescence in tissues, thus reaching deeper penetration depths inside the body. Fluorescent imaging of biological systems in the NIR-II window can probe centimetre tissue depth and achieve micron-scale spatial resolution at millimetre depth (
      • Diao S.
      • Hong G.
      • Antaris A.L.
      • Blackburn J.L.
      • Cheng K.
      • Cheng Z.
      • Dai H.
      Biological imaging without autofluorescence in the second near-infrared region.
      ,
      • Welsher K.
      • Sherlock S.P.
      • Dai H.
      Deep-tissue anatomical imaging of mice using carbon nanotube fluorophores in the second near-infrared window.
      ). Single-wall carbon nanotubes embedded with fluorophores capable of emitting near-infrared light upon activation by laser were generated. Due to the avascular nature of granulosa cells in ovarian follicles and presence of vessels in the theca layer surrounding follicles, outlines of individual follicles could be identified (Figure 1G). However, this approach lacks specificity because the nanotubes were found in the general circulation.
      FSH is a heterodimeric glycoprotein essential for gonadal development by binding to specific receptors in granulosa cells of ovarian follicles and Sertoli cells of testicular seminiferous tubules (
      • Dierich A.E.
      • Sairam M.R.
      • Monaco L.
      • Fimia G.M.
      • Gansmuller A.
      • LeMeur M.
      • Sassone-Corsi P.
      Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.
      ,
      • Sprengel R.
      • Braun T.
      • Nikolics K.
      • Segaloff D.L.
      • Seeburg P.H.
      The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA.
      ,
      • Tapanainen J.S.
      • Aittomäki K.
      • Min J.
      • Vaskivuo T.
      • Huhtaniemi I.T.
      Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility.
      ). This study used the near-infrared approach by conjugating FSH to a small molecular weight near-infrared fluorophore (CH1055) to allow the visualization of FSH receptors in live rodents (Figure 1H). The strong near-infrared signals emitted from the fluorophore conjugated to FSH allowed specific imaging of granulosa cells expressing FSH receptors (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      ).
      Because FSH receptors are expressed in primary and larger follicles upon their recruitment from the dormant primordial stage (
      • Oktay K.
      • Briggs D.
      • Gosden R.G.
      Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles.
      ), the near-infrared approach allowed imaging of primary, secondary, antral and pre-ovulatory follicles in intact ovaries. Using prepubertal mice, near-infrared signals were detected in ovaries containing only preantral follicles (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      ). For infertile female patients, it is important to assess the presence of preantral follicles because recent studies have indicated the possibility of promoting preantral follicle growth for infertility treatment (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.
      ). Because the CH1055 fluorophore used has minimal cytotoxicity and a short in-vivo half-life (
      • Antaris A.L.
      • Chen H.
      • Cheng K.
      • Sun Y.
      • Hong G.
      • Qu C.
      • Diao S.
      • Deng Z.
      • Hu X.
      • Zhang B.
      • Zhang X.
      • Yaghi O.K.
      • Alamparambil Z.R.
      • Hong X.
      • Cheng Z.
      • Dai H.
      A small-molecule dye for NIR-II imaging.
      ), further improvement of the FSH-CH1055 approach and design of a portable transvaginal NIR-II probe could allow live imaging of preantral follicles in infertile female patients as a diagnostic tool. Furthermore, the same approach is applicable for in-vivo imaging of LH receptors in thecal, mature granulosa and luteal cells as well as for receptors interacting with diverse peptide and protein hormones in different target tissues.

      Imaging of ovarian tissues in vitro: from two-dimensional histology and electron microscopy to the three-dimensional CLARITY approach

      Histological analyses allow detailed investigation of the sectional morphology of the ovary (Figures 2A and 2B). These studies serve as the basis for most of our present-day understanding of ovarian morphology. However, histological analyses at either light or electron microscopic levels are restricted to two dimensions and it is difficult for investigators to reconstruct the exact structure and location of entire follicles or corpora lutea inside the ovary. It is also difficult to investigate relationships among diverse ovarian structures. Using the synchrotron X-ray microtomography technique (
      • Kim J.
      • Choi Y.H.
      • Chang S.
      • Kim K.-T.
      • Je J.H.
      Defective folliculogenesis in female mice lacking Vaccinia-related kinase 1.
      ) (Figure 2C) and 3D X-ray microscopy approach (Figure 2D), imaging of ovarian follicles and tracing of vessels in fixed mouse ovaries without clearing has been performed. However, these facilities are not widely accessible.
      Figure 2
      Figure 2Imaging 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 J.
      • Choi Y.H.
      • Chang S.
      • Kim K.-T.
      • Je J.H.
      Defective folliculogenesis in female mice lacking Vaccinia-related kinase 1.
      )). (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 Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      ). (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 Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ). (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 Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ).
      Taking advantage of the availability of fluorescence protein markers, transgenic mice have been generated to allow tracing of follicular cell lineages following induction of marker genes in a time- and stage-dependent manner (
      • Zhang H.
      • Zheng W.
      • Shen Y.
      • Adhikari D.
      • Ueno H.
      • Liu K.
      Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries.
      ). Using this approach, Kui Liu's laboratory traced the in-vivo development of two classes of primordial follicles in mice. The first wave of follicles exists in the ovaries for 3 months and contributes to the onset of puberty and to early fertility. For the second wave, primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life (
      • Zhang H.
      • Zheng W.
      • Shen Y.
      • Adhikari D.
      • Ueno H.
      • Liu K.
      Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries.
      ). Using a similar tracing approach, Liu's group also demonstrated that Ddx4 (also known as VASA)-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis, thus providing evidence for the absence of female germ line progenitors in mice (
      • Zhang H.
      • Zheng W.
      • Shen Y.
      • Adhikari D.
      • Ueno H.
      • Liu K.
      Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries.
      ) and refuting the existence of ovarian germ stem cells in mammalian ovaries (
      • White Y.A.R.
      • Woods D.C.
      • Takai Y.
      • Ishihara O.
      • Seki H.
      • Tilly J.L.
      Oocyte formation by mitotically-active germ cells purified from ovaries of reproductive age women.
      ).
      Using traditional histological analyses, only limited information was derived regarding interrelationships among developing follicles and corpora lutea due to uncharacterized three-dimensional architecture. Many organs in vertebrates consist of compartmentalized branching units such as lobules in the lung, liver and mammary gland or tubules in the liver, testis and kidney. Although lobule-like ovarian cords are present in the fetal stage, adult ovaries contain primordial follicles situated in the cortical region together with growing follicles arranged in a centripetal fashion without a clear pattern of organization (
      • Hirshfield A.N.
      • DeSanti A.M.
      Patterns of ovarian cell proliferation in rats during the embryonic period and the first three weeks postpartum.
      ).
      FSH conjugated to a near-infrared dye, as described in the previous section, allowed either ex-vivo confocal scanning or in-vitro whole ovary scanning (Figure 2E) to obtain 3D images (
      • Feng Y.
      • Zhu S.
      • Antaris A.L.
      • Chen H.
      • Xiao Y.
      • Lu X.
      • Jiang L.
      • Diao S.
      • Yu K.
      • Wang Y.
      • Raya S.H.
      • Yue J.
      • Hong X.
      • Hong G.
      • Cheng Z.
      • Hsueh A.J.
      • Dai H.
      Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.
      ). However, this approach is limited due to the labelling of only one marker for follicles. Likewise, the SeeDB method, using a saturated fructose solution to clear tissues (
      • Migone F.F.
      • Cowan R.G.
      • Williams R.M.
      • Gorse K.J.
      • Zipfel W.R.
      • Quirk S.M.
      In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.
      ), and the ScaleA2 method based on whole-mount immunofluorescence following tissue clearing with the ScaleA2 reagent (ScaleA2) (
      • Malki S.
      • Tharp M.E.
      • Bortvin A.
      A whole-mount approach for accurate quantitative and spatial assessment of fetal oocyte dynamics in mice.
      ), have been introduced for imaging of whole ovarian tissues. However, these protocols of tissue clearing are only appropriate for one immunostaining step.
      The recently developed CLARITY approach makes intact tissues transparent and enables immunostaining of multiple markers for imaging detailed structures of organs (
      • Chung K.
      • Wallace J.
      • Kim S.-Y.
      • Kalyanasundaram S.
      • Andalman A.S.
      • Davidson T.J.
      • Mirzabekov J.J.
      • Zalocusky K.A.
      • Mattis J.
      • Denisin A.K.
      • Pak S.
      • Bernstein H.
      • Ramakrishnan C.
      • Grosenick L.
      • Gradinaru V.
      • Deisseroth K.
      Structural and molecular interrogation of intact biological systems.
      ). Using up to three specific immunostaining markers/steps and advanced computer algorithms after clearing with the CLARITY approach, ovarian follicles and corpora lutea were imaged at different developmental stages in the same intact ovaries and generated 3D digital maps of ovarian organelles (Figure 2F) (
      • Feng Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ,
      • Hu W.
      • Tamadon A.
      • Hsueh A.J.W.
      • Feng Y.
      Three-dimensional reconstruction of vascular architectures of passive CLARITY-cleared mouse ovary using Imaris software.
      ). Three-dimensional architecture of the murine ovary was evaluated, including the quantitation of follicle diameters, volumes, surface areas and locations in the ovary after clearing of whole mouse ovaries using confocal and light-sheet microscopies (Figures 2G and 2H). As shown in Figure 3, murine follicle volume increased ~45-fold during transition from the primordial to the primary stage whereas secondary follicle development encompassed 125-fold increases in volume. From early antral to the largest pre-ovulatory follicles, ~ 15-fold increases in volume were found. Throughout folliculogenesis, from the smallest primordial to largest pre-ovulatory follicles, ~ 3 × 105-fold increases in follicle volume were found. Furthermore, oocytes from primordial to pre-ovulatory follicles are characterized by 88,000-fold increases in volume, making oocytes the largest cell in the body. During follicle growth, oocytes reached maximal growth at the early antral stage, whereas the overall volume of follicles continues to expand until the pre-ovulatory stage. Because human pre-ovulatory follicles are much bigger than their rodent counterparts, it is anticipated that future studies using human samples will show even bigger changes in follicle volume.
      Figure 3
      Figure 3Development 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.

      Intraovarian architecture revealed by imaging ovary using CLARITY

      Relationships among follicles and corpora lutea

      Using the CLARITY approach, 3D images were obtained indicating that individual follicles were in close contact with other follicles or corpora lutea with minimal surrounding interstitial tissues (
      • Feng Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ). Follicles of the same type were clustered together with primordial follicles aggregated in the cortical region. Follicle clustering was calculated based on distances between the same types of follicles, which found that clustering indexes decreased as primordial follicles developed into primary, secondary and antral stages in ovaries from mice at all ages. After digitally isolating individual pre-ovulatory follicles and corpora lutea together with their surrounding structures, the number of follicles at different developmental stages neighbouring individual follicles was estimated. Interestingly, corpora lutea were in direct contact with follicles at all stages of development, whereas pre-ovulatory follicles had fewer neighbouring follicles. Quantitative analyses using ovaries from mice at different stages indicated that pre-ovulatory follicles had fewer neighbouring primordial, primary and secondary follicles compared with those adjacent to the corpora lutea. In contrast, comparable numbers of antral and pre-ovulatory follicles were found near pre-ovulatory follicles and corpora lutea.
      The observed close relationships between follicles at different stages suggest paracrine or even juxtacrine interactions among follicles at different stages of development (
      • Feng Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ). Highly clustered primordial follicles probably produce ‘dormancy’ factors via juxtacrine or paracrine mechanisms to suppress each other from activation to reach the primary stage. Earlier data indicated that primordial follicles could secrete a diffusible inhibitor for follicle activation as predicted by 2D spatial analysis of histological sections (
      • Da Silva-Buttkus P.
      • Marcelli G.
      • Franks S.
      • Stark J.
      • Hardy K.
      Inferring biological mechanisms from spatial analysis: prediction of a local inhibitor in the ovary.
      ). After initiation of growth following activation, primary follicles reach secondary and antral stages and begin to disperse from each other. Interestingly, earlier follicle culture studies showed that two secondary follicles in direct contact invariably led to the dominance of one and growth suppression of its neighbour via juxtacrine mechanisms (
      • Spears N.
      • de Bruin J.P.
      • Gosden R.G.
      The establishment of follicular dominance in co-cultured mouse ovarian follicles.
      ). Secondary follicles also secret anti-Müllerian hormone (AMH) and AMH promotes secondary follicle growth in murine (
      • McGee E.A.
      • Smith R.
      • Spears N.
      • Nachtigal M.W.
      • Ingraham H.
      • Hsueh A.J.W.
      Müllerian inhibitory substance induces growth of rat preantral ovarian follicles.
      ) and primate (
      • Xu J.
      • Bishop C.V.
      • Lawson M.S.
      • Park B.S.
      • Xu F.
      Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates.
      ) models. Furthermore, our findings showing the presence of all types of follicles adjacent to individual corpus luteum but not pre-ovulatory follicles suggested that high levels of progesterone produced by corpora lutea are unlikely to affect folliculogenesis, whereas high levels of oestrogen, inhibin or other factors secreted by pre-ovulatory follicles could confer unfavourable environments for preantral follicles (
      • Dong G.
      • Guo Y.
      • Cao H.
      • Zhou T.
      • Zhou Z.
      • Sha J.
      • Guo X.
      • Zhu H.
      Long-term effects of repeated superovulation on ovarian structure and function in rhesus monkeys.
      ).

      Interactions between local vasculature and follicles

      The CLARITY approach allows investigation of the 3D architecture of ovarian vasculature. Using the CLARITY approach followed by immunostaining for platelet endothelial cell adhesion molecule 1 (PECAM1), a marker for endothelial cells (
      • Cao G.
      • Fehrenbach M.L.
      • Williams J.T.
      • Finklestein J.M.
      • Zhu J.-X.
      • DeLisser H.M.
      Angiogenesis in platelet endothelial cell adhesion molecule-1-null mice.
      ,
      • Newman P.J.
      The biology of PECAM-1.
      ), the vasculature in ovaries from mice at different ages was traced, which found age- and follicle stage-dependent increases in local vasculature (
      • Feng Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ). Of particular interest, gonadotrophin-dependent increases in ovarian vasculature were found within 2 days after equine chorionic gonadotrophin (eCG) treatment in immature mice (
      • Feng Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ). These findings are consistent with earlier reports indicating intense neo-angiogenesis and increased permeability of blood vessels during follicular development, ovulation and subsequent formation of the corpus luteum (
      • Fraser H.M.
      Regulation of the ovarian follicular vasculature.
      ,
      • Hazzard T.M.
      • Stouffer R.L.
      Angiogenesis in ovarian follicular and luteal development.
      ). It is also clear that follicle development is accompanied by increases in vasculature branching. Instead of being randomly distributed inside the ovary, 3D analyses indicated that follicles are located along vascular branches. Growing follicles near vascular branches probably acquire more nutrients and oxygen via local vessels to allow rapid follicle growth (Figure 4). Growing follicles, in turn, secrete angiogenesis factors such as vascular endothelial growth factor (VEGF) (
      • Fraser H.M.
      Regulation of the ovarian follicular vasculature.
      ), basic fibroblast growth factor (bFGF) (
      • Chaves R.N.
      • Tavares de Matos M.H.
      • Buratini J.
      • Ricardo de Figueiredo J.
      The fibroblast growth factor family: involvement in the regulation of folliculogenesis.
      ), prokineticin (
      • Kisliouk T.
      • Friedman A.
      • Klipper E.
      • Zhou Q.-Y.
      • Schams D.
      • Alfaidy N.
      • Meidan R.
      Expression pattern of prokineticin 1 and its receptors in bovine ovaries during the oestrous cycle: involvement in corpus luteum regression and follicular atresia.
      ), angiopoietin1 (
      • Hazzard T.M.
      • Molskness T.A.
      • Chaffin C.L.
      • Stouffer R.L.
      Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval.
      ,
      • Tal R.
      • Seifer D.B.
      • Grazi R.V.
      • Malter H.E.
      Angiopoietin-1 and angiopoietin-2 are altered in polycystic ovarian syndrome (PCOS) during controlled ovarian stimulation.
      ), and other factors to promote local neo-angiogenesis and vasculature development. This positive feedback loop leads to rapid blood vessel formation during development and after gonadotrophin stimulation. The adult ovary can be pictured as a vascular tree with three or four main branches each bearing fruits (follicles) of different sizes. It is becoming clear that intraovarian distribution of follicles is not a random event but is ‘constructed’ around the local vasculature.
      Figure 4
      Figure 4Interactions 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.
      Although the CLARITY approach (
      • Feng Y.
      • Cui P.
      • Lu X.
      • Hsueh B.
      • Billig F.M.
      • Yanez L.Z.
      • Tomer R.
      • Boerboom D.
      • Carmeliet P.
      • Deisseroth K.
      • Hsueh A.J.W.
      CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.
      ) required long duration for tissue clearing, two reports (
      • Ku T.
      • Swaney J.
      • Park J.Y.
      • Albanese A.
      • Murray E.
      • Cho J.H.
      • Park Y.G.
      • Mangena V.
      • Chen J.
      • Chung K.
      Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.
      ;
      • Murray E.
      • Cho J.H.
      • Goodwin D.
      • Ku T.
      • Swaney J.
      • Kim S.Y.
      • Choi H.
      • Park Y.G.
      • Park J.Y.
      • Hubbert A.
      • McCue M.
      • Vassallo S.
      • Bakh N.
      • Frosch M.P.
      • Wedeen V.J.
      • Seung H.S.
      • Chung K.
      Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.
      ) indicated that samples could be cleared in a shorter time (from 4–8 weeks to 3–4 days for adult mouse ovaries). Also, freely accessible 3D software is now available for image analysis, including Vaa3D (http://vaa3d.org) and POV-Rayv (www.povray.org). It is expected that this valuable method will soon be applied to elucidate follicle and corpora lutea dynamics in human ovaries.

      Future directions

      Increasing sophistication of imaging and tissue clearing tools, as well as advances in computer-based analyses, allow ovarian physiologists to gain new perspectives on ovarian structures in both fixed samples and live animals. Availability of the 3D analyses of ovarian architecture could allow analyses of human ovaries to reveal the physiological basis of follicle interactions and pathological basis of ovarian dysfunctions. Live imaging of follicles at preantral stages paves the way for future clinical applications in monitoring ovarian reserve for predicting the reproductive potential of patients. Rapid technical advances in anatomical imaging have occurred in parallel with developments in physiologic imaging (Raichle, 1998). Future physiologic imaging of the ovary could permit evaluation of changes in metabolic processes, including blood flow, follicular oxygenation, metabolic rate for glucose, and biochemical changes such as functional FSH receptors. New non-invasive live imaging approaches in real time could provide easy diagnosis of polycystic ovarian syndrome, primary ovarian insufficiency, and the highly lethal ovarian surface epithelial cancer at early stages.

      Acknowledgements

      The authors are grateful to researchers (their researches or names are mentioned in the paper) for their assistance in providing images.
      This study was supported by grants from the National Natural Science Foundation of China (No. 81673766 to YF), the Chinese Special Fund for Postdocs (No. 2014T70392 to YF), the New Teacher Priming Fund, the Zuoxue Foundation of Fudan University, and the Development Project of Shanghai Peak Disciplines-Integrative Medicine (20150407).

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      Biography

      Aaron Hsueh is an ovarian physiologist and has published in the field for decades, with over 380 refereed papers. His laboratory has contributed to the understanding of ovarian follicle growth and atresia, intraovarian mechanisms of oocyte maturation, and autocrine/paracrine regulation of early embryonic development, and established the Ovarian Kaleidoscope Database.
      Key message
      The ovary is a unique organ with hormonally regulated folliculogenesis, follicle rupture, luteal formation/regression and associated vasculature changes, leading to tissue remodelling during each reproductive cycle. Advances in ovarian imaging techniques provide a new understanding of folliculogenesis and vasculature to allow better diagnosis and treatment of ovarian diseases.