CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions

Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 105-fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.

of the oocyte as an indicator of follicles, a recent whole mount immunofluorescence 3D imaging study begun to provide follicle dynamics from neonate to adult mice 7 . However, there is no analysis on follicular somatic cells together with oocytes in 3D architecture. The vascular system is generally quiescent in adult mammals and the ovary and endometrium are the only normal adult tissues capable of undergoing neo-angiogenesis 8 . Furthermore, the relationship between folliculogenesis and ovarian vasculature is unclear.
Recently developed CLARITY approach makes intact tissues transparent and enables immunostaining of markers to elucidate detailed 3D structure of organs 9 . Using specific markers and advanced computer algorithms, we imaged ovarian follicles and corpora lutea in intact ovaries and generated 3D digital maps of ovarian organelles in relation to vasculatures to reveal changes in follicle sizes, location, roundness, clustering, interrelationships, and vasculature throughout reproductive life. In mutant mice with deletion of the hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter, defective ovarian vasculature was found to be associated with lower ovulatory responses following gonadotropin stimulation. Following treatment with an inhibitor for VEGF receptor kinases, we also suppressed gonadotropin-induced ovarian neo-angiogenesis, leading to defective vasculature and ovulation. The purpose of the current study was to use the CLARITY approach to elucidate folliculogenesis and the relationship between vasculature and follicles in mice after birth to adult life.

Results
Identification of follicles and corpora lutea at different developmental stages. We used the CLARITY approach to render ovaries transparent (Figure S1A), followed by staining with different markers before digital imaging ( Figure S1B). Our preliminary data ( Figure S2) indicated that staining with antibodies against tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, led to prominent staining in theca cells, ganglia, and corpora lutea as confirmed using antibodies against neuron-specific class III beta-tubulin (Tuj1) 10 and brain-derived neurotropic factor (BDNF), consistent with earlier findings 11,12 . In addition, nonspecific background TH staining was found around all follicles, allowing for identification of follicular structures. As shown in Figure S1B, primordial and primary follicles both showed TH staining but could be separated based on shapes of granulosa cells (squamous for primordial; cuboidal for primary). In secondary follicles, anti-Müllerian hormone (AMH) staining was evident in granulosa cells as reported 13 . Also, BID (a pro-apoptosis protein) and CYP19 (the enzyme for estrogen biosynthesis) marked granulosa cells in atretic 14 and antral/preovulatory follicles 15 , respectively ( Figure S1B). Preovulatory and antral follicles were detected semi-manually according to their sizes and the antral shape and location. In addition to their unique homogenous texture, corpora lutea showed staining for VEGF important for angiogenesis 8 . Also, digital slicing of adjacent images showed an ovulating follicle ( Figure S1B).

Three-dimensional determination of follicle numbers throughout life.
We analyzed follicle dynamics of intact ovaries from mice at different ages following CLARITY processing and immunostaining for TH together with AMH or CYP19 staining ( Figure 1). Scanned images were analyzed following 3D rendering, digital slicing, and Spot transformation to identify primordial, primary, secondary, antral, and preovulatory follicles ( Fig. 1A and Figure S3). In addition, corpora lutea were distinguished based on their unique homogenous texture in ovaries of adult and aging (day 360) mice. Three-D images of a day-10 mouse ovary were generated for Spot transformation, followed by manual improvement, to identify all follicles (Movie S1). Using specific markers, follicles at all developmental stages were traced together with corpora lutea in the ovary of an adult proestrous animal to construct follicle relationship maps (Movie S2).
Based on specific markers, we estimated number of follicles at different stages of development ( Fig. 1) and determined their diameters, surface areas, and volumes (Table S1). Follicle volume increased ~45 fold during transition from primordial to primary stage whereas secondary follicle development encompassed 125-fold increases in volume, indicating major growth (Tables S1 and S2). From early antral to the largest preovulatory follicles, ~15-fold increases in volume were found. Throughout folliculogenesis from the smallest primordial to largest preovulatory follicles, ~3 × 10 5 -fold increases in follicle volume were found, accompanied by 88,000-fold increases in oocyte volume. Analyzing lifetime changes in follicle numbers indicated an age-dependent decline in total follicles, together with major decreases in primordial follicles (Fig. 1B) as previously reported 16 . Of interest, percentages of both primordial and primary follicles increased with age (Fig. 1C) accompanied by a drop of secondary follicles, suggesting age-dependent follicle arrest before the secondary stage.
Clustering and migration of follicles based on 3D mapping. We identified locations of follicles in ovaries from mice at different ages ( Fig. 2A). Clustering of follicles at the same developmental stage was monitored by measuring spatial homogeneity using the Ripley's K function that quantifies the degree of object clustering/dispersion as compared to a random distribution 17 . As shown in Fig. 2B, clustering indexes decreased as primordial follicles developed into primary, secondary and later stages in ovaries from animals at all ages. Of interest, there is also an age-dependent increase in follicle clustering from ovaries of day 10 to day 60 animals. We also analyzed distances of developing follicles from geometric center of the ovary using Imaris Spot and Surface algorithms. As shown in Fig. 2C, Spot plotting of distances of individual follicles from the center in adult ovaries suggested inward migration of primary, secondary, and antral follicles, consistent with earlier identification of primordial follicles near the periphery of ovaries in prepubertal and adult mice 6 . Analyses of follicles neighboring preovulatory follicles and corpora lutea. Three-D imaging analyses indicated that individual follicles were in close contact with other follicles or corpora lutea with minimal interstitial tissues. We digitally isolated individual preovulatory follicles and corpora lutea to estimate neighboring follicles in ovaries from proestrous mice (Fig. 3A). Corpora lutea were in direct contact with follicles of all stages whereas preovulatory follicles had fewer neighboring follicles. Quantitative analyses using ovaries from Scientific RepoRts | 7:44810 | DOI: 10.1038/srep44810 mice at different stages ( Fig. 3B) indicated that preovulatory follicles had fewer neighboring primordial, primary, and secondary follicles as compared with those adjacent to the corpora lutea. In contrast, comparable numbers of antral and preovulatory follicles were found near preovulatory follicles and corpora lutea. By monitoring corpora lutea throughout all estrous stages by daily vaginal smears, we also found decreases in volumes of corpora lutea from proestrus to diestrus ( Fig. 3C and D), suggesting rapid luteolysis within one reproductive cycle. Sphericity index analysis in follicles during development. We calculated sphericity of individual follicles. In ovaries of adult proestrous animals, roundness of follicles decreased as follicles developed into more advanced stages ( Fig. 4A; left panel: whole ovary image; right panel: representative follicles at different stages). Unlike adult ovaries, no changes in sphericity were found during follicle development in ovaries of immature mice treated with equine chorionic gonadotropin (eCG) for 24 h ( Fig. 4B and C), suggesting differences in ovarian rigidity. In adult, sphericity indices decreased from 0.91 in nearly round primordial follicles to 0.73 in irregularly shaped preovulatory follicles (Fig. 4C).
Ovarian vasculature networks surrounding follicles and corpora lutea. We investigated ovarian vasculature changes following immunostaining for platelet-endothelial cell adhesion molecule 1 (PECAM1), a marker for endothelial cells 18,19 . PECAM1 staining in ovaries from mice at different ages was traced using the Imaris Filament algorithm and showed age-dependent increases in vasculature (Fig. 5A, upper panel: 3D rendering; lower panel: blood vessel tracing). In day 3 ovaries containing mainly primordial follicles, no PECAM1 staining outlining individual follicles were found despite the existence of major branches. In day 10 ovaries, growing secondary follicles showed strong staining in the theca layer whereas primordial/primary follicles in the cortical region had negligible PECAM1 signal. From day 3 to day 60 ovaries, major increases in vasculature branching were accompanied by a 40% increase in diameters of the largest vessel originating from the helium.

Figure 2. Construction of 3D follicle maps indicates follicle clustering and migration. (A) Spot
representation shows location of follicles in ovaries from mice at 10, 21, and 60 days of age. Follicles at primordial (green), primary (pink), secondary (purple), antral (orange), and preovulatory (red) stages are shown. Note absence of antral and preovulatory follicles in day 10 ovary whereas day 21 ovary lacks preovulatory follicles. This graph has been drawn by Imaris Vantage, plot type: XYZ 3D color and scale; different sizes in each follicular type denote different depths of follicles (smaller follicles are located the deeper from the surface). (B) Clustering analyses. Ovaries from animals at different ages were processed using Spot identification before analyzing clustering of follicles at the same developmental stage using the Ripley's K function. The Y-axis depicts deviations of the follicle distribution (K(d)) from randomness (E(K(d)) over a given distance (X-axis) between center of follicles of the same size. Higher values indicate clustering whereas negative values indicate dispersion. (C) Follicle development from primary to antral stages was characterized by movement toward the center of the ovary. Distances from geometric center of the ovary to the center of individual follicles were determined in ovaries from mice at different ages (n = 3 ovaries). All values were normalized using location of the most peripheral primordial follicles (set at 1.0). Means + SE are shown.  Ovaries from proestrous mice were processed to reveal follicles in direct contact with preovulatory follicles (red color) and corpora lutea (CL, yellow color). Upper panel: 3D cropping of representative graphs showing selected preovulatory follicle and corpus luteum together with neighboring follicles. Lower panel: follicles shown as Spot graphs (red color: preovulatory; orange color: antral; blue color: secondary). Because preovulatory follicles and corpus lutea have irregular shapes, the Surface algorithm in Imaris was used to define their exact borders before neighboring analysis. Smaller and generally round follicles were reconstructed by using the Spot algorithm in Imaris. (B) Number of follicles neighboring individual preovulatory follicles or corpora lutea was plotted based on stages of follicle development (n = 10 from 5 ovaries). Stars indicate significant differences between numbers of follicles neighboring preovulatory follicles and CL (P < 0.05). Means + SE are shown. (C) Changes in volumes of corpora lutea during the estrous cycle. Upper panel: representative Spot graphs of corpora lutea (yellow color) and antral/preovulatory follicles (red color) at different stages of the estrous cycle. Lower panel: Decreases in volume of individual corpus luteum (CL) from proestrus to diestrus. Means ± SE are shown.
Scientific RepoRts | 7:44810 | DOI: 10.1038/srep44810 found (Fig. 5B, lower panel). Following human chorionic gonadotropin (hCG) treatment of eCG-primed animals to induce ovulation, follicle luteinization was accompanied by rapid increases in vasculature inside preovulatory follicles and increases in volume of individual corpus luteum (Fig. 5C). In a representative adult ovary (Fig. 5D), PECAM1 staining in primordial/primary follicles was negligible whereas theca layers surrounding secondary, antral, and preovulatory follicles showed strong staining with no signal in inner granulosa cells. Serial sections of a representative corpus luteum (Fig. 5E) showed continuous PECAM1 staining throughout the solid structure. Supplementary Movie S3 showed relationship of blood vessels and antral/preovulatory follicles after eCG stimulation whereas Movie S4 showed invasion of PECAM1-stained endothelial cells into ruptured follicles during corpora lutea formation in hCG-treated animals. Movie S5 traced major vessels showing surrounding follicles in an ovary at 24 h after eCG treatment. After genotyping of wild type and heterozygous animals (Fig. 6A), we performed vaginal smearing of adult (~12 weeks of age) females and found heterozygous mutant animals were acyclic, showing prolonged diestrus. We then injected wild type and mutant animals at diestrus with eCG for 48 h, followed by hCG. At 16 h later, ovarian weights and number of ovulated oocytes were determined. As shown in Fig. 6B, mutant mice showed decreases in ovarian weights and lower number of oocytes released, together with a small decrease in body weight. CLARITY processing and immunostaining using PECAM1 antibodies indicated defective ovarian vasculature as shown by decreased total vasculature (Fig. 6C), presumably underlying the defective ovulatory responses. Furthermore, we uni-laterally injected axitinib, an inhibitor for all three VEGF receptor tyrosine kinases 22 , into ovarian bursa of immature wild type mice, before intraperitoneally treating the mice with eCG to induce follicle development. Forty-eight h later, axitinib pre-treatment decreased ovarian weights by ~30% (Fig. 7A and B) when compared with untreated contralateral ovaries in the same animal. Image analyses showed that axitinib treatment led to decreased total vasculature and lower antral/preovulatory follicle numbers (Fig. 7C). Following subsequent treatment with hCG to induce ovulation, axitinib-treated ovaries released fewer numbers of oocytes into oviducts (Fig. 7D).

Discussion
Using the CLARITY approach, we performed marker staining, digital imaging, and spatial analysis to obtain 3D ovarian architecture throughout life. This method transforms intact tissues into a nanoporous hydrogel-hybridized form that is optically transparent and macromolecule-permeable 9 . Unlike classical 2D sectional histological analyses, CLARITY provides 3D mapping of interrelationships among follicles and corpora lutea at different developmental stages. Marker staining of ovarian structures revealed changes in follicle numbers, volume, clustering, migration, and sphericity, as well as previously unappreciated association among follicles and relationship between local vasculature and follicles/corpora lutea.
We found ~3 × 10 5 -fold increases in follicle volume from primordial to the preovulatory stage. This was accompanied by ~88,000-fold increases in oocyte volume, forming the largest cell in the body with a volume of  Figure S4). (B) Wild type and heterozygous mice at 10-11 weeks of age were treated with eCG, followed by hCG to induce ovulation. Ovarian and body weights, together with numbers of ovulated oocytes were determined. WT: wild type. Stars indicate significant differences between wild type and Vegfa +/delta mutant mice (P < 0.01). Means + SE are shown. (C) Tracing of ovarian vasculature in wild type and Vegfa +/delta mutant mice. "N" represents number of total antral/preovulatory follicles in individual ovaries whereas Φ denotes the diameter of vascular trunks originating from the helium.

Figure 7. Unilateral intrabursal injection of a VEGF receptor inhibitor axitinib suppressed ovarian neoangiogenesis, ovarian weight, and ovulation.
Immature mice at 21 days of age were intrabursally injected with axitinib into the right ovary with the left side serving as controls. Control animals were injected unilaterally with the vehicle (DMSO). Animals were then treated with eCG to stimulate follicle growth. At 48 h after eCG injection, weights (A), morphology (B), and vasculature (C) of control and axitinib-treated ovaries were recorded. Stars indicate significant differences between untreated and treated sides (*P < 0.05 and **P < 0.05). Means + SE are shown. In (C),"n" represents number of total antral/preovulatory follicles in individual ovaries whereas Φ denotes the diameter of vascular trunks originating from the helium. (D) Some animals were further treated with hCG to induce ovulation before recording number of ovulated eggs retrieved. Stars indicate significant differences between untreated and treated sides (P < 0.001). Means + SE are shown.
Scientific RepoRts | 7:44810 | DOI: 10.1038/srep44810 3 × 10 6 um 3 . Spot transformation of 3D images of stained follicles facilitated quantitation of follicle numbers and confirmed age-dependent decreases of follicle numbers 5 . Findings of increases in percentages of primordial and primary follicles during senescence further suggested an age-related decline in follicle activation and development. Our findings related to age-dependent decline of number of primordial follicles are comparable to prior estimates of primordial follicle loss using a stereological method 23 and whole mount immunofluorescence analyses 7 . For intact ovarian imaging, other methods such as X-ray imaging and ultrasound imaging have been developed in rodents 24,25 . Using a synchrotron X-ray imaging method, whole ovaries was evaluated to monitor Graffian and antral follicles at various ages in mice 26 . Although the exact age evaluated in that study were not comparable to our study, their findings showed lower number of antral follicles as observed here. Although X-ray and ultrasound imaging methods allowed imaging of antral follicles in the same animal overtime, these approaches do not allow evaluation of smaller follicles. On the other hand, traditional histological counting of follicles suffers from variability in section thickness, imprecise correction for total ovary volume, and a lack of consideration for follicle clustering, leading to large variations of follicle numbers differing up to ten-fold 27 . Using a whole-mount approach, a recent study minimized sectional errors and quantified oocyte dynamics in murine prenatal ovaries of small sizes 28 . The present approach using large adult ovaries provides a standardized way to determine follicle dynamics with minimal loss.
In ovaries from mice at different ages, we found clustering of follicles at same stages of development but clustering decreased as follicle development advanced. Earlier studies demonstrated clustering of preantral/early antral follicles in groups of up to 20-50 follicles 6,29 whereas whole mount immunofluorescence analyses showed decreases in clustering as follicles develop based on nuclear staining in oocytes 7 . Highly clustered primordial follicles could secret follicle activation inhibitors as predicted by 2D spatial analysis 30 whereas decreased clustering of secondary follicles is consistent with culture studies showing that two secondary follicles in direct contact invariably led to the dominance of one and growth suppression of its neighbor 31 . Consistent with rat studies 6 , small, non-growing primordial follicles were found under the surface epithelium in prepubertal and adult mice. Once primordial follicles are activated by local factors, they migrate away from the cortical region (Fig. 2C). This inward movement of follicles is likely due to the stiffer cortical enclosure as compared with the softer inner medulla. Although primordial and primary follicles are round in adult ovaries, sphericity indexes decreased in larger follicles. In contrast, advanced follicles maintained their roundness in ovaries of gonadotropin-treated immature mice. These data suggested that repeated tissue remodeling during recurring reproductive cycles could decrease tensile strength/elasticity in adult ovaries, rendering larger follicles into irregular shapes.
Our 3D imaging analyses indicated that follicles are in direct contact with each other. Although follicles at all stages were found adjacent to corpora lutea, preovulatory follicles were in close contact with antral, but fewer primordial, primary, and secondary follicles. These findings suggest that high levels of progesterone produced by the corpora lutea do not affect folliculogenesis whereas high levels of estrogen, inhibin, or other factors secreted by preovulatory follicles could confer unfavorable environments for preantral follicles. Also, we observed decreases in average volumes of corpora lutea from proestrous to the diestrous stage, suggesting rapid tissue remodeling and dissolution of the regressing structure within one estrous cycle.
In adult tissues, angiogenesis mainly occurs during wound healing and fracture whereas abnormal capillary growth is associated with pathological tumor growth, retinopathies, fibroses, and rheumatoid arthritis 32 . However, cyclic changes in ovarian and endometrial vasculature represent exceptions 4,8 . Consistent with the essential role of PECAM1 in vasculogenesis and angiogenesis 33 , we found major increase in PECAM1 staining during folliculogenesis and luteinization. Instead of randomly distributed inside the ovary, follicles are located along vascular branches. Growing follicles near vascular branches likely acquire more nutrients and secret more angiogenesis factors to promote local vasculature, resulting in a positive feedback loop. The adult ovary can be pictured as a vascular tree with 3 to 4 main branches each bearing fruits (follicles) of different sizes. Although the ovary does not have branching organelles similar to many other organs, intraovarian distribution of follicles is not a random event but is "constructed" around local vasculature.
We confirmed the avascular nature of cortical primordial follicles 8 . Because mTOR signaling regulated by nutrient availability is pivotal for the activation of dormant primordial follicles 34,35 , our data underscore the important role of local neo-angiogenesis in regulating follicle dormancy. In growing secondary and larger follicles, surrounding thecal cells have rich blood vessels that do not penetrate the basal membrane 32 . Extensive angiogenesis takes place following follicle rupture during luteinization and invasion of vasculature into corpora lutea is regulated by local pro-and anti-angiogenic factors 8 .
FSH stimulation of follicle growth is likely associated with hypoxia in the avascular granulosa cell compartment, leading to increased secretion of angiogenesis factors to promote neo-angiogenesis. The roles of ovarian VEGFA and neo-angiogenesis during gonadotropin induction of ovulation are demonstrated by our studies on ovulatory responses and vasculature changes in mutant mice with deletion of the hypoxia-response element in the VEGFA promoter. Because all three VEGF receptors are expressed in the ovary 36-38 , we unilaterally injected immature mice with axitinib, a pan-specific inhibitor for VEGF receptors, into ovarian bursa. Findings of defective ovarian vasculature and ovulatory responses after inhibitor treatment further underscore importance of endogenous VEGFs during gonadotropin-induced follicle maturation and ovulation.
Most ovarian research deals with molecular, cellular, and follicular scales, revealing roles of gonadotropins, paracrine factors, and local Hippo signaling [39][40][41] . The present approach facilitates studies at the whole ovary scale, highlighting the roles of vasculature, follicles interactions, and follicle locations in intact ovaries. The CLARITY approach provides opportunities to investigate hormonally regulated folliculogenesis, interrelationships among follicles and corpora lutea as well as remodeling of ovarian architecture during each reproductive cycle. Three-D imaging could allow elucidation of local tissue remodeling, including roles of invading macrophages during follicle rupture 42 as well as fimbrial stem cells during surface epithelium repair 43 . CLARITY studies on cyclic ovarian remodeling confirm the theory that blood vessels direct the configuration of organs, first proposed by Aristotle 44 .
Scientific RepoRts | 7:44810 | DOI: 10.1038/srep44810 Due to wide interests in using the CLARITY approach, recent modifications of this method has shortened the duration needed for tissue processing from weeks used here to days 45 . In addition, processing of larger samples is now possible 46 , thus allowing future evaluation of 3D architecture of the human ovary. In addition to providing models to investigate roles of neo-angiogenesis during organogenesis and tumorigenesis, future studies using the CLARITY approach could reveal ovarian architecture and vasculature underlying ovarian diseases, including polycystic ovarian syndrome, primary ovarian insufficiency, and ovarian tumorigenesis.

Methods
Study design. The experiments presented here were designed to investigate relationships among ovarian follicles, corpora lutea, and vasculatures as well as to test the hypothesis that ovarian vasculature dynamics are playing an important role in folliculogenesis and ovarian development. Using a newly established three-dimensional imaging technique, we provided evidences on vasculature control of folliculogenesis with the help of whole tissue imaging, together with immunostained markers in mouse ovaries. Furthermore, genetic and pharmacological mouse models were employed to regulate angiogenesis induced by VEGF signaling for evaluating folliculogenesis and ovulation. All experiments and analyses were repeated at least three times and were in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised in 1996) and the "Principles of Laboratory Animal Care" (National Society for Medical Research). These experimental protocols and animal care were approved by regional committees (Stanford University, Project Nr. 10347 and Fudan University, Project Nr. 20150119-019).
Animals and experimental design. Female C57BL/6 mice at different ages were obtained from Charles River Laboratories (Wilmington, MA). Aging C57BL/6 mice were from Shanghai Research Center for Model Organisms (Shanghai, China). Animals were housed in animal facilities at Stanford University or Fudan University under 12 h dark/light with free access to food and water. To investigate gonadotropin regulation of folliculogenesis, immature mice at 21 days of age were injected i.p. with 2 IU eCG (Peamex), followed at 48 h later by an injection of 10 IU hCG (Asuka Pharma) for up to 72 h. Animals were sacrificed at different times to obtain ovaries for CLARITY processing. To analyze atretic follicles, immature mice were treated with a single injection of 5 IU eCG for 96 h 47 . For studies using axitinib (Tocris), a pan-specific inhibitor for all three VEGF receptor tyrosine kinases 22 , mice at 21 days of age were anesthetized using isoflurane (Piramal Group) and injected with axitinib (5 or 10 μ g dissolved in 10 μ l DMSO; Tocris Inc.) using a 31-gauge insulin syringe (BD Inc.) through the ovarian fat pad into the bursa of the right ovary. This was followed by an injection of 5 IU eCG to stimulate follicle development, before collection of ovaries at 48 h after eCG treatment for weighing and CLARITY processing. To estimate ovulatory potency, animals were injected with 10 IU hCG at 48 h after eCG treatment before counting numbers of oocytes in oviducts at 16 h later. Each group included 6 to 12 mice.
Genotyping and ovulation induction of mutant mice. Vegfa delta/delta mice were generated by mating male and female heterozygous animals for the transgenic allele and maintained on a C57BL/6 genetic background. Wild type C57BL/6 mice were obtained from the Charles River Laboratory. Genotyping analyses were performed by PCR on DNA obtained from tail biopsies using the oligonucleotide primers 5′-GCTTATCTGAGCCCTTGTCTGATC-3′ ( Ve g fa genot yp e s ens e prob e); 5′ -GATGAAC C GTAAGC CTAGGCTAG-3′ ( Ve g fa genotype antisense probe); 5′-GAGTGAGACGACCTGTGGAAATG-3′ (mutant-sense probe); and 5′-AGACTACACAGTGCATACGTGGGT-3′ (wild type-antisense probe) based on the following PCR conditions: 1 min at 94 °C for one cycle, 30 sec at 94 °C, 30 sec at 60 °C, and 30 sec at 72 °C for 29 cycles, and 10 min at 72 °C for one cycle. Primers were designed to generate PCR products of 482 bp and 208 bp for the wild type Vegfa allele, or 482, 303, and 208 bp for the heterozygous Vegfa +/delta allele. Adult (10-11 weeks of age) wild type and heterozygous mutant mice were injected i. p. with 7.5 IU of eCG on the diestrous day, followed by a single injection of hCG (10IU) at 48 h later. At 16 h after hCG injection, ovarian and body weights were determined together with counting the number of ovulated oocytes in oviducts to estimate ovulatory potential. Some ovaries were further processed using CLARITY and immunostaining to evaluate ovarian vasculature.
CLARITY processing of ovaries. Mice were anesthetized i.p. with 2, 2, 2-Tribromoethanol (250 mg/kg body weight, Sigma-Aldrich) before transcardially perfused with 10 (postnatal day 3 or 10) or 20 ml (postnatal day 21 or older) of ice-cold 1 × PBS (Invitrogen GIBCO), followed by 10 or 20 ml of ice-cold hydrogel solution with 4% (weight/volume) paraformaldehyde (Electron Microscopy Sciences), 4% acrylamide (Bio-Rad), 0.05% bis-acrylamide (Bio-Rad), 0.25% VA-044 (Wako), 0.05% Saponin (Sigma) and 1 × PBS for 2 min as described 9 . Fixed ovaries were immediately extracted and immersed in 10 ml conical tubes containing 6-7 ml of the hydrogel solution for 3-4 days at 4 °C. Then, tubes were de-gassed with dry ice to remove oxygen and submerged in a 37 °C water bath for 3 h to polymerize hydrogel monomers. Embedded samples were then transferred into 10 ml of the clearing solution (200 mM boric acid (Sigma), 4% (wt/vol) sodium dodecyl sulfate (SDS) (Sigma) dissolved in distilled H 2 O, with the addition of NaOH (EMD) to reach pH 8.5 9 . The clearing solution was replaced 2-3 times/ week and ovaries gradually became transparent after 4-8 weeks depending their sizes and ages of the animal. We performed CLARITY without the electrophoretic step to allow better antibody staining and to minimize damage of small and fragile murine ovaries.
Immunofluorescence staining. Cleared ovaries were transferred into 10 ml glass bottles containing PBST (PBS with 0.1% Triton-X (Sigma)) before shaking slowly in PBS for 1 day to remove residual SDS. Samples were then incubated with primary antibodies for two days, washed in buffer for 1 day, followed by secondary antibodies for 2 days. Before mounting and imaging, samples were incubated in PBST for at least 1 day in the FocusClear solution (CelExplore, Taiwan) for 1 h to correct for refractive index. All procedures were conducted with shaking at 37 °C. Detailed antibody information is shown in Table S3.