Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.
The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P <0.01) between the length of the follicular capillaries and volume of the follicular wall.
The follicle is the fundamental structural and functional unit of the ovary, and its development is highly related to the vasculature within the ovary. Blood vessels supply nutrition and hormones to the follicles and thus play important roles in the growth and maturation of follicles1.
A combination of technologies, including selective blood vessel markers, transgenic mouse models, and pharmaceutical development, have increased our knowledge about ovarian vascular networks, angiogenesis, and the function of blood vessels in folliculogenesis. The ovary is known as an active organ because it remodels various tissues and vascular networks during folliculogenesis and ovulation. Such active remodeling in the size and structure of vessels is required for the biological function of developing and recruiting follicles.
Traditional histological and histomorphometric methods using ovarian sections and immunolabeling of blood vessels are limited to two-dimensional (2D) images2. With the development of three-dimensional (3D) reconstruction technologies, 2D images of tissue slices can be overlapped to make a 3D structure, but this method still has some limitations — sectioning of the tissue can destroy the microstructures, some parts of the tissue are often missing, and significant labor is involved in making 3D reconstructions from images obtained from slices. Whole-tissue 3D imaging with confocal microscopy can overcome many of these limitations, but these methods are limited to the evaluation of angiogenesis in the embryonic ovary3. Using whole tissue clearing methods such as CLARITY4 can increase the visualized volume so as to solve these problems in postnatal and adult ovaries, and such methods provide optical clearance of the ovary without any structural deformations. Imaging of the 3D architecture of the intact ovary provides an accurate image database for image analysis software, such as the Imaris software package used in this work.
Remodeling of the ovary throughout adulthood is part of a dynamic physiological system, and this makes the ovary an excellent model for investigations into the regulation of angiogenesis. Furthermore, evaluating the role of ovarian blood vessels in pathologic conditions of the female reproductive system such as polycystic ovary syndrome or ovarian cancers can be studied through whole ovarian tissue imaging. The development of the passive CLARITY method and the use of advanced image analysis software have provided detailed spatial information on the relationships between blood vessels and ovarian structures such as follicles.
All procedures involving animal subjects followed the guidelines of the Animal Ethics Committee at Shanghai Medical College, Fudan University (Approval number 20160225-013).
1. Preparation of the Transparent Mouse Ovary
2. Immunostaining
3. Confocal Microscopy
4. 3D Reconstruction of Individual Follicles and Their Vasculatures
5. Statistical Analysis
NOTE: Image analysis data are presented as means ± standard errors.
We adapted the passive CLARITY method into a quick and simple method for passive ovary clearing while preserving the follicular and vascular architecture and obtaining the highest fluorescent signal from labeled markers of vessels and follicles. The 3D architecture of the follicular vasculature was determined by immunostaining for CD31, a marker for endothelial cells6. CD31 staining in the ovaries of adult mice was traced using the Filament algorithm and showed interrelationships between follicular capillaries and primary and secondary follicles (Figure 1).
The Spot transformation and Surface algorithms were used to detect the tyrosine hydroxylase-immunostained oocytes and granulosa and thecal layers and to calculate the total volume of follicles7,8. Because of the compaction of the follicles, some parts of cropped follicles were identified semi-manually. After Spot transformation of the individual follicular volume and oocytes and Surface reconstruction of the follicular wall layers and Filament reconstruction of the vessels, clear relationships between individual follicular vasculatures and the other measured indices, especially granulosa and thecal layer volumes, were observed (Figure 2).
Figure 1: High-resolution imaging of an intact adult mouse ovary after clearing with the adapted passive CLARITY protocol. (A-B) Sections and XYZ large-scan acquisition. Three dimensional (3D) cropped secondary (C) and primary (D) follicles (top row) and 3D reconstructed capillaries, oocytes, and granulosa/thecal cells of the same follicle (bottom row) are shown as identified by the Filament, Surface, and Spot transformation algorithms. Scale bars = 200 µm (A-B), 50 µm (C), 30 µm (D). Please click here to view a larger version of this figure.
Figure 2: Comparative indices of the relationships between primary and secondary ovarian follicles and their vasculatures. The oocyte volume (A) was extracted after 3D reconstructions of follicular volume and oocytes in individual follicles as determined by the Spot transformation algorithm, and the follicular wall volume (B) was determined by the Surface algorithm and the capillaries were traced by the Filament algorithm. The follicular volume (C), the oocyte volume/capillary length ratio (D), the follicular wall volume/capillary length ratio (E), the follicular volume/capillary length ratio (F), the capillary length (H), and the correlation of capillary length and follicular wall volume (G) were calculated using the appropriate tools in the software package. Error bars represent the standard errors of the means. n = 6; * P <0.05; ** P <0.01. Please click here to view a larger version of this figure.
Figure 3: Three-dimensional reconstruction of the capillaries in secondary follicles of the mouse ovary. This reveals a hollow space above the oocyte side of the follicles (white squares show the borders of the space). Please click here to view a larger version of this figure.
In the current study, we present 3D imaging to evaluate the relationships between capillaries and individual growing follicles. In our previous work using the same protocol 9, we studied the roles of large vasculature, interactions between follicles, and the location of follicles in intact mouse ovaries. The passive CLARITY approach allowed us to study micro- and macro-vasculatures, folliculogenesis, and the interrelationships between the corpora lutea and follicles as well as to reconstruct the ovarian architecture at different developmental stages.
To obtain 3D image data of intact tissues, the clearing process is the first important step. There are two main strategies — dehydration methods and hydrophilic reagent-based methods, such as passive CLARITY10. Because dehydration methods often lead to a decrease in fluorescent signals and often produce toxic organic solution waste, we have focused our attention on simplifying some steps of the passive CLARITY method for use with various tissue types.
The first critical procedure in the clearing process is the transcardial perfusion. During the perfusion, heart pumping plays an important role in the whole-body circulation of PBS and hydrogel solution, which can flush out the blood so as to decrease the background fluorescence. Thus, the perfusion surgery should be performed accurately and quickly to avoid the premature death of the animal before the hydrogel is completely distributed throughout the animal. All agents must be kept on ice during the entire experiment, as mentioned by Liang et al.11, or they will begin to polymerize before the perfusion is complete. The next important step is removing the hydrogel, and it is necessary to remove as much excess gel as possible because it will retain antibodies. Once the tissue is cleared, it should be removed from the clearing solution and stored in PBS at 4 °C because too much time in the clearing solution will cause too much protein to be lost. In the confocal microscopy imaging, the Z-settings should always be set before the X-Y range. If the X-Y range is set down first, the Z-settings cannot be synchronized to the entire X-Y field.
Passive CLARITY was first introduced by Yang et al.12 In their protocol, they did not perform the perfusion step and instead degassed with nitrogen. In addition, their clearing solution did not include bisacrylamide in the hydrogel solution, they used 8% SDS in the clearing solution, and they used RIMS for refractive index matching media. This method was subsequently improved by increasing the SDS concentration13, adding the perfusion step and the nitrogen-free degassing procedure14, and combining the method with other clearing methods such as PARS-mPACT15. Here, we have further developed the protocol by combining transcardial perfusion and nitrogen-free degassing, decreasing the SDS in the clearing solution to 4%, and using FocusClear as the refractive index matching media, which provides better-quality microscopy images.
After passive CLARITY, confocal multiphoton microscopy and image analysis can be performed to obtain the intact 3D architecture of the ovarian follicular vasculature. This protocol transforms a non-light-permeable intact ovary into an optically transparent hydrogel-hybridized nanoporous form that is appropriate for penetration by macromolecules such as antibodies16. Unlike classical 2D sectional histological analyses, passive CLARITY and staining with specific markers allows the 3D mapping of relationships among capillaries and individual growing follicles.
In adult tissues, active vascular remodeling and angiogenesis mainly occurs during pathological processes such as wound healing, fracture repair, tumor and metastasis formations, retinopathies, fibroses, and rheumatoid arthritis17. However, cyclical changes during folliculogenesis represent a unique capacity for vasculature remodeling. After formation of the antrum in secondary follicles, follicular fluid fills it and surrounds the oocyte, which provides a suitable microenvironment for oocyte maturation and follicular development18. Previous 2D imaging studies showed that ovarian follicles are surrounded by a network of capillaries in the theca interna19. The granulosa layer is nonvascular, and each follicle is surrounded by its own capillaries. Consistent with the essential role of CD31 in vasculogenesis6, we found an increase in capillary length during folliculogenesis in the transition from primary to secondary follicles (Figures 2G and 2H). Instead of a random distribution of capillaries around the individual primary or secondary follicles, 3D reconstruction of capillaries demonstrated that capillary branches distributed in follicles were detectable in larger follicles, and in most cases these capillary branches formed a hollow space (Figure 3).
One major concern is that the adapted method takes a relatively long time to clear the ovary, but the long time is required for the best clearing effect. To save some time, we recommend collecting all of the needed samples at one time and clearing them together because the cleared tissues can be stored in PBS and 0.02% sodium azide at 4 °C for several months. Another limitation is the working distance of the confocal microscope. Higher-magnification objective lenses have shorter working distances, which could be an obstacle for imaging of thick samples.
Several protocols have been introduced for the imaging of transparent and intact tissues, including BABB20, Scale21, 3DISCO22, ClearT23, SeeDB24, CLARITY16, passive CLARITY (with4 or without hydrogel25), PACT12, CUBIC26,27, FASTClear28, and FACT29, but only three of them have been used for whole ovarian tissue clearing and evaluation9,30,31. SeeDB30 and ScaleA231 have been introduced for imaging of whole ovarian tissues, but these protocols only allow a single immunostaining step. Passive CLARITY, however, allows for multiple protein-labeling reactions, as shown in the present study and in our previous study9.
In conclusion, our adapted protocol for determining the 3D structural relationship between follicular layers and blood vessels using whole clarified ovarian tissue provides a new approach for studying angiogenesis in different ovarian diseases such as polycystic ovary syndrome and ovarian cancers.
The authors have nothing to disclose.
This study was supported by grants from the Chinese Special Fund for Postdocs (No. 2014T70392 to YF), the National Natural Science Foundation of China (No. 81673766 to YF), the New Teacher Priming Fund, the Zuoxue Foundation of Fudan University, and the Development Project of Shanghai Peak Disciplines-Integrative Medicine (20150407).
Acrylamide | Vetec | v900845 | http://www.sigmaaldrich.com/catalog/product/vetec/v900845 |
Alexa Flour 488 (Dilution 1:50) | Life Technologies | A11039 | https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039 |
Alexa Flour 594 (Dilution 1:50) | Life Technologies | A11012 | https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012 |
Bisacrylamide | Amresco | 172 | http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx |
Black wall glass bottom dish (Willco-Dish) | Ted Pella | 14032 | http://www.tedpella.com/section_html/706dish.htm#black_wall |
Boric acid | Sinopharm Chemical Reagent | 10004818 | http://en.reagent.com.cn/enshowproduct.jsp?id=10004818 |
Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O) | Sinopharm Chemical Reagent | 10020318 | http://en.reagent.com.cn/enshowproduct.jsp?id=10020318 |
FocusClear | Celexplorer | FC-102 | http://www.celexplorer.com/product_list.asp?MainType=107&BRDarea=1 |
Parafilm | Bemis | PM996 | http://www.parafilm.com/products |
Paraformaldehyde | Sinopharm Chemical Reagent | 80096618 | http://en.reagent.com.cn/enshowproduct.jsp?id=80096618 |
PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10) | Abcam | ab28364 | http://www.abcam.com/cd31-antibody-ab28364.html |
Photoinitiator VA044 | Wako | va-044/225-02111 | http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm |
Sodium azide | Sigma | S2002 | http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en®ion=US |
Sodium chloride (NaCl) | Sinopharm Chemical Reagent | 10019318 | http://en.reagent.com.cn/enshowproduct.jsp?id=10019318 |
Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O) | Sinopharm Chemical Reagent | 20040718 | http://en.reagent.com.cn/enshowproduct.jsp?id=20040718 |
Sodium dodecyl sulfate | Sinopharm Chemical Reagent | 30166428 | http://en.reagent.com.cn/enshowproduct.jsp?id=30166428 |
Sodium hydroxide (NaOH) | Sinopharm Chemical Reagent | 10019718 | http://en.reagent.com.cn/enshowproduct.jsp?id=10019718 |
Triton X-100 | Sinopharm Chemical Reagent | 30188928 | http://en.reagent.com.cn/enshowproduct.jsp?id=30188928 |
Tyrosine hydroxylase (TH, Dilution 1:50) | Abcam | ab76442 | http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html |