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Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.
Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.
Most mammalian females are born with a finite number of meiotically arrested oocytes stored within primordial follicles, constituting the ovarian reserve (OR)1,2. The OR determines the overall female reproductive lifespan and health3. The OR normally declines in size with aging and can be prematurely depleted upon exposure to certain genotoxic agents (radiation/chemotherapy) and environmental stresses (malnutrition), leading to infertility4,5,6. Idiopathic female infertility can often be attrib....
All mice used were of the genetic strain C57BL/6J (see the Table of Materials). This strain has been fully sequenced and is standard for many studies on ovarian structure and function. Mice were housed according to NIH guidelines, and procedures performed were approved by the Institutional Animal Care and Use Committee of The Jackson Laboratory. Reagents and compositions used in this protocol are listed in Table of Materials and Table 1, respectively.
1. Preparation of reagents
Immunostaining and imaging of the whole ovary enables the visualization and quantification of oocytes or protein expression in ovaries at different developmental stages using the same technique and markers (Figure 3). This protocol was developed for a large-scale project in which analysis of ovaries at multiple stages and from multiple mouse strains was required. Here, we present data gathered for the C57BL6/J strain, a standard strain for genetic analysis. The technique presented here is st.......
This article presents a detailed 3D immunostaining and imaging protocol for prenatal and postnatal ovaries for high-throughput and comparative studies for germ cell quantification and protein localization. We developed this protocol to analyze oocyte numbers in ovaries (N=6-12) at six developmental time points in 10-16 different strains, where 2-4 24-well plates are typically processed at one time. This method can be adapted for other organs or cellular markers. For example, this protocol can be used to label and visuali.......
The authors have no conflicts of interest to disclose.
This work was supported by the National Institutes of Health grants (R01 HD093778 to E.B-F and T32 HD007065 to R.B). We thank Zachary Boucher for his assistance with radiation experiment. We thank Mary Ann Handel for critical reading of the manuscript. We gratefully acknowledge the contribution of Sonia Erattupuzha and the Microscopy Core Service at The Jackson Laboratory for expert assistance with the microscopy work described in this publication and Jarek Trapszo from the Scientific Instrument Services at The Jackson Laboratory for designing the 3D-printed adaptor slide.
....Name | Company | Catalog Number | Comments |
Benchtop Incubator | Benchmark Scientific | H2200-H | 37 °C incubator |
Bovine Serum Albumin (BSA) | VWR | 97061-416 | |
C57BL/6J | The Jackson Laboratory | 000664 | mouse inbred strain |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D1435 | Hazardous material |
D-Sorbitol | Sigma-Aldrich | S6021 | |
Dumont #5 Forceps | FST | 91150-20 | |
FastWells Reagent Barriers | GraceBio | 664113 | Sticky and flexible silicone gasket (adhesive well) |
Fine Scissors | FST | 91460-11 | |
Glycerol | Sigma-Aldrich | G2025 | |
Glycine | ThermoFisher Scientific | BP381-500 | |
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A-21246 | Dilution 1:1000 |
Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A-21434 | Dilution 1:1000 |
Goat serum | Sigma-Aldrich | G9023 | |
IMARIS Software | Oxford Instruments | Version 9.7.0 | Image visualization and analysis software |
Insight X3 | Spectra-Physics | InSight X3 Tunable Ultrafast Laser | Laser for Multiphoton Imaging |
LASX software | Leica | Version 3.5.6 | Image acquisition software |
Leica DIVE/4TUNE/FALCON | Leica | Leica Dmi8, 2P-M-ready: # 158005406 | Multiphoton Microscope |
MaiTai HP | Spectra-Physics | Mai Tai DeepSee One Box Ultrafast Laser | Laser for Multiphoton Imaging |
Masterflex Pump Controller | SPW Industrial | Model: 7553-50 | Peristaltic pump for perfusion |
Mayo Scissors | FST | 14010-17 | 5” –7” blunt/blunt scissors for decapitation |
Micro Cover Glasses, Square, No. 1.5 25x25mm | VWR | 48366-249 | |
Mini BioMixer | Benchmark Scientific | B3D1020 | shaker/nutator for 37 °C incubator |
Nikon Ergonomic SMZ1270 | Leica | SMZ1270 | stereomicroscope |
Paraformaldehyde 16% (formaldehyde aqueous solution) | Electron Microscopy Sciences | 15710 | Hazardous material |
PBS Tablets, Phosphate-buffered Saline | ThermoFisher Scientific | BP2944100 | Dissolve in Milli-Q water |
Penicillin-Streptomycin, 200x, Dual Antibiotic Solution | ThermoFisher Scientific | ICN1670249 | |
Polyvinyl alcohol (PVA) | Sigma-Aldrich | P8136 | |
Quadrol (N,N,N′,N′-Tetrakis(2-Hydroxypropylethylenediamine) | Sigma-Aldrich | 122262 | |
Rabbit anti-DDX4/MVH | Abcam | ab27591 | Dilution 1:500 |
Rabbit anti-LINE-1 ORF1p | Abcam | ab216324 | Dilution 1:500 |
Rat anti-TRA98/GCNA | Abcam | ab82527 | Dilution 1:500 |
Sodium azide | Sigma-Aldrich | S2002 | Hazardous material |
Sodium borohydride | Sigma-Aldrich | 452882 | Hazardous material |
Sucrose | ThermoFisher Scientific | S0389 | |
Tekmar Orbital Shaker | Bimedis | VXR-S10 | shaker for room temperature |
Triethanolamine | Sigma-Aldrich | 90279 | |
Triton X-100 | Sigma-Aldrich | X100 | |
UNOLOK Infusion Set | MYCO Medical | 7001-23 | needles for perfusion |
Urea | Amresco | 97061-920 | |
X-Cite 120LED | Excelitas | S/N XT640-W-0147 | low-power LED fluorescence lamp |
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