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.

The authors have no conflicts of interest to disclose.

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...

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 attributed to the genetic and physiological quality of eggs developing from the OR and remains poorly understood7,8. Because female follicle endowment is largely predetermined by birth, it is essential to understand the regulatory mechanisms involved in the OR establishment and maintenance.
In mice, OR formation starts with the specification of primordial germ cells (PGCs) around Embryonic day (E) 7.52. The PGCs migrate to the genital ridges, where they will reside by approximately E10.59. The following extensive proliferation occurs with incomplete cytokinesis resulting in the formation of cysts that will be broken down later in development10,11. At approximately E12.5, gonadal sex is determined, and PGC proliferation halts in ovaries. In females, PGCs, now oocytes, enter meiotic prophase I (MPI) at approximately E13.512,13.&#160;Oocytes progress through extended MPI and arrest at the dictyate stage around the time of birth. During the first week after birth, each arrested oocyte is surrounded by granulosa cells, thereby forming a primordial follicle.
The number of primordial follicles in the OR of a female depends on how many oocytes survived the waves of oocyte elimination that occur before and during MPI arrest through apoptosis, autophagy, or necrosis14,15. The first wave occurs during fetal development and is known as FOA. FOA is an evolutionarily conserved process in females (mammalian and non-mammalian), whereby an estimated 50-80% of the oocytes are eliminated depending on the female species16,17,18,19. In mice, FOA occurs during E15.5 to E18.5 and has been attributed to the reactivation and expression of retrotransposon LINE-1 sequences causing oocyte death20,21. The second wave of oocyte elimination occurs through a meiotic checkpoint that eliminates oocytes with meiotic defects such as unrepaired DNA double-strand breaks (DSBs)22,23. The next wave of oocyte elimination occurs during cyst breakdown, culminating during the formation of primordial follicles, each of which contains a single oocyte10,11,24,25.
In mice, the primordial follicle reserve is largely established by puberty, after which it decreases as primordial follicles are activated for growth during regular reproductive cycles. The OR size varies among individual women and among different genetic strains of mice; yet, the genetic regulation of OR size is not well understood26,27,28,29. Genetic studies of OR regulation are hampered by the lack of standardized protocols to study the waves of oocyte elimination during prenatal and postnatal development. Several oocyte quantification methodologies have been developed in mice, with the most common and widely used being histomorphometric evaluation of histological sections30,31. In this technique, oocytes are identified on serial sections with histological stains, such as hematoxylin and eosin (H&#38;E) and periodic acid-Schiff (PAS) or fluorescent markers. This technique is reliable if all conditions remain constant, including section thickness, efficient recovery of all sections throughout the ovary, and the counting schemes of individual laboratories. However, numbers reported by different laboratories often differ significantly and thus are not easily comparable.
Moreover, given genetic differences, the use of different mouse strains can also influence oocyte counts. Additional computational approaches have been developed for histomorphometric evaluation and include the automated detection of oocytes using the fractionator approach, automatic counting using computational algorithms, and 3D reconstruction of histological images to prevent multiple counts of the same oocyte31,32,33,34,35,36. Even with these improvements added to histomorphometric evaluation, the technique is relatively labor-intensive, particularly for large-scale and high-throughput studies. The data collected may not be reproducible and comparable between studies due to differences in counting schemes, computer algorithms, and software used.
Recently, accelerated by the development of new medium-resolution multiphoton and light sheet microscopy and optical tissue clearing methods, 3D modeling and analysis techniques for intact ovaries are becoming the method of choice to efficiently quantify oocyte numbers and study protein localization and dynamics37,38. These 3D methods are typically advantageous compared to histological methods as tissues and organs are better preserved and kept intact. Moreover, 3D analysis and modeling provide additional insights into function and interactions within and between cell niches or substructures within the organ that may be missed in 2D analysis.
3D analysis of whole organs requires optimization of fixation, immunostaining, and optical clearing protocols for individual organs, such as ovaries, without tissue distortion or damage. Additional optimization of sample mounting for imaging is required for high-resolution microscopy and may depend on the imaging platform available. Finally, imaging of the whole intact ovary generates a large amount of data for subsequent computational analyses. Therefore, there is a need to develop standardized 3D methods for counting oocytes for comparative studies and across developmental stages.
This protocol uses standard immunostaining and previously reported clearing protocols, focusing on a simple, user-friendly, and high-throughput approach38,39,40,41. The protocol is optimized to analyze large numbers of prenatal and postnatal ovaries up to postnatal day 28 (P28) and varying sizes of ovaries from different mouse genetic backgrounds. The immunostaining steps are similar for all stages; however, the clearing protocols differ for pubertal ovaries due to their larger size, ScaleS4(0) and CUBIC for small and large ovaries, respectively40,41. Further, whole-body perfusion is performed in P28 mice before fixation to prevent autofluorescence from blood cells. A multiphoton microscope was built on the Leica DIVE/4Tune platform as an alternative to light sheet microscopy to acquire images, and IMARIS 3D Visualization and Analysis software with various analytical tools was chosen for this protocol. This protocol is simple to follow and less hands-on, hence time-saving. Moreover, oocyte quantification is relatively quick, depending on the size of the ovary and arrangement of oocytes.

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			<td>Benchtop Incubator</td>
			<td>Benchmark Scientific</td>
			<td>H2200-H</td>
			<td>37 &#176;C incubator</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>mouse inbred strain</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D1435</td>
			<td>Hazardous material</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S6021</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>FastWells Reagent Barriers</td>
			<td>GraceBio</td>
			<td>664113</td>
			<td>Sticky and flexible silicone gasket (adhesive well)</td>
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			<td>Fine Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td></td>
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			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>ThermoFisher Scientific</td>
			<td>BP381-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A-21246</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-21434</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>IMARIS Software</td>
			<td>Oxford Instruments</td>
			<td>Version 9.7.0</td>
			<td>Image visualization and analysis software</td>
		</tr>
		<tr>
			<td>Insight X3</td>
			<td>Spectra-Physics</td>
			<td>InSight&#160;X3 Tunable Ultrafast Laser</td>
			<td>Laser for Multiphoton Imaging</td>
		</tr>
		<tr>
			<td>LASX software</td>
			<td>Leica</td>
			<td>Version 3.5.6</td>
			<td>Image acquisition software</td>
		</tr>
		<tr>
			<td>Leica DIVE/4TUNE/FALCON</td>
			<td>Leica</td>
			<td>Leica Dmi8, 2P-M-ready: # 158005406</td>
			<td>Multiphoton Microscope</td>
		</tr>
		<tr>
			<td>MaiTai HP</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai DeepSee One Box Ultrafast Laser</td>
			<td>Laser for Multiphoton Imaging</td>
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		<tr>
			<td>Masterflex Pump Controller</td>
			<td>SPW Industrial</td>
			<td>Model: 7553-50</td>
			<td>Peristaltic pump for perfusion</td>
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		<tr>
			<td>Mayo Scissors</td>
			<td>FST</td>
			<td>14010-17</td>
			<td>5&#8221; &#8211;7&#8221; blunt/blunt scissors for decapitation</td>
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		<tr>
			<td>Micro Cover Glasses, Square, No. 1.5 25x25mm</td>
			<td>VWR</td>
			<td>48366-249</td>
			<td></td>
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		<tr>
			<td>Mini BioMixer</td>
			<td>Benchmark Scientific</td>
			<td>B3D1020</td>
			<td>shaker/nutator for 37 &#176;C incubator</td>
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		<tr>
			<td>Nikon Ergonomic SMZ1270</td>
			<td>Leica</td>
			<td>&#160;SMZ1270</td>
			<td>stereomicroscope</td>
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		<tr>
			<td>Paraformaldehyde 16% (formaldehyde aqueous solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Hazardous material</td>
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		<tr>
			<td>PBS Tablets, Phosphate-buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2944100</td>
			<td>Dissolve in Milli-Q water</td>
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		<tr>
			<td>Penicillin-Streptomycin, 200x, Dual Antibiotic Solution</td>
			<td>ThermoFisher Scientific</td>
			<td>ICN1670249</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadrol (N,N,N&#8242;,N&#8242;-Tetrakis(2-Hydroxypropylethylenediamine)</td>
			<td>Sigma-Aldrich</td>
			<td>122262</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-DDX4/MVH</td>
			<td>Abcam</td>
			<td>ab27591</td>
			<td>Dilution 1:500</td>
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		<tr>
			<td>Rabbit anti-LINE-1 ORF1p</td>
			<td>Abcam</td>
			<td>ab216324</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Rat anti-TRA98/GCNA</td>
			<td>Abcam</td>
			<td>ab82527</td>
			<td>Dilution 1:500</td>
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			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Hazardous material</td>
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			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>Hazardous material</td>
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			<td>Sucrose</td>
			<td>ThermoFisher Scientific</td>
			<td>S0389</td>
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			<td>Tekmar Orbital Shaker</td>
			<td>Bimedis</td>
			<td>VXR-S10</td>
			<td>shaker for room temperature</td>
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			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>90279</td>
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			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
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		<tr>
			<td>UNOLOK Infusion Set</td>
			<td>MYCO Medical</td>
			<td>7001-23</td>
			<td>needles for perfusion</td>
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		<tr>
			<td>Urea</td>
			<td>Amresco</td>
			<td>97061-920</td>
			<td></td>
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		<tr>
			<td>X-Cite 120LED</td>
			<td>Excelitas</td>
			<td>S/N XT640-W-0147</td>
			<td>low-power LED fluorescence lamp</td>
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whole ovary immunofluorescence, clearing, and multiphoton microscopy for quantitative 3d analysis of the developing ovarian reserve in mouse

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.

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...

Watch this Scientific Journal Video about Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse at JoVE.com

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

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 ...

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4.3K Views.  The Jackson Laboratory. 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.