Whole Mount Immunostaining of Fetal and Neonatal Mouse Ovaries

Summary

This protocol describes a method for whole mount immunolabeling of developing mouse ovaries, with applications to other small tissues.

Abstract

The ovarian reserve consists of the oocytes available to a female for reproduction and are found in primordial follicles, each containing one oocyte surrounded by several granulosa cells. The oocytes start out as primordial germ cells that form outside the gonad and migrate to the developing ovary during embryonic development. Once they reach the ovary, they become oogonia and divide by mitosis, with incomplete cytokinesis, forming clusters of interconnected cells called germ cell cysts. While in cysts, the cells enter meiosis and become oocytes. In the mouse, two days before birth, the cysts begin to break apart, and each oocyte is packaged into a primordial follicle. Only some of the oocytes survive during this process, the rest undergo apoptosis. The regulation of cyst breakdown and primordial follicle formation is only partially understood, and identifying the molecules involved is crucial to understanding this process. Whole mount immunostaining of developing mouse ovaries can be used to determine the localization of potential regulatory molecules and to assess effects on primordial follicle formation due to mutation or other molecular changes using oocyte and somatic cell markers. Ovaries are harvested from fetal or neonatal mice, fixed in formaldehyde, and blocked. The ovaries are then exposed to antibodies against a protein of interest, followed by secondary antibodies conjugated to a fluorescent tag that detect the primary antibodies. Finally, the ovaries are mounted and examined by confocal microscopy. This procedure can detect multiple proteins of interest and can be applied to other small tissues, such as fetal mouse testes.

Introduction

Whole mount immunostaining combined with confocal imaging is a powerful method for analyzing protein expression and cell morphology in small tissues without sectioning¹. In this method, tissues are first harvested, fixed, and blocked, and then exposed to antibodies against proteins of interest. The antibodies are either directly conjugated to fluorophores or followed by labeling with secondary antibodies conjugated to fluorophores that recognize the primary antibodies. The tissue is then incubated with the fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. This step is important to provide spatial orientation when imaging the tissue. Finally, the tissue is mounted and visualized by confocal microscopy. Another technique that has been used to study tissue morphology involves embedding the tissue in paraffin, sectioning, and either labeling with dyes such as hematoxylin and eosin or with antibodies. Alternatively, frozen sections can be prepared and immunostained. Unfortunately, both these techniques involve sectioning, which results in the loss of three-dimensional information. While serial sections can help, analysis is tedious and involves viewing a large number of sections. Thus, the whole mount method is advantageous as it preserves the three-dimensional structure of the tissue that can be visualized using optical sectioning.

The development of primordial germ cells (PGCs) into oocytes and the assembly of oocytes into primordial follicles are essential for female fertility. PGCs form extraembryonically and must migrate to the ovary during embryonic development. Once the PGCs arrive at the ovary, starting at approximately embryonic day (E) 10.5 in the mouse, they are classified as oogonia². The oogonia undergo mitosis but with incomplete cytokinesis, forming interconnected clusters of cells called germ cell cysts³. Oogonia enter meiosis starting at E 13.5 to become oocytes and progress through prophase I to the diplotene stage. As the oocytes develop, the cysts break apart, allowing each oocyte to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte⁴. There is a loss of a large number of oocytes coincident with cyst breakdown, and this is believed to be important for quality control during primordial follicle formation. The number of diplotene arrested oocytes in primordial follicles makes up the ovarian reserve of oocytes available for reproduction and is directly related to fertility. If follicle formation is disrupted, this could lead to a smaller ovarian reserve, limiting female reproductive life span.

Whole mount immunostaining was used to identify and analyze the expression of proteins during embryonic, fetal, and neonatal oocyte development. Molecules examined include MSY2, SYCP3, ESR1, ESR2, CYP19A1, 3βHSD, KIT, and STAT3^5,6,7,8,9. This technique has also been used extensively to uncover how primordial follicle formation is regulated. Fetal or neonatal ovaries were cultured and exposed to signaling molecules or inhibitors. Oocytes in cultured ovaries undergo primordial follicle formation similar to in vivo¹⁰. Ovary culture has been successfully used to examine steroid hormone^6,10,11, KIT⁸, and PI3K signaling¹² during this stage of ovary development. Following culture, whole mount immunostaining is employed using antibodies against DEAD box helicase 4 (DDX4, also known as Mouse vasa homolog (MVH)), a protein expressed in the cytoplasm of oocytes. Ovaries are then examined by confocal microscopy to assess primordial follicle formation.

Protocol

All procedures using mice were reviewed and approved by the Syracuse University Institutional Animal Care and Use Committee (IACUC). The following protocol is for labeling neonatal mouse ovaries on post-natal days 1–5 (see Figure 1 for schematic). The reagents and the equipment used are listed in the Table of Materials.

1. Harvesting and fixing ovaries

1. Euthanize neonatal C57BL/6 mice following the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals” and IACUC-approved animal protocols. Dissect ovaries in 1x phosphate-buffered saline (PBS).
   NOTE: For this procedure, neonatal mice were euthanized by decapitation as approved in our IACUC animal protocol. Males and females of the C57BL/6 mouse strain can be distinguished in neonates by the presence of a small dark spot between the anogenital openings in males. To dissect the ovaries, first cut at the stomach and scoop out the intestines. Then, open up the abdomen with a midline incision to the tail, and then make two horizontal cuts. Locate the ovaries under each kidney, isolate each ovary, and clean off excess tissue.
2. Place ovaries directly into 600 μL 5.3% paraformaldehyde (PFA; 16% EM grade PFA diluted in 1x PBS) (fix) in a microfuge tube at room temperature (RT). Incubate the ovaries in fix on a nutator overnight (O/N) at 4 °C.
   ​CAUTION: Paraformaldehyde is toxic. Use in a fume hood or well-ventilated area. Wear gloves, eye protection, and protective clothing to prevent skin and eye contact. Dispose of as hazardous waste.
3. Remove fix and wash ovaries two times in 1 mL 0.1% Triton X-100 in 1x PBS (PT) quickly, then once in 1 mL PT on nutator for at least 30 min at RT. Incubate in 1 mL PT + 5% BSA (Block) for at least 30 min at RT on nutator or store at 4 °C for up to 2 weeks.

2. Incubating with primary and secondary antibodies

1. Incubate with primary antibody diluted in 500 μl PT + 5% BSA O/N at 4 °C on nutator. Wash three times in 1 mL PT + 1% BSA, 30 min at RT on nutator.
   NOTE: Dilution depends on the antibody. Representative data shown here is labeled with DDX4 diluted 1:200. Antibodies against two different proteins can be used at the same time as long as the primary antibodies are made in two different species and one secondary isn’t made in the species the other secondary recognizes (don’t use goat anti-mouse-Alexa 488 and donkey anti-goat-Alexa 568).
2. While incubating ovaries with primary antibody, incubate diluted secondary antibody with a pinch of embryo powder in 500 μL PT + 5% BSA O/N at 4 °C on nutator (covered with foil) to reduce nonspecific binding of the secondary antibody. Centrifuge the secondary antibody at 6000 x g for 30 s to collect the residue powder at the bottom. Make sure not to draw up this residue when transferring secondary to the sample tube.
   ​NOTE: Dilution depends on the antibody. Representative data shown here is labeled with goat anti-rabbit Alexa 488 diluted 1:200.
3. Incubate with pre-absorbed fluorescent secondary antibody (made in step 2.2) for 2–4 h at RT on nutator. Cover in foil from this point on. Wash three times in 1 mL PT + 1% BSA, for 30 min at RT on a nutator.

3. Labeling nuclei with DAPI and mounting ovaries on a slide

1. Incubate in DAPI 1:1000 in PBS for 90 min at RT. Wash once with 1 mL 1x PBS.
2. Transfer ovaries to a microscope slide using a wide 200 μL micropipette tip. Remove excess PBS with needle/syringe. Add 3 drops of mounting media onto the ovaries.
3. Gently apply a coverslip on top of the ovaries, ensuring the mounting media spreads to the edges of the coverslip. Seal edges of coverslip with nail polish and allow nail polish to dry, then store at -20 °C.

4. Preparation of embryo powder

1. Homogenize ~ E 12.5–E 14.5 mouse embryos in a minimum volume of ice-cold 1x PBS. Add 4 volumes of ice-cold acetone, mix, and incubate on ice for 30 min.
   ​CAUTION: Acetone is flammable and an eye and skin irritant. Use in a fume hood or well-ventilated area. Wear gloves, eye protection, and protective clothing to prevent skin and eye contact. Dispose of as hazardous waste.
2. Centrifuge at 10,000 x g for 10 min, remove the supernatant, and then wash the pellet with ice-cold acetone and spin again.
3. Spread the pellet out on a sheet of filter paper, place it into a plastic tray, and allow drying O/N in a chemical hood. Grind into a fine powder, allow to air-dry completely, and store at -20 °C.

5. Analysis of protein expression by confocal microscopy

1. After labeling for the protein of interest, visualize it by confocal microscopy. For examining protein expression, we typically use 3–4 animals (6–8 ovaries) and take several images of each ovary.
   ​NOTE: Images described in the representative results section were obtained on either a spinning disk confocal microscope or a confocal laser scanning microscope. Parameters used are described in the representative results section and will vary depending on the microscope used.

6. Analysis of primordial follicle formation and development by confocal microscopy

1. After labeling ovaries for a cytoplasmic oocyte marker such as DDX4, for each ovary, randomly assign two areas or “cores” for visualization. 
   NOTE: A core consists of 4 main optical sections that are 212 × 212 μm and are each separated by a depth of 15–20 μm. The 4 optical sections span both the cortex and medulla to ensure that all regions of the ovary are assessed. For each ovary, there will be 2 cores with 4 optical sections for a total of 8 sections (see Figure 2).
2. After imaging the main optical section for analysis, take a Z-stack of 5 images above and 5 images below the main section, each 1 μm apart. For each main section, count the total number of oocytes in the section
3. Then, for each oocyte in the main section, determine if it is associated with another oocyte or not by examining the main section and also the stack of sections to determine if oocytes in the main section were associated with oocytes above or below the plane of focus. Finally, for every single oocyte, determine what stage of follicle development it is (primordial, primary, or secondary).
   ​NOTE: It is recommended to analyze at least 4 animals (8 ovaries) to obtain statistically significant data.

7. Using propidium iodide as an alternative nuclear marker

1. After step 2.3, incubate for 30 min in 0.1 mg/ml RNase A (10 μL of 10 mg/ml in 1 mL of PT + 1% BSA). Remove RNase A and incubate in 10 μg/mL propidium iodide (10 μL of 1 mg/mL in 1 mL PT + 1% BSA) for 20 min at RT on a nutator. Cover the tube with foil from this point on.
   CAUTION: Propidium iodide is toxic and mutagenic. Use in a fume hood or well-ventilated area. Wear gloves, eye protection, and protective clothing to prevent skin and eye contact. Dispose of as hazardous waste.
   NOTE: Toto-3 can also be used as a nuclear marker.
2. Remove propidium iodide and wash for 30 min in 1 mL PT + 1% BSA. Skip step 3.1 (DAPI staining).

Results

Typical results from the immunostaining method described here are shown. Images in Figure 3, Figure 4, Figure 5, and Figure 6 were taken on a spinning disk confocal microscope using a 40x oil objective, NA 1.3, with an exposure time between 100–250 ms and laser power between 0.01 and 0.1%. In Figure 3, neonatal mouse ovaries are labeled with antibodies agai...

Discussion

This report describes a method to fluorescently label whole ovaries for proteins of interest. This procedure has been extensively used to determine the expression of proteins during primordial follicle formation and also to characterize changes in follicle formation under different conditions^4,5,6,7,8,9,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti Human VASA (made in goat)	R&D	AF2030
anti-DDX4/MVH (made in rabbit)	Abcam	ab13840
anti-GCNA1/TRA98 (made in rat)	Abcam	ab82527
Bovine Serum Albumin (BSA)	Fisher	BP-1600
Cover slips 22 x 22 #1.5	Warner Instruments	64-0721
DAPI (powder)	Sigma Aldrich	D-9542	prepare a 5 mg/ml solution in dH2O
Dissection scope
Donkey anti-Goat Alexa 568	Invitrogen	A11057
Donkey anti-Mouse Alexa 488	Invitrogen	A21202
Donkey anti-Rabbit Alexa 568	Invitrogen	A10042
Donkey anti-Rabbit Alexa 647	Invitrogen	A31573
Donkey anti-Rat Alexa 488	Invitrogen	A21208
Ethanol 200 proof	Pharmco	111000200
Fine Scissors - Sharp	Fine Science Tools	14060-09	for fine work removing ovary and trimming excess tissue
Forceps (Dumont Tweezer, Style 5)	Electron Microscopy Science	72705-01 (0209-5-PO)
Goat anti-Rabbit Alexa 488	Invitrogen	A11008
Microcentrifuge Tubes 1.5 mL	Thermo Scientific	3451PK
Microscope Slides	Fisherbrand	12-550-123
Nutating Mixer	LabNet	S0500
Paraformaldehyde 16%	Electron Microscopy Science	15710
Petri Dishes 100 x 15 mm	Bioplast Manufacturing	940-10774
Phosphate Buffered Saline 1X		(Fisher) S271-1;          (Sigma) P9541-500G; (Fisher) BP332-500; (Fisher) P285-500	800 mL MQ H2O               80 g NaCl                     2.0 of KCl                         14.4 g Na2HPO4          2.4 g KH2PO4          Adjust pH to 7.4 with HCl; final volume 1 L Then dilute from 10X to 1X for use
Pin holder	Fine Science Tools	26016-12	to hold straight needle bent in a slight curve
Pipette Filter Tips	OneTip	(10uL) 1121-3810;              (20 uL) 1120-1810;          (200 uL) 1120-8810;     (1000 uL XL) 1122-1830
Pipette tips	OneTIp	(10 uL) 1111-3800;          (200 uL) 1111-1800;     (1250 uL) 1112-1820
Pipettes 0.2-2, 2-20, 20-200, 100-1000	Gilson	(0.1-2 uL) F144054M;    (2-20 uL) F144056M;   (20-200 uL) F144058M;    (100-1000 uL) F144059M
Propidium iodide, 1mg/ml in dH2O	Invitrogen	P3566
RNase A powder	Sigma Aldrich	RG513-50mg	prepare a 10 mg/ml solution in 0.1 M sodium acetate (pH 5.2). Heat to 100 oC for 15 min. Cool to RT and adjust pH by adding 0.1 volume of 1 M Tris-Cl (pH 7.4)
Straight Needle			straight needle bent into a curve for severing excess tissue from the ovary
Triton X-100	Thermo Scientific	X100-100ML
Tuberculin Syringe 28G x 1/2	Tyco/Healthcare Kendall Monoject	1180128012
Vectashield	Vector Labratories	H-1900
Wagner Scissors	Fine Science Tools	14068-12	For decapitation, and larger cuts
Wide bore pipette tip 20 ul	LabCon	1026-965-008-9