Reagents and chemicals
Deoxyribonuclease I, collagenase (Type IV), and 5-bromo-2′-deoxyuridine (Brdu) were purchased from Sigma Aldrich (St Louis, MO). Glycine, N, N-dimethyl formamide, and other general chemicals were of tissue culture grade and purchased from Fisher Scientific (Nepean, ON). Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM) alpha, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), dithiothreitol (DTT), propidium iodide (PI), 4′, 6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin D (7-AAD), and 5-chloromethylfluorescein di-b-D-galactopyranoside (CMFDG) kit were purchased from Invitrogen (Burlington, ON). Bovine serum albumin (BSA) and fetal bovine serum (FBS) were purchased from Wisent (St-Bruno, QC), Hoechst 33342 was purchased from Roche (Laval, QC), Tween-20 from Bio Basic Inc (Markham, ON), mowiol from Calbiochem (La Jolla, CA), and N–Hydroxysulfosuccinimide (Sulfo-NHS)-biotin from Pierce Thermo Fisher (Nepean, ON). Strepavidin phycoerythrin-Cy5 (PE-Cy5) was obtained from Biolegend (San Diego, CA). The mouse monoclonal antibodies against cytokeratin-8 (TROMA-1) and BrdU (G3G4) were obtained from Developmental Studies Hybridoma Bank (DSHB; Iowa City, IA), Cy3-labeled goat anti-mouse IgG antibody (Jackson Immuno Research, West Grove, PA) and avidin-FITC (EY Laboratories, San Mateo, CA) were obtained through Cedarlane Laboratories (Burlington, ON). The In Situ Cell Death Detection Kit, Fluorescein was purchased from Roche (Laval, QC)
All animal procedures followed the guidelines established by the Canadian Council of Animal Care and approved by the Animal Care Committee of the Royal Victoria Hospital, McGill University. CD1 mice bearing the β-catenin/Tcf-responsive lacZ reporter gene (TopGal mice) have been described . Dr. Daniel Dufort (Department of Obstetrics and Gynecology, McGill University, Montreal, Canada) provided us with female and male CD1 mice, homozygous for the transgene, for colony establishment. Dr Makoto C. Nagano (Department of Obstetrics and Gynecology, McGill University, Montreal, Canada) provided us with wild type CD1 mice. Female mice were examined daily for vaginal plugs. The day of plug detection was considered day 0.5 of gestation and the day after birth designated postnatal day 1 (P1). Gonads were isolated from a minimum of three mice for analysis and all experiments replicated at least thrice.
Mice were sacrificed by cervical dislocation. Gonads were isolated, washed in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and fixed for 5–15 min in freshly prepared 4% paraformaldehyde (PFA) in PBS. After rinsing in wash buffer (PBS containing 2 mM MgCl2, 0.1% Triton, 0.05% sodium deoxycholate), gonads were stained in the dark overnight at 37°C in wash buffer supplemented with 1 mg/ml X-gal, 0.04% N, N-dimethyl formamide, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide to disclose β-galactosidase activity . Following staining, gonads were washed in PBS and photographed or processed for histology.
X-gal-stained ovaries were post-fixed in 4% PFA overnight at room temperature (RT), rinsed in PBS, and embedded in paraffin. Sections were cut 6 μm thick, mounted on glass slides and counterstained with hematoxylin and eosin. In studies where OSE cell numbers were estimated, sections were stained with periodic acid Schiff (PAS) to define the basement membrane and counterstained with Hoechst 33342 to identify cell nuclei. The total number of OSE cells was estimated by applying the nucleator and fractionator principle described by Gundersen . Only cells with large visible nuclei were counted. Every fourth ovary section was evaluated and an estimate of the total number of OSE cells per ovary determined by multiplying the cell counts by four. LacZ-positive OSE cells were determined by evaluating every section of the ovary since these cells were not uniformly distributed.
OSE labeling and isolation
OSE cells were isolated as follows: ovaries (2 ovaries/0.5 ml DMEM) were placed in a 1.5 ml capped tube and incubated for 60 min at 37°C in DMEM containing 1 mg/ml Type IV collagenase, 1 mg/ml deoxyribonuclease I, and 0.53 mM EDTA. Ovaries were agitated every 10 min by swirling the tube for a few seconds. Released cells were transferred to a fresh tube. The ovaries were rinsed in fresh DMEM and additional released cells combined with the previously isolated cell suspension. The cell suspension was vortexed and cells pelleted by centrifugation at 500 g for 5 min. The cell pellet was washed with PBS and resuspended in PBS.
We needed to confirm that our isolation procedure yielded primarily OSE cells with minimal contamination by other ovarian cells. Additionally, we wanted to assess the efficiency of the OSE isolation procedure. This was accomplished by labeling OSE cells in situ. We took advantage of a water-soluble and membrane impermeable biotinylation reagent (Sulfo NHS-biotin) that reacts chemically with exposed amine groups of cell surface proteins. Intact ovaries were incubated in 1 mg/ml Sulfo NHS-biotin in PBS for 1 min at 4°C. The reaction was quenched by incubating the ovary in ice-cold PBS containing 0.1 M glycine for 1 min. Selective labeling of OSE cells was confirmed by preparing sections of NHS-biotin-labeled ovaries followed by incubation with avidin-conjugated FITC (1:200 in PBS). Sections of enzymatically-treated ovaries were also examined for the extent of OSE removed by avidin-FITC staining.
Cytokeratin 8 (CK8) staining was performed on paraffin-embedded sections and isolated OSE cells. Six μm thick paraffin tissue sections were deparaffinized with xylene and rehydrated in graded ethanol. Antigen retrieval was performed by boiling the sections in 10 mM sodium citrate buffer, pH 6.0, for 25 min. After rinsing in PBST (PBS + 0.5% Tween), nonspecific binding was blocked for 30 min in blocking solution (5% BSA in PBST). Sections were incubated with primary antibody (TROMA-1) in blocking solution at 4°C overnight. The primary antibody was omitted for negative control slides. Slides were subsequently rinsed in PBST, incubated with Cy3-labeled goat anti-mouse antibody diluted 1:200 in blocking solution for 60 min in the dark, counterstained with DAPI, and mounted in mowiol.
Isolated OSE cells (50,000 cells/ml) were cytospun onto slides, fixed in 4% PFA for 5 min, and processed for CK8 staining as described above. Cell counts were made in five microscopic fields and approximately 100 cells were counted per field.
Fluorescence-activated cell sorting (FACS) analysis
LacZ expression in isolated OSE cells was detected using the DetectaGene Green CMFDG LacZ gene expression kit as outlined by the supplier (Invitrogen). OSE cells were washed in PBS and incubated with pre-warmed 0.1 M CMFDG in PBS at 37°C for 15 min. PI was added to label dead cells and FACS analysis performed on a Becton-Dickinson FACScan. Wild type CD1 OSE cells were used as controls. Dual parametric analysis of forward versus side scatter was the primary gate for identification of cells in the appropriate size range and to eliminate cell debris.
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining
Six μm thick paraffin-embedded lacZ-stained ovary sections from each age under investigation were examined for the presence of fragmented DNA indicating apoptosis using the In Situ Cell Death Detection Kit, Fluorescein, according to the manufacturer’s instructions (Roche). Sections were counterstained with PI to label cell nuclei and mounted in mowiol.
A solution of 1 mg 5′-bromo-2′-deoxyuridine (BrdU)/100 μl PBS was freshly prepared and filter sterilized (0.45 μm filter). Mice received BrdU by injection (i.p.) of 100 μl of sterile preparation. Ovaries were collected 48 hrs later and stained for X-gal, post fixed in 4% PFA overnight and embedded in paraffin. To detect proliferating cells, 6 μm thick paraffin sections were analyzed using a monoclonal mouse antibody (DSHB) specific for BrdU. Sections were dewaxed, washed in PBST (PBS + 0.5% Tween 20) and incubated with 2 N HCl at RT for 1 hr. Subsequently, slides were rinsed 3x5 mins in PBST and blocked in a blocking solution (3% BSA in PBST) for 30 mins. Sections were then incubated with anti-BrdU antibody diluted 1:100 in blocking solution at 4°C overnight. Following primary antibody incubation, slides were rinsed 3×5 mins in PBST, incubated with an FITC-labeled goat anti-mouse secondary antibody diluted 1:100 in blocking solution overnight at 4°C, counterstained with DAPI, and mounted in mowiol.
Total RNA was isolated from FACS-sorted OSE cells using the miRNeasy Mini kit (Qiagen, Toronto, ON) and incorporating on-column RNase-free DNase digestion. Quantity and quality of mRNA samples were assured by analysis with the Thermo Scientific NanoDrop 2000 (Thermo Scientific, Wilmington, DE). To survey the expression of wnt signaling components, total RNA (100 ng) was reverse transcribed using M-MLV reverse transcriptase (Invitrogen). Polymerase chain reactions (PCRs) were conducted as described previously . Wnts for RT-PCR analysis were selected based on previous studies [15, 19]. A PCR reaction for a known housekeeping gene, GAPDH, was generated as an internal control. The annealing temperature for each primer pair was optimized using positive control tissues to generate single bands corresponding to correct product sizes. Information regarding positive control tissue in which the genes of interest are expressed was obtained from: (i) Mouse Genome Database (MGD) (http://www.informatics.jax.org 09/2009), RT-PCR database and included brain (for wnt2, wnt 4, wnt 7a, wnt 8a, wnt 11), eye (wnt5a), testis (wnt3a); and (ii) a previous study . The RT-PCR protocol was performed on two separate RNA preparations. For each sample, a RT-minus control was included to provide for a negative control for subsequent PCR. All minus RT controls were negative. PCR products were visualized on a UV transilluminator after electrophoresis on a 1% agarose gel in TAE buffer (40 mM Tris, 1 mM EDTA and 20 mM acetic acid) and SafeView nucleic acid staining.
Side population (SP) analysis
OSE cells were labeled with DyeCycle Violet (DCV) according to a modified protocol . Briefly, OSE cells were suspended in the appropriate medium (DMEM containing 2% FBS and 2 mM HEPES) at 1 x 106 cells/ml. Before DCV incubation, cells were pre-incubated for 15 mins with or without 50 μM verapamil. DCV was added to the cell suspension at a final concentration of 5 μM and the mixture incubated for 30 minutes at 37°C in the dark. Propidium iodide was added to a final concentration of 1 μg/mL to identify dead cells. FACS analysis and sorting were performed on a dual laser flow cytometer (Becton Dickinson). The SP was defined as described previously .
OSE cells isolated from postnatal day 5 (P5) to P9 mice were grown in MEM alpha  supplemented with 4% FBS and 20 U/ml PenStrep at a density of ~3000 cells/well in 96-well plates. The cells were grown in a humidified incubator at 37°C and 5% CO2.
Data were analyzed using SYSTAT 10.2 statistical software (SYSTAT Software, Richmond, CA). Analysis of variance (ANOVA) was used with Tukey’s test in the post hoc analysis for cell counts to ascertain group mean differences. Data for the percentage of lacZ-positive OSE cells obtained following CMFDG labeling was subjected to ANOVA after arcsine transformation . Data are presented as mean ± standard error of the mean. P ≤ 0.05 was considered significant.