Collection of ovarian samples
A total of 27 small samples of human ovarian tissue were collected and analyzed from pre- and post-pubertal girls entering a program of ovary cryopreservation. All patients had suffered extra-gonadal malignant disease and 8 of them received chemotherapy before surgery (Table 1). In all these 8 patients, samples were taken one month after the end of treatment. At the time of cryopreservation, the patients were aged between 7 and 19 years. Pre- and post-pubertal ovarian tissue samples were obtained by laparoscopic surgery at the “Hospital de Niños Dr. Ricardo Gutiérrez”, Buenos Aires, Argentina, including pre-menarcheal patients of 7 (n = 1), 9 (n = 3), 10 (n = 2), 11 (n = 1) and 12 (n = 1) years old and post-menarcheal patients of 12 (n = 2), 13 (n = 3), 14 (n = 5), 15 (n = 4), 16 (n = 2), 17 (n = 1), 18 (n = 3) and 19 (n = 1) years old (Table 1). The hospital submitted anonymized medical records with samples indicating the menarcheal condition of the patients. Samples were grouped into patient who received (Group 1; treated-patients 4, 5, 10, 11, 13, 23, 26, 27) or not (Group 2; untreated-patients 1–3, 6–9, 12, 14–22, 24, 25) chemotherapy before surgery (Table 1). Ovarian tissue collected at the time of surgery under sterile conditions was preserved in 10% formaldehyde until embedded in paraffin, serially sectioned at 5 μm thickness, mounted onto cleaned slides, and kept at room temperature until used. When possible (Table S1), two small fresh-tissue fragments were preserved either in an RNAse/DNAse-free sterile cryotube or in 1 ml RNA later (QiaGen, Ambion Inc., Austin, TX, USA) and stored at -80 °C until used. The present study was reviewed and approved by the Institutional Research Ethics Committee, Universidad Maimónides, Buenos Aires, Argentina, and Research Ethics Committee from the collaborating hospital. Samples were utilized only upon obtaining informed consent from patients or, in cases where patients were under the age of sixteen, from their respective parents or legal representatives, in compliance with Argentinean regulations.
Immunohistochemistry
Immunohistochemistry was performed according to Albamonte et al. [24] with some modifications. Briefly, mounted paraffin sections were dewaxed in xylene, rehydrated in graded alcohols, and washed in distilled water. Endogenous peroxidase activity was inhibited with 0.5% H2O2/methanol (v/v) for 20 min at room temperature. The sections were treated for one hour with 15% normal horse serum or normal rabbit serum in phosphate-buffered saline (PBS) followed by overnight incubation at room temperature with primary antibodies that were diluted 1:100. The primary antibodies used were mouse monoclonal anti-FKHRL1 (FOXO3) (sc-48348), goat polyclonal anti-p-FKHRL1 (pFOXO3) (sc-12357), and mouse polyclonal anti-PTEN (sc-7974), all from Santa Cruz Biotechnology, Dallas, TX, USA. After the incubation period, the slides were rinsed three times in PBS and incubated for one hour with the appropriate 1:200-diluted biotinylated secondary antibody (Vector Labs, Peterborough, UK) at room temperature. The sections were washed again in PBS and then incubated for 30 min with 1:100 diluted streptavidin-peroxidase complexes (ABC kit, Vector Labs, UK). Following this step, the sections were washed twice with PBS and the peroxidase activity was detected with 0.05% 3,3´-diaminobenzidine (w/v) and 0.1% H2O2 (v/v) in Tris–HCl. Finally, the sections were washed with distilled water and mounted in Canada balsam (Biopack, Buenos Aires, Argentina). Negative controls were processed simultaneously by omitting the primary antibody and/or preincubating the primary antibody with the specific commercial synthetic peptide.
Sections were examined in an Olympus BX40 microscope. To determine the prevalence of primordial follicles expressing or not the antibody used and their cellular localization, the entire processed sections were screened and all primordial follicles counted.
Double immunofluorescence
Mounted paraffin sections were dewaxed in xylene, rehydrated in graded alcohols, and washed in distilled water. Sections were then blocked for 1 h with bovine serum albumin 15% + bovine fetal serum 10% (w/v) in PBS + tween 0,1% (v/v) and incubated overnight at room temperature with the 1:100 diluted primary antibody mouse polyclonal anti-PTEN (sc-7974). The next day, sections were then washed twice with PBS and incubated for 1 h with secondary antibody anti-mouse Alexa Fluor 488 (1/100, Life Technology). Sections were then washed twice with PBS and incubated overnight at room temperature with the 1:100 diluted primary antibody goat polyclonal anti-p-FKHRL1 (pFOXO3) (sc-12357). The next day, sections were washed twice with PBS and incubated for 1 h with secondary antibody anti-goat Alexa Fluor 555 (1/100, Life Technology). Finally, sections were washed with distilled water and mounted in Vectashield Mounting Medium with DAPI (Vector Laboratories). Negative controls were processed simultaneously by omitting the primary antibody and/or preincubating the primary antibody with the specific commercial synthetic peptide. Sections were examined in a confocal Eclipse Ti microscope (Nikon Instruments Inc. NY, USA).
Western blot analysis of PTEN
This procedure was performed according to Albamonte et al. [24] with some modifications. Ovarian fragments preserved at -80°C were homogenized in ice-cold lysis buffer containing a protease inhibitor cocktail [0.5 mM phenylmethylsulfonyl fluoride (PMSF); 10 mM leupeptin; 10 mM pepstatin; 10 mM aprotinin], and centrifuged at 1.200 g at 4°C for 10 min. The supernatant was collected and proteins were quantified using the Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total proteins (20 µg) from tissue extracts were separated by one-dimensional SDS-PAGE 15% and then transferred onto polyvinylidene fluoride (PVDF) membrane (Amersham Hybond-P, GE Healthcare). The membrane was then blocked for 1 h in PBS + 0.1% Tween20 with 5% nonfat dry milk. After that, it was incubated for 1 h at room temperature with the mouse polyclonal anti-PTEN diluted 1/100 (A2B1: sc-7974 Santa Cruz Biotechnology. Dallas, TX, USA). After washing, the membrane was incubated with a goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Bio-Rad, 1:5000). The immunoreactive product was visualized using the enhanced chemiluminescence system ECL plus GE (Amersham, Fairfield, Connecticut, USA) and Image Quant 350. To confirm equal loading, each membrane was analyzed for β-actin protein expression demonstrating that the band intensities did not show significant changes between the samples analyzed. Briefly, the membrane was incubated with mouse monoclonal anti-β-actin (Sigma, Saint Louis, Missouri, USA) diluted 1/1000. After washing, the membrane was incubated with a goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Bio-Rad, 1:5000). Stained protein molecular weight markers were used as standards (Fermentas, Vilnius, Lithuania). Densitometry was performed on Scion Image for Windows software (Scion Corporation, Frederick, MD, USA) and PTEN expression was normalized to β-actin. The human uterine fibroblast (HUF) cell line was utilized as a positive control for PTEN expression [25].
RNA isolation and real time-PCR
The present protocol was performed according to Albamonte et al. 2020 [24] with some modifications. Samples recovered in RNA later in the surgery room were maintained in that solution for 48 h and then stored a -80 °C until used. RNA was extracted from the ovary using Trizol (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Subsequently, DNAse I (Invitrogen, Waltham, MA, USA) was applied to 3 μg of total RNA, followed by reverse transcription using a 20 μl-reaction containing M-MLV reverse transcriptase (200 U/μl, Promega, Madison, WI, USA) and random hexamers primers (Biodynamics, Buenos Aires, Argentina). Finally, quantitative polymerase chain reaction (PCR) was conducted utilizing reverse-transcribed cDNA, specific forward (F) and reverse (R) primers, and SYBR Green PCR Master Mix in a Stratagene MPX500 cycler (Stratagene, La Jolla, CA, USA). Specific primers used were: PTEN; F 5´- CCAATGTTCAGTGGCGGAACT-3´, R 5´- GAACTTGTCTTCCCGTCGTGT-3´; FOXO3: F 5´-TCTACGAGTGGATGGTGCGTT-3´, R 5´- CGACTATGCAGTGACAGGTTGT-3´; ACTIN: F 5´- CTTCCCCTCCATCGTGGG-3´, R 5´- GTGGTACGGCCAGAGGCG-3´). Primers were used at a concentration of 0.3 μM in each reaction. The cycling conditions were as follows: step 1, 10 min at 95 °C; step 2, 15 s at 95 °C; step 3, 30 s at 60 °C; step 4, 30 s at 72 °C, repeating steps 2 to 4 forty-five times. Data from the reaction were collected and analyzed by the complementary computer software (MxPro3005P v4.10 Build 389, Schema 85, Stratagene, La Jolla, CA, USA). Melting curves were run to confirm the specificity of the signal. Relative quantitation of gene expression was performed using standard curves and normalized to β-actin in each sample. To evaluate quantitative differences in the cDNA target among samples, we employed the Pfaffl mathematical model. The expression ratio was determined for each sample by calculating (Etarget)ΔCt(target)/(EGβACTIN)ΔCt(βactin), where E is the efficiency of the primer set and CT is threshold cycle with ΔCt = Ct (normalization cDNA)—Ct (experimental cDNA). The amplification efficiency of each primer set was calculated from the slope of a standard amplification curve of log (ng cDNA) per reaction vs. Ct value (E = 10-(1/slope)). Efficiencies of 2 ± 0.1 were considered optimal.
Statistical analysis
Analysis of the data entailed calculating the mean and standard error (SEM), with one-way analysis of variance being conducted using InfoStat Software (Version 2012, Grupo InfoStat, Universidad Nacional de Córdoba, Córdoba, Argentina). A log10 transformation of the data was performed, and Tukey’s test was conducted when comparing differences between more than two groups. A p-value of less than 0.05 was considered statistically significant.
