FOXL2 in Human Endometrium

Patients and Specimens

The patients (age range 25-42 years) who participated in this study were classified as (1) healthy women (n = 52), with regular menstrual cycle, undergoing laparoscopy for tubal sterilization or due to a benign, nonendometriotic ovarian cyst and (2) women with ovarian endometriosis (n = 38) who underwent laparoscopic surgery. The study was approved by the local Human Investigation Committee and informed consent was obtained from all patients before inclusion. A complete medical history was obtained and physical examination was performed for each patient. Exclusion criteria for the study included prior or current infections, endocrine disorders, or the use of hormonal treatment and GnRH analogs within the past 3 months. Patients included in the study were classified at stage III or IV endometriosis according to the American Society for Reproductive Medicine classification of endometriosis. ³⁰

Endometrial specimens were classified as proliferative or secretory according to the last menstrual period and confirmed by transvaginal ultrasound scans by a transvaginal probe at 4.5 to 7.0 MHz and by the histological criteria of Noyes et al. ³¹

Endometrial specimens were obtained by hysteroscopy while specimens of endometriotic tissue were collected during the laparoscopic approach. A fragment was immediately submerged in liquid nitrogen to allow subsequent RNA extraction and real-time polymerase chain reaction (RT-PCR) and another was fixed in paraformaldehyde for immunohistochemical analysis. In case of endometrioma, endometrial tissue samples were carefully stripped from the lining cyst wall avoiding taking any of the normal ovarian cortex, as confirmed by histological evaluation.

RNA Extraction and Complementary DNA Preparation

Total RNA was extracted from frozen tissue samples as follows. Briefly, frozen samples were disrupted and homogenized. Then, liquid nitrogen and ground tissue were transferred to an appropriately sized sterile tube, allowing the liquid nitrogen to evaporate and then were immediately processed for RNA extraction using the RNeasy Protect Mini kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany).

The RQ1 RNase-free DNase (Promega, Milan, Italy) was used to degrade single-stranded or double-stranded genomic DNA. Absence of residual genomic DNA was verified by polymerase chain reaction (PCR) on total RNA without reverse transcription (RT). Genomic human DNA was used as a positive control for PCR reactions. RNA quantity was assessed using an ND-1000 Nanodrop Spectrometer (Thermo Fisher Scientific, Wilmington, Delaware). RNA integrity was checked prior to downstream analysis with the FlashGel System (Lonza Group, Ltd, Switzerland) to verify total RNA quality and check for RNA degradation. Complementary DNA (cDNA) was generated from 1 μg of each RNA sample using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, California).

Polymerase Chain Reaction and Sequencing for FOXL2

Polymerase chain reaction was performed by adding 2 µL of each cDNA sample to a 20 µL reaction containing 1× Pfx amplification buffer, 2× enhancer solution, 1 mmol/L MgSO₄, 200 μmol/L deoxyribonucleotides, 300 nmol/L forward (5′-CGG CTT TGT CAT GAT GGC CAG-3′) and reverse (5′-GGC CAC GAG TTG TTG AGG AAG-3P′) primers suitable for amplifying the human FOXL2 gene, and 1 U of Platinum Pfx DNA polymerase (Invitrogen, Life Technologies Italia, Monza, Italy). Polymerase chain reaction cycling conditions were as follows: 5 minutes at 98°C; 35 cycles of 30 seconds at 98°C, 30 seconds at 60°C, and 1 minute at 72°C; 10 minutes at 72°C. Nuclease-free water was used as negative control. Polymerase chain reaction products were run on a 2% agarose gel for confirmation of predicted fragment size (624 bp) and, once purified, were labeled using dye terminator cycle sequencing (DTCS) kit (Beckman-Coulter, Milan, Italy) according to the manufacturer’s instructions. DNA sequence was determined using CEQ 8000 automated DNA analyzer (Beckman-Coulter, Milan, Italy).

Quantitative Real Time-PCR

Quantification of FOXL2 and activin A (INHBA) mRNA expression was measured in quadruplicate on 1/10 dilutions of each cDNA in 20 µL reactions using the 1× MESA GREEN qPCR MasterMix Plus for SYBR Assay No ROX (Eurogentec, Seraing, Belgium) on Rotor Gene 6000 (Corbett) with appropriate 300 nmol/L forward and reverse primers.

Nucleotide sequences of FOXL2 (NM_023067.3), INHBA (NM_002192.2), and the housekeeping hypoxanthine phosphoribosyltransferase 1 (HPRT1, NM_000194.2) genes were downloaded from GenBank library files. Polymerase chain reaction primers for real-time assays were designed using Beacon Designer 2.06 (Premier Biosoft International) or Primer3 software, giving special attention to primer length, annealing temperature, base composition, and 3′-end stability. FOXL2 primers were forward 5′-TCC AAT AAA AGA TGG TTT CGT CT-3′ and reverse 5′-TCT TCA GAT AGG GAG AGG GTG A-3′; INHBA primers were forward 5′-GAG ATC ATC ACG TTT GCC GAG-3′ and reverse 5′-GAA GAG CCA GAC TTC TGC ACG-3′; HPRT1 primers were forward 5′-CGT GAT TAG TGA TGA TGA ACC AG-3′ and reverse 5′-CGA GCA AGA CGT TGA GTC CT-3′. All the RT-PCR primer pairs used in this study span exon–exon junctions or are located on different exons, with the exceptions of those for the FOXL2 gene, which is encoded by a single exon.

During preliminary quantitative RT-PCR (qRT-PCR) assays, the optimal primer concentration was determined for each primer pair (300 nmol/L final). For each pair of primers, the efficiency of qRT-PCR (e%) and the correlation coefficients (R ² ) were determined using serial 1:4 dilutions of template cDNA.

Products were subsequently run on 2% agarose gel to check for size specificity and eventually sequenced. Rotor gene runs consisted of a hot start at 95°C for 5 minutes, followed by 45 cycles of a 2-step protocol: 10 seconds at 95°C and 30 seconds at 59°C, with fluorescence detection at the end of each extension step. Melt curve analysis was also performed to confirm specific products and standard curves were performed to confirm efficient amplification of each gene before final analysis of all samples. HPRT1 should be considered as reliable internal control gene for qRT-PCR analysis in this study based on geNorm analysis results, hence FOXL2 relative mRNA expression levels were determined by comparison to the HPRT1 and plotted as ratio to HPRT1 expression values. Final results were expressed as fold differences in gene expression relative to the normalized calibrator, calculated by the ΔΔCt method as follows: n-fold = 2^{−(ΔCtsample−ΔCtcalibrator)} where ΔCt values of the sample and calibrator were determined by subtracting the average threshold cycle (Ct) value of the transcript under investigation from the average Ct value of the HPRT1 gene for each sample. Raw Ct values were imported into GenEx Pro (version 4.3.5) and analyzed. All the primer pairs used in this study showed good overall efficiency (>96% and <106%) and excellent reproducibility of the amplification reactions.

Immunohistochemistry And Immunofluorescence

Immunohistochemistry was performed on 7-μm sections of paraffin-embedded samples including healthy endometrium, endometrium from patients with endometriosis, and endometrioma. Samples were washed in phosphate-buffered saline (PBS) 3 times, followed by endogenous peroxidase blockage by the use of 3% H₂O₂ in methanol solution. Again, samples were washed in PBS and incubated with 5% bovine serum albumin (BSA) in PBS for 1 hour to block nonspecific binding sites. The sections were incubated overnight at 4°C with the primary goat immunoglobulin (Ig) G anti-FOXL2 polyclonal antibody, diluted 1:40 in 0.1% BSA/PBS. Subsequently, sections were washed in PBS and then treated with HPR-conjugated antigoat IgG secondary antibody for 1 hour at room temperature (RT), followed by peroxidase streptavidin incubation in avidin–biotin complex reagent (Vectastain Elite Universal kit—Vector Laboratories, Burlingame, California). Diaminobenzidine (Sigma Chemicals CO, St Louis, Missouri) was subsequently used in order to visualize the immunolocalization of the primary antibodies. Tissue slices were counterstained with hematoxylin (Sigma Chemicals CO). Negative control slices were incubated with normal serum instead of primary antibodies.

For immunofluorescence, the samples were fixed in Histochoice (Amresco, Solon, Ohio) at RT for 4 hours. Sections were treated with 3% H₂O₂ for 1 hour and unmasked with citrate buffer solution (Thermo Fisher Scientific, Cheshire, United Kingdom) and 0.01 mol/L EDTA, pH 8. Slides were blocked with Dako Cytomation Protein Block Serum-free (Dako, Glostrup, Denmark) for 30 minutes at RT and then incubated overnight at 4°C with goat IgG anti-FOXL2 polyclonal antibody (ABCAM, Cambridge, United Kingdom) and mouse IgM anti-GMP-1 (SUMO-1) monoclonal antibody (Zymed Laboratories, San Francisco, California) diluted 1:40 and 1:20, respectively, in 0.1% BSA/PBS. After washing in PBS, the samples were incubated with secondary antibodies: Alexa Fluor 633 antimouse IgM and Alexa Fluor 488 antigoat IgG (Molecular Probes, Lifetechnologies Italia, Monza) diluted 1:500. Immunofluorescence analysis was performed using a Leica DMRB microscope (Leica Microsystems, Milano) equipped with fluorescence apparatus (488-633 excitation wavelength).

Statistical Analysis

The data were normally distributed and the results of the studies are reported as mean ± standard error of the mean. Statistical analysis was performed using the GenEx Pro and GraphPad Prism (GraphPad 4.0 Software Inc, San Diego, California). The selected reference gene HPRT1 was used to normalize Ct values, and quantities were calculated relative to the maximum Ct value. The comparison of 2 groups was performed with a Student t test while for analysis of more than 2 groups, a 1-way analysis of variance was performed, followed by the post hoc Tukey test. Correlation analyses were performed between FOXL2 and activin A expression levels, respectively, using the Pearson test. Statistical significance was set at P ≤ .05.

Sequencing of FOXL2 Gene and Protein Localization in Healthy Endometrium

FOXL2 gene expression in healthy human endometrium as well as in endometrium of patients with endometriosis was demonstrated by qRT-PCR. The PCR products were first sequenced to confirm the expression of FOXL2 mRNA by the comparison with the reference sequence (NM_023067.3) from the National Center for Biotechnology Information database (data not shown).

Quantitative RT-PCR was performed to quantify mRNA levels. FOXL2 mRNA was expressed in both the groups, throughout menstrual cycle without any significant difference (Figure 1). When FOXL2 protein expression was evaluated in healthy endometrial tissue by immunohistochemistry, a FOXL2 staining was observed in the cytoplasm of glandular cells with a scattered distribution in stroma (Figure 2, healthy panel). Double-fluorescence staining revealed a colocalization of FOXL2 with SUMO-1, consistent with FOXL2 protein being SUMOylated and stabilized in the cytoplasm (Figure 2, healthy panel). Negative controls did not stain at all.

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Figure 1.

FOXL2 mRNA expression in human endometrium quantified by qRT-PCR during different phases of the endometrial cycle of healthy individuals or patients with endometriosis (healthy proliferative [HP]; healthy secretory [HS]; endometriosis proliferative [EP]; endometriosis secretive [ES]). Fold change (y axis) represents FOXL2 expression normalized to HPRT1, relative to P phase of healthy endometrium considered to be equal to 1. Bars indicate the standard error of the mean (SEM). Statistical analysis was performed using t test. mRNA indicates messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; FOX, Forkhead box.

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Figure 2.

FOXL2 is expressed in human endometrium and colocalizes with SUMO-1. Representative micrographs of immunohistochemical staining of FOXL2 in human endometrium from women without (healthy panel) and with endometriosis (endometriosis panel) showed cytoplasmic staining in glandular and stromal cells. Immunofluorescence localization of FOXL2 and SUMO-1. Blue: DAPI; green: FOXL2; red: SUMO-1; yellow: merge (scale bars 100 µm). DAPI indicates 4′,6-diamidino-2-phenylindole; FOX, Forkhead box.

FOXL2 mRNA Expression And Protein Localization in Endometrium of Patients With Endometriosis and in Endometriotic Tissue

FOXL2 mRNA endometrial expression was not different in patients with endometriosis during endometrial cycle (Figure 1). Immunostaining of eutopic endometrium showed a cytoplasmic localization more concentrated in the glandular epithelium while diffuse in the stromal cells in endometriotic samples. SUMO-1–FOXL2 double staining showed a specific colocalization with the same pattern (Figure 2).

As shown in Figure 3, FOXL2 mRNA level was significantly higher in ovarian endometriosis than in healthy endometrium (2.63-fold induction, P < .05) or in eutopic endometrium with endometriosis (3.27-fold induction, P < .01; Figure 3, panel A).

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Figure 3.

FOXL2 mRNA expression in healthy endometrium, eutopic endometrium from women with endometriosis, ectopic endometrium from women with endometrioma. A, Fold change (y axis) represents FOXL2 expression normalized to HPRT1, relative to healthy endometrium considered to be equal to 1. A statistical significance was found for endometrioma when compared with healthy endometrium (*P < .05) and endometrium from endometriosis (°P < .01). Bars indicate the standard error of the mean (SEM). Statistical analysis was performed using t test. FOXL2 protein localization in endometriotic lesions. B, Representative micrographs of immunohistochemical staining of FOXL2 in endometrioma, cytoplasmic FOXL2 staining was observed in glandular and stromal cells (A, scale bar 100 µm; B, scale bar 25 µm). mRNA indicates messenger RNA; HPRT1, hypoxanthine phosphoribosyltransferase 1; FOX, Forkhead box.

The presence of FOXL2 protein in endometriotic lesions was confirmed by immunohistochemistry (Figure 3, panel B). FOXL2 mRNA expression levels correlated with the activin A expression in endometriosis (r = .635).