Cells were diluted 1:1 in Trypan blue (Sigma-Aldrich, Italia) and counted. Cell cycle and cell death Analysis was performed in duplicate. 100.000 cells were re-suspended in the staining solution containing RNAse A, Propidium Iodide (PI) (50 mg/mL), MK0683 sodium citrate (0.1%), and NP40 (0.1%) in PBS 1X for 30 min in the dark and room temperature. Cell cycle distribution was assessed with an FACScalibur flow cytometer (Becton Dickinson), and 10,000 cells were analyzed by ModFit version 3 Technology (Verity) and Cell Quest (Becton Dickinson) . RNA, RT-PCR Total RNA was extracted with TRIzol (Life selleck Technologies) and converted into cDNA using SuperScript VILO kit according
to the manufacturer’s protocol. (Invitrogen). Converted cDNA was amplified using EuroTaq (Euroclone). selleckchem Amplified DNA fragments were loaded on 2.0% agarose gel and photographed on a Gel Logic 200 Imaging system
UV transilluminator (Kodak). Levels of AMH, AMH type II Receptor (AMHR-II) and CYP19 expression were quantified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Real-Time PCR was performed using iQ_ SYBR_ Green Supermix (Bio-Rad) in a DNA Engine Opticon2 thermal cycler (MJ Research Incorporated). Primers: AMH gene (1) (Forward 5′-CAC CCG CTA CCT GGT GTT AG-3′, Reverse 5′-GGT CAT CCG TGT GAA GCA G-3′). AMH gene (2) (Forward 5′-AAG CTG CTC ATC AGC CTG TC-3′, Reverse 5′-TGG GGT CCG AAT AAA TAT GG-3′). AMHR-II gene (1) (Forward 5′-CCC TGC TAC AGC GAA AGA AC-3′, Reverse
5′-ATG GCA ACC AGT TTT CCT TG-3′). AMHR-II gene (2) (Forward 5′-AAC TGG CCT ATG AGG CAG AA-3′, Reverse 5′-GGT CTG CAT CCC AAC AGT CT-3′). GAPDH gene (Forward 5′-GGA GTC AAC GGA TTT GGT CGT-3′, 3-oxoacyl-(acyl-carrier-protein) reductase Reverse 5′-GCT TCC CGT TCT CAG CCT TGA-3′). Results Histologic examination of endometriosis lesions of the rectovaginal septum showed the typical presence of both endometriotic glands and stroma. Immunohistochemical staining demonstrated that both epithelial and stromal component expressed significant levels of AMH. Figure 1 depicts some exemplary cases of the immunohistochemical staining for AMH in cases of endometriosis of the rectovaginal septum. Figure 1 Immunohistochemical expression of AMH in endometriosis tissues. (A) AMH expression in the epithelium of an endometriosis gland (Original magnification X20). (B) The immunohistochemical expression of AMH is clearly visible also in the stromal cells of the endometriosis gland (Original magnification X20). We were able to demonstrate the effects induced by Recombinant Human Mullerian-Inhibiting Substance (rhMIS)/anti-Mullerian hormone (AMH)E. Coli derived on endometriosis stromal and epithelial cell growth, cell cycle progression and apoptosis induction. We have treated cultured human endometriosis stromal and epithelial cells with rhMIS at different concentrations (10-100-1000 ng/mL) and analyzed the effects induced after 24-48-72 hours of treatment.