BackgroundEndometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1.ResultsUpstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified.ConclusionThese data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

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The role of the tumor necrosis factor (TNF) system in endometriosis is not completely clear and therefore was the focus of this study. In eutopic endometria obtained from women with (n = 41) and without (controls; n = 34) endometriosis during laparoscopy, the subcellular location and the percentages for TNF, TNFR1, TNFR2 and CD45 positive-cells were determined (immunohistochemistry); the protein concentration (ELISA) of the soluble receptors (sTNFR1 and sTNFR2) were measured in 4h-explants culture media and the TNF concentration was performed in ex vivo endometrial homogenates. The TNF, TNFR1 and TNFR2 mRNAs were analyzed by real-time PCR. In control endometria, TNF, TNFR1 and sTNFR1 proteins increased during the late secretory phase. In endometriosis endometria, the protein highest level of TNFR1 was reached during the early and the mid secretory phases and of sTNFR1 concentration during the proliferative phase; however, during the late secretory phase, both TNFR1 and sTNFR1 protein levels were reduced compared to the control endometria (p < 0.05). TNFR2 was scarcely immunodetected in some CD45-positive stromal cells. The TNFand CD45-positive cell percentages, the TNF and sTNFR2 concentrations, and TNF and TNFR2 mRNAs were similar in control and endometriosis endometria. Although TNFR1 mRNA was highly expressed, no significant differences were found between control and endometriosis endometria. In summary, this study establishes that TNF and TNFR1 protein expressions and the sTNFR concentrations in eutopic endometria from women with and without endometriosis are dependent on the menstrual cycle. The differences on the TNFR1 and sTNFR1 protein pattern between both groups, mainly during the mid and late secretory phases may play a role in the lower implantation rates observed in women with endometriosis, and also could be related to the altered cell death, which favor the augmented cell viability facilitating the characteristic endometrial implantation and growth outside the uterus in this disease.

RESUMENEl presente estudio tuvo por objetivo inmunolocalizar la distribución de las células enteroendocrinas L, productoras del péptido similar al glucagón (GLP-1), en el intestino de alpacas. Se utilizaron 36 crías de alpaca de 0-45 días de edad y adultas, distribuidas en siete grupos etarios. Se tomaron muestras de intestino delgado y grueso que fueron procesados mediante inmunohistoquímica, empleando el anticuerpo monoclonal anti-GLP-1. Se contó el número de células positivas por eje cripta-vellosidad de cada porción intestinal en cada animal. En el grupo de recién nacidos (RN), el yeyuno fue la porción que contenía más células L, mientras que en los demás grupos fue en el yeyuno e íleon. Respecto a la ubicación, en el duodeno se aprecia un aumento sostenido de células L a partir de los 8 días. El mayor número de células L en el yeyuno y el íleon se presentó en los grupos etarios de RN y 35-45 días de edad, respectivamente, mientras que en el ciego y en el colon se observó en el grupo de animales adultos. Se concluye que las células L en alpacas se encuentran presentes desde el nacimiento, mayoritariamente en yeyuno e íleon.Palabras clave: GLP-1, intestino, células L, alpaca ABSTRACTThe aim of this study was to immunolocalize the distribution of producer enteroendocrine cells of glucagon-like peptide (GLP-1) on the epithelial intestine of alpacas. A total of 36 young (0-45 days old) and adult alpacas were used and distributed in seven age groups. The intestinal samples were processed by immunohistochemistry using an

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