RESUMEN
Chorionic gonadotropin (CG) is an early embryo-derived signal that is known to support the corpus luteum. An in vivo baboon model was used to study the direct actions of human CG (hCG) on the endometrium, during the periimplantation period. Endometrial gene expression was analyzed using microarrays. The endometrial biopsies were taken from hCG-treated (n = 5) and control (n = 6) animals on d 10 after ovulation. Class comparison identified 61 genes whose transcript levels differed between control and hCG-treated samples (48 increased, 13 decreased in mean expression level more than 2.5-fold; P < 0.01). Real-time PCR of transcript abundance confirmed up-regulation of several of these, including SerpinA3, matrix metalloproteinase 7, leukemia inhibitory factor (LIF), IL-6, and Complement 3 (P = 0.05). Analysis of protein abundance in endometrial flushings showed increased LIF and IL-6 protein in uterine flushings from hCG-treated animals compared with controls. Complement C3 and Superoxide dismutase 2 that were also up-regulated, were further evaluated by immunocytochemistry. Complement C3 showed a marked increase in stromal staining in response to hCG, whereas and superoxide dismutase 2 localization was most markedly increased in the glandular epithelial cells. Expression of Soluble Frizzled Related Protein 4, the most highly down-regulated gene, was also validated by PCR. Our experiments have shown that hCG induces alterations in the endometrial expression of genes that regulate embryo attachment, extracellular matrix remodeling and the modulation of the immune response around the implanting blastocyst. Several of these genes, including LIF and gp130, have been shown to be essential for implantation in other species. This study provides strong evidence that the preimplantation embryo itself influences the development of the receptive endometrium via secreted paracrine signals.
Asunto(s)
Gonadotropina Coriónica/fisiología , Implantación del Embrión/fisiología , Endometrio/metabolismo , Regulación de la Expresión Génica/fisiología , Expresión Génica , Papio/metabolismo , Animales , Gonadotropina Coriónica/farmacología , Complemento C3/metabolismo , Sistemas de Computación , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Interleucina-6/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/metabolismo , Superóxido Dismutasa/metabolismo , Distribución Tisular , Regulación hacia Arriba , Útero/metabolismoRESUMEN
Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in tissue localization and developmental stage. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Here we examined the possibility that interleukin-6 (IL-6), a trophoblastic and endometrial cytokine, represents such a regulatory factor. Purified first trimester cytotrophoblastic cells (CTB) were cultured for 4 days in presence or absence of increasing concentrations of IL-6. MMP-2 and MMP-9 bioactivity (zymography) and immunoactivity were measured in the culture supernatants together with total human chorionic gonadotrophin (HCG), fetal fibronectin (FFN) and leptin. IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG. In contrast, this cytokine induced a dose-dependent stimulation of the leptin secretion and increased the activity, but not the immunoreactivity, of MMP-9 and MMP-2. These results indicate that IL-6 could be considered as an endometrio-trophoblastic regulator of cytotrophoblastic gelatinases.
Asunto(s)
Interleucina-6/farmacología , Trofoblastos/efectos de los fármacos , Aborto Inducido , Células Cultivadas , Femenino , Humanos , Interleucina-1/metabolismo , Leptina/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Embarazo/metabolismo , Trofoblastos/enzimología , Trofoblastos/metabolismoRESUMEN
To investigate the role of leptin during pregnancy, we assessed leptin production by pure cultured human cytotrophoblastic cells (CTB), its regulation by cytokines and 17beta-oestradiol and its effects on human chorionic gonadotrophin (HCG) secretion. Purified CTB from first trimester placenta were incubated in duplicates in the presence or absence of cytokines or 17beta-oestradiol. Medium was harvested on day 2 and the culture stopped on day 4. Results were corrected for protein content of each individual well and expressed as percent of controls per day (mean +/- SEM). Basal CTB leptin production was 25.2 +/- 2.6 (ng/mg prot). In comparison with controls, leptin production was stimulated to 320 +/- 16% (P < 0.0001) and 195 +/- 3.2% (P < 0.0004) by 3 and 10 ng/ml of interleukin-1alpha respectively. 17beta-oestradiol 10(-6) to 10(-9) mol/l increased basal leptin production 5-9-fold, while 10(-5) mol/l had no such effect. Basal CTB HCG secretion was 5722 +/- 1055 (mIU/mg prot). There was a dose-dependent leptin-induced increase in HCG secretion (P = 0.0039) reaching a 5-fold increase with a leptin concentration of 1 microg/ml (P < 0.006). Gonadotrophin-releasing hormone (GnRH) 8.5 x 10(-8) mol/l significantly increased HCG secretion to 140 +/- 21% of controls (P = 0.031). Cetrorelix (0.1 microg/ml) inhibited leptin-induced HCG secretion (P = 0.0028).
Asunto(s)
Gonadotropina Coriónica/metabolismo , Estradiol/metabolismo , Interleucina-1/metabolismo , Leptina/metabolismo , Trofoblastos/metabolismo , Células Cultivadas , Femenino , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Embarazo , Proteínas Recombinantes/metabolismoRESUMEN
Circulating embryotoxic factors could be responsible for reproductive failures observed in patients suffering from recurrent spontaneous abortions (RSA) and endometriosis. The mouse bioassay has been widely used to detect such factors, since sera from these patients inhibit early embryonic development. This bioassay consists in the in-vitro culture of two-cell mouse embryos in the presence of different sera up to the blastocyst stage (72 h of culture). In the present study experiments were performed over long culture times (3-7 days), from two-cell to spreading stages, to determine the in-vitro effect of sera obtained from RSA or endometriosis patients, as well as the effect of interferon (INF)-gamma on embryo development. An embryotoxicity cut-off value of 45% blastocyst formation was established using control sera. When development to the blastocyst stage was considered only 25% of RSA and 20% of endometriosis sera were embryotoxic. However, all RSA sera significantly inhibited hatching (P < 0.05) and spreading stages (P < 0.01). IFN-gamma (10 micrograms/ml) (P < 0.001) did not impair early embryo development, but significantly inhibited blastocyst spreading. These observations suggest that culture to advanced embryonic stages increases the sensitivity of the bioassay and that IFN-gamma alters in-vitro peri-implantation mouse embryo development.