RESUMEN
Humans have evolved along with the millions of microorganisms that populate their bodies. These microbes (10(14)) outnumber human cells by 10 to 1 and account for 3 × 10(6) genes, more than ten times the 25,000 human genes. This microbial metagenome acts as our "other genome" and like our own genes, is unique to the individual. Recent international efforts such as the Human Microbiome Project (HMP) and the MetaHIT Project have helped catalog these microbial genomes using culture-independent, high-throughput, next-generation sequencing. This manuscript will describe recent efforts to define microbial diversity in the female reproductive tract because of the impact that microbial function has on reproductive efficiency. In this review, we will discuss current evidence that microbial communities are critical for maintaining reproductive health and how perturbations of microbial community structures can impact reproductive health from the aspect of infection, reproductive cyclicity, pregnancy, and disease states. Investigations of the human microbiome are propelling interventional strategies from treating medical populations to treating individual patients. In particular, we highlight how understanding and defining microbial community structures in different disease and physiological states have lead to the discovery of biomarkers and, more importantly, the development and implementation of microbial intervention strategies (probiotics) into modern day medicine. Finally this review will conclude with a literature summary of the effectiveness of microbial intervention strategies that have been implemented in animal and human models of disease and the potential for integrating these microbial intervention strategies into standard clinical practice.
RESUMEN
Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.
Asunto(s)
Ciclo Celular , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Leiomioma/patología , Miocitos del Músculo Liso/patología , Actinas/metabolismo , Proliferación Celular , Colágeno/química , Citoesqueleto/metabolismo , Femenino , Colágenos Fibrilares/química , Colágenos Fibrilares/metabolismo , Adhesiones Focales/metabolismo , Humanos , Leiomioma/metabolismo , Sistema de Señalización de MAP Quinasas , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Uterine leiomyomas, or fibroids, are the most frequent gynecological tumors in premenopausal women with as many as 65% of women becoming clinically symptomatic. Uterine fibroids are benign myometrial tumors that produce large quantities of extracellular matrix proteins. Despite its high morbidity, the molecular basis underlying the development of uterine leiomyomas is not well understood. Domestic hens of Gallus gallus domesticus develop oviductal leiomyomas similar to those found in humans. We investigated the natural history of chicken leiomyomas, in vivo expression of protein biomarkers, and in vitro expression of ovarian steroid receptors. Based on the analysis of 263 hens, tumor prevalence, tumor number per hen, and tumor size increased as the hens aged. Immunohistochemistry for alpha-smooth muscle actin (SMA) and desmin confirmed the smooth muscle phenotype of the chicken leiomyomas. Intense collagen expression was detected in these oviductal leiomyomas by Mason's trichrome, and the tumors also showed increased expression of TGFB3 and collagen type I mRNAs. Consistent with human leiomyomas, chicken fibroids displayed increased BCL2 and estrogen (E) and progesterone (P) receptor expression. Chicken leiomyomas were dissociated for in vitro culture. Cells from explants were positive for SMA, desmin, and E and P receptors until the fourth passage. These cells also displayed a response similar to human cells when challenged with halofuginone, an antifibrotic agent. Our findings indicate that the chicken is an excellent complementary model for studies involving the pathophysiology of human uterine leiomyomas.
Asunto(s)
Envejecimiento/fisiología , Pollos , Modelos Animales de Enfermedad , Leiomioma/patología , Neoplasias Uterinas/patología , Envejecimiento/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Leiomioma/tratamiento farmacológico , Leiomioma/epidemiología , Leiomioma/veterinaria , Oviductos/patología , Piperidinas/farmacología , Piperidinas/uso terapéutico , Enfermedades de las Aves de Corral/tratamiento farmacológico , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/etiología , Enfermedades de las Aves de Corral/patología , Prevalencia , Cultivo Primario de Células , Quinazolinonas/farmacología , Quinazolinonas/uso terapéutico , Células Tumorales Cultivadas , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/epidemiología , Neoplasias Uterinas/veterinariaRESUMEN
PROBLEM: Endometriosis is the presence of ectopic uterine endometrial tissue in the peritoneal cavity. Peritoneal fluid samples of women with endometriosis show elevated interleukin-1 (IL-1)beta, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta(1) levels, indicating that an altered immune system may play an important role in the pathogenesis of endometriosis. The invasion of ectopic endometrium into peritoneal mesothelium requires matrix metalloproteinases (MMPs) for tissue remodeling. Several MMPs are differentially expressed in human uterine endometrium with menstrual endometrium showing the highest level of expression. MMPs are stimulated by cytokines and also by the protein Extracellular Matrix Metalloproteinase Inducer (EMMPRIN). METHOD OF STUDY: To determine the role of cytokines in ectopic endometrial invasion, we investigated whether cytokines could regulate MMP production by endometrial fibroblast cells and whether this stimulation occurred through an effect on EMMPRIN expression. Human uterine fibroblasts (HUF) were treated with IL-1beta, TGF-beta(1) and TNF-alpha in a dose dependent and time dependent manner (C, 0.1, 1, 10 ng/mL IL-1beta or TGF-beta(1); C, 2, 10, 50 ng/mL TNF-alpha) for 0, 6, 12, and 24 hr. Cell conditioned medium samples were collected and concentrated at each timepoint for immunoblot analysis. Cellular RNA was collected for real time PCR analysis of MMPs-1, -2, -3 and EMMPRIN mRNA levels. RESULTS: Our results showed that IL-1beta stimulated MMP-1 protein secretion and mRNA levels in a time dependent manner (P < 0.05), MMP-2 mRNA in a time dependent manner and MMP-3 in a time and dose dependent manner. TNF-alpha stimulated MMP-1 and -3 protein secretion in a time dependent manner and stimulated MMP-1, -2 and -3 mRNA levels in a time dependent manner (P < 0.05). Neither IL-1beta nor TNF-alpha treatment affected MMP-2 protein secretion. TGF-beta(1) inhibited MMP-1 and MMP-2 mRNAs at the highest treatment dose after 24 hr but there was no effect on protein secretion. TGF-beta(1) exerted no effect on MMP-3 mRNA or protein secretion (P < 0.05). Neither of the cytokines affected EMMPRIN protein or mRNA levels but the 10 ng/mL TGF-beta(1) treatment did cause a reduction in EMMPRIN mRNA levels. CONCLUSIONS: These data show that elevated cytokines may play a role in the establishment of ectopic endometrium in the peritoneal cavity by stimulating MMPs to remodel the mesothelial lining of the peritoneum thus allowing for tissue invasion. The stimulation of MMPs by cytokines occurred without any change in EMMPRIN expression whereas the inhibitory effect of TGF-beta(1) involved a reduction in EMMPRIN mRNA levels.
Asunto(s)
Basigina/metabolismo , Citocinas/fisiología , Endometrio/metabolismo , Metaloproteinasas de la Matriz/genética , Basigina/genética , Línea Celular , Separación Celular , Células Cultivadas , Citocinas/farmacología , Endometriosis/inmunología , Endometriosis/metabolismo , Endometrio/citología , Endometrio/enzimología , Femenino , Fibroblastos/enzimología , Humanos , Interleucina-1/inmunología , Interleucina-1beta , Metaloproteinasas de la Matriz/metabolismo , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/fisiología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Despite the development of many new techniques, laboratory assays still do not predict male fertility accurately. To identify targets for laboratory assessment, we first need to determine which steps in fertilization are most often defective in subfertile males. We developed a competitive in vitro fertilization assay in which spermatozoa from 2 different males, stained with different lipophilic dyes, are incubated together with oocytes in a droplet. By exposing mixed spermatozoa to the same oocytes, this assay controls for many of the variables of in vitro fertilization and should allow identification of the most common faulty steps in fertilization. The relationship of zona-binding ability to fertility is controversial. Therefore, as a first step, we determined if zona pellucida-binding ability, measured by this competitive assay, was related to bovine spermatozoal fertility. Fertility data were collected from 2 groups of bulls by 2 means of evaluation, nonreturn to estrus rates postinsemination and competitive insemination. In the nonreturn to estrus study, semen samples from 15 bulls were effectively ranked by zona-binding ability, using pairwise competitive in vitro zona-binding assays (R(2) = 0.84). However, this ranking was not significantly correlated with nonreturn rates (r = -0.04). In the competitive insemination study, semen samples from 8 bulls were effectively ranked by pairwise comparison using the competitive zona-binding assay (R(2) = 0.67). Again, this ranking was not significantly correlated to the competitive insemination index calculated for these bulls (r = 0.29). In the third study, we tested 3 bulls to determine if in vivo zona binding, assessed by the number of accessory spermatozoa, was correlated with in vitro zona binding. The number of accessory spermatozoa on oocytes recovered from cows after mating was not correlated with in vitro competitive binding of the spermatozoa. In conclusion, in vitro competitive zona binding was not correlated with bovine fertility or binding of accessory spermatozoa to oocytes in vivo.