In vitro secretion and activity profiles of matrix metalloproteinases, MMP-9 and MMP-2, in human term extra-placental membranes after exposure to Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Premature rupture of fetal membranes (PROM) complicated with intrauterine infection has been associated to alterations of the extracellular matrix (ECM) homeostasis. The aim of this work was to evaluate the integral/functional response of the amnion (AMN) and choriodecidua (CHD) to synthesis, secretion, and activity of MMP-2 and MMP-9 and of their inhibitors TIMP-1, -2, and -4, after stimulation with <it>Escherichia coli</it>.</p> <p>Methods</p> <p>Full-thickness membranes were mounted on a Transwell device, constituting two independent chambers, <it>Escherichia coli </it>(1×10 (6) CFU/mL) were added to either the amniotic or the choriodecidual face or to both. Secretion profiles of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-4 were quantified by ELISA and gelatinolytic activity by zymography. Immunoreactivity for MMP-2 and MMP-9 was revealed by immunohistochemistry and the collagen content was assessed by the hydroxyproline assay.</p> <p>Results</p> <p>Levels of MMP-9 in CHD and AMN increased 4- and 8-fold, respectively, after simultaneous infection. MMP-2 secreted to the medium by CHD increased a mean of 3 times after direct stimulation. Secretion profiles of TIMP-1, TIMP-2, and TIMP-4 remained without significant changes. Collagen content was significantly decreased (4-fold) in infected membranes, and was associated with loss of structural continuity and co-localization with immunoreactive forms of MMP-2 and MMP-9.</p> <p>Conclusions</p> <p>Infection of chorioamniotic membranes with <it>E. coli </it>induces an increase in the secretion of inactive forms and an association to ECM of active forms of MMP-2 and MMP-9 without changes in TIMP-1, -2, and -4. These changes could explain the significant decrease of collagen content and loss of structural continuity.</p

Light and focused ion beam microscopy workflow for resin-embedded tissues

Although the automated image acquisition with the focused ion beam scanning electron microscope (FIB-SEM) provides volume reconstructions, volume analysis of large samples remains challenging. Here, we present a workflow that combines a modified sample protocol of the classical transmission electron microscope with FIB-SEM volume imaging. The proposed workflow enables efficient 3D structural surveys of rabbit ovaries collected at consecutive developmental stages. The precise trimming of the region of interest adds the time dimension to the volume, constructing a virtual 4D electron microscopy. We found filopodia-like processes emitted by oocyte cysts allowing contact between oocytes not previously observed

In vitro secretion profiles of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and TNF alpha after selective infection with Escherichia coli in human fetal membranes

<p>Abstract</p> <p>Background</p> <p>Chorioamniotic membranes infection is a pathologic condition in which an abnormal secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the present study was to evaluate the functional response of human chorioamniotic membranes, as well as the individual contribution of the amnion and choriodecidua after stimulation with Escherichia coli, a pathogen associated with preterm labor.</p> <p>Methods</p> <p>Explants of chorioamniotic membranes from 10 women (37–40 weeks of gestation) were mounted and cultured in a Transwell system, which allowed us to test the amnion and choriodecidua compartments independently. Escherichia coli (1 × 10 6 CFU/mL) was added to either the amniotic or the choriodecidual regions or both; after a 24-h incubation, the secretion of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10 in both compartments was measured using a specific ELISA. Data were analyzed by Kruskal-Wallis one-way analysis of variance.</p> <p>Results</p> <p>After stimulation with Escherichia coli, the choriodecidua compartment showed an increase in the secretion of IL-1beta (21-fold), IL-6 (2-fold), IL-8 (6-fold), and IL-10 (37-fold), regardless of which side of the membrane was stimulated; TNFalpha secretion augmented (22-fold) also but only when the stimulus was applied simultaneously to both sides. When the amnion was stimulated directly, the level of IL-1beta (13-fold) rose significantly; however, the increase in IL-8 secretion was larger (20-fold), regardless of the primary site of infection. TNFalpha secretion in the amnion compartment rose markedly only when Escherichia coli was simultaneously applied to both sides.</p> <p>Conclusion</p> <p>Selective stimulation of fetal membranes with Escherichia coli results in a differential production of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10. These tissues were less responsive when the amnion side was stimulated. This is in agreement with the hypothesis that the choriodecidua may play a primary role during an ascending intrauterine infection, being the main barrier to progression of the infection into the amniotic cavity. Therefore, the tissue-specific capacities for the secretion of these immune modulators could be a determining factor for the degree of severity of the inflammation process in fetal membranes.</p

GLI1(+) progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-like tissue

As certain strains of mice age, hyperplastic lesions resembling gonadal tissue accumulate beneath the adrenal capsule. Gonadectomy (GDX) accelerates this heterotopic differentiation, resulting in the formation of wedge-shaped adrenocortical neoplasms that produce sex steroids. Stem/progenitor cells that reside in the adrenal capsule and retain properties of the adrenogonadal primordium are thought to be the source of this heterotopic tissue. Here, we demonstrate that GLI1(+) progenitors in the adrenal capsule give rise to gonadal-like cells that accumulate in the subcapsular region. A tamoxifen-inducible Cre driver (Glil-creER(T2)) and two reporters (R26R-lacZ, R26R-confetti) were used to track the fate of GLI1(+) cells in the adrenal glands of B6D2F2 mice, a strain that develops both GDX-induced adrenocortical neoplasms and age-dependent subcapsular cell hyperplasia. In gonadectomized B6D2F2 mice GLI1(+) progenitors contributed to long-lived adrenal capsule cells and to adrenocortical neoplasms that expressed Gata4 and Foxl2, two prototypical gonadal markers. Pdgfra, a gene expressed in adrenocortical stromal cells, was upregulated in the GDX-induced neoplasms. In aged non-gonadectomized B6D2F2 mice GLI1(+) progenitors gave rise to patches of subcapsular cell hyperplasia. Treatment with GANT61, a small-molecule GLI antagonist, attenuated the upregulation of gonadal-like markers (Gata4, Foxl2) in response to GDX. These findings support the premise that GLI1(+) progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic tissue. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

The remarkable reproductive diversity of teleost fishes

Teleosts display the most striking reproductive diversity of all vertebrates. However, no convincing hypothesis has yet been proposed to explain why they have evolved this remarkable variability in their modes of reproduction. Some of the features of the reproductive biology of teleosts are briefly reviewed and unique characteristics of the group that may have made possible the evolution of their remarkable reproductive diversity are considered. These include whole genome duplication, the mode of differentiation of the gonads, and the organisation of the brain–pituitary relationship

Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility

Ten to 15% of couples are infertile, with the most common causes being linked to the production of few or no oocytes or sperm. Yet, our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility

Sex Determination:Why So Many Ways of Doing It?

Sexual reproduction is an ancient feature of life on earth, and the familiar X and Y chromosomes in humans and other model species have led to the impression that sex determination mechanisms are old and conserved. In fact, males and females are determined by diverse mechanisms that evolve rapidly in many taxa. Yet this diversity in primary sex-determining signals is coupled with conserved molecular pathways that trigger male or female development. Conflicting selection on different parts of the genome and on the two sexes may drive many of these transitions, but few systems with rapid turnover of sex determination mechanisms have been rigorously studied. Here we survey our current understanding of how and why sex determination evolves in animals and plants and identify important gaps in our knowledge that present exciting research opportunities to characterize the evolutionary forces and molecular pathways underlying the evolution of sex determination

Nuevo avance hacia la clonación terapéutica humana: se reaviva la polémica

EstanciasLa clonación terapéutica como una opción para el tratamiento de pacientes con padecimientos genéticos recibió un nuevo impulso. Un equipo de la Universidad Nacional de Seúl, Corea del Sur, generó por primera vez una línea de células troncales derivadas de la masa celular interna de un blastocisto humano. El blastocisto fue desarrollado in vitro a partir de un cigoto “reconstruido” por la fusión de una célula somática del ovario con un ovocito enucleado. De manera que las células del blastocisto desarrollado poseen copias del genoma de la célula somática. Como las células troncales obtenidas son genéticamente idénticas a la célula somática, se asume que pudieran ser usadas para transplantarse ya que el rechazo inmunológico sería nulo o muy leve. Si además de células ováricas usadas por el equipo coreano, se derivan células troncales clonadas a partir de otras células de individuos adultos, el camino hacia una clonación terapéutica confiable estará abierto. Sin embargo, el manejo de ovocitos humanos “reconstruídos” para generar blastocistos plantea cuestiones éticas porque de ser implantados en el útero de una mujer, podrían desarrollarse como individuos clonados. Sin embargo, hay una importante diferencia entre las clonaciones terapéutica y reproductiva en términos de “intencionalidad”. Mientras la primera intenta clonar células para aliviar a un paciente, en la segunda se busca clonar a un individuo. Como en ambos tipos de clonación se requiere del ovocito humano, la discusión puede centrarse en la potencialidad única de esa célula para desarrollar a un ser humano. En el presente artículo expongo una opinión al respecto de acuerdo con el actual contexto biológicoTherapeutic cloning as an alternative clinical method to treat genetic illness has received a new impulse. In South Corea, a team working in the National University of Seul has established for the first time a line of stem cells derived from the inner cell mass of a cloned human blastocyst. The blastocyst developed from a “reconstructed” egg made by fusing an enucleated oocyte with a somatic ovarian cell. Thus, the cells of the developed blastocyst contained copies of the somatic cell genome. Since the derived stem cells are genetically identical to the somatic cell, it is assumed that they can be used for transplantation because the immunological rejection may be null or highly reduced. If besides ovarian somatic cells used by the Corean team, stem cells can also be derived from other adult somatic cells, the way for a reliable therapeutic cloning in humans will be open. However, handling human oocytes “reconstructed” posse ethical concerns since they may develop as a cloned baby if transferred to a woman womb. Thus, distinction between therapeutic and reproductive cloning is in terms of “intentionality”. While the former intends to clone cells of a patient, the latter seeks to clone individuals. Since for both, therapeutic and reproductive cloning human oocytes are required, discussion is centered on the potential of these cells to develop as human beings. In this article a personal opinion is given considering the actual biological contex