ABSTRACT
The union between mammalian gametes begins with the sperm binding to the zona pellucida (ZP). We studied the interaction between human sperm and ZP by using recombinant human ZP proteins (rhZP). The cDNAs coding for human ZP2, ZP3, and ZP4 were expressed in Sf9 cells and proteins were characterized to determine their competence for sperm binding. Capacitated human sperm binding abilities were analyzed using immobilized rhZP and a well-characterized antihuman sperm antiserum. The results demonstrated that all rhZP proteins were structurally similar to their native counterparts and were specifically recognized, in a dose and time dependent manner, by human sperm. The rhZP4 was the main sperm binder followed by rhZP3 and rhZP2, although combinations of rhZP proteins enhanced sperm adhesion. Moreover, this experimental approach may represent a useful model to study sperm-ZP interactions for research and clinical purposes.
Subject(s)
Egg Proteins/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Sperm-Ovum Interactions/physiology , Spermatozoa/metabolism , Zona Pellucida/metabolism , Female , Humans , Male , Protein Binding , Recombinant Proteins/metabolism , Zona Pellucida GlycoproteinsABSTRACT
Several proangiogenic/proinflammatory factors involved in endometrial cancer are regulated by leptin, but the signaling mechanisms responsible for these leptin-induced actions are largely unknown. Here, we report that in benign (primary and HES) and cancerous-endometrial epithelial cells (EEC) (An3Ca, SK-UT2 and Ishikawa), leptin in a dose-dependent manner regulates vascular endothelial growth factor, (VEGF); interleukin-1 beta, (IL-1beta); leukemia inhibitory factor, (LIF) and their respective receptors, VEGFR2, IL-1R tI and LIFR. Remarkably, leptin induces a greater increase in VEGF/VEGFR2 and LIF levels in cancer than in benign cells. However, IL-1beta was only increased by leptin in benign primary-EEC. Cancer-EEC expressed higher levels of leptin receptor (full-length OB-Rb and short isoforms) in contrast to benign primary-EEC. Leptin-mediated activation of JAK2 (janus kinase 2) was upstream to the activation of PI-3K (phosphatidylinositol-3 kinase) and/or MAPK (mitogen-activated protein kinase) signaling pathways. Leptin induction of cytokines/receptors generally involved JAK2 and MAPK activation, but PI-3K phosphorylation was required for leptin increase of LIF, IL-1/IL-1R tI. Leptin-mediated activation of mTOR (mammalian target of Rapamycin), mainly linked to MAPK, played a central role in leptin regulation of all cytokines and receptors. These results suggest that leptin's effects are cell-specific and could confer a proliferative or cell survival advantage or possibly promote endometrial thickness. Leptin's effects on proangiogenic molecules were more evident in malignant versus benign cells and may imply that there is an underlying shift in leptin-induced cell signaling pathways in endometrial cancer cells.
Subject(s)
Adenocarcinoma/metabolism , Endometrial Neoplasms/metabolism , Endometrium/metabolism , Leptin/metabolism , Neovascularization, Pathologic/metabolism , Protein Kinases/metabolism , Adenocarcinoma/blood supply , Blotting, Western , Endometrial Neoplasms/blood supply , Endometrium/blood supply , Female , Humans , Interleukin-1beta/metabolism , Leukemia Inhibitory Factor/metabolism , Receptors, Interleukin-1/metabolism , Receptors, OSM-LIF/metabolism , Signal Transduction , TOR Serine-Threonine Kinases , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
To gain insight into the mechanism(s) by which leptin contributes to mammary tumor (MT) development we investigated the effects of leptin, kinase inhibitors, and/or leptin receptor antagonists (LPrA2) on 4T1 mouse mammary cancer cells in vitro and LPrA2 on 4T1-MT development in vivo. Leptin increases the expression of vascular endothelial growth factor (VEGF), its receptor (VEGF-R2), and cyclin D1 through phosphoinositide 3-kinase, Janus kinase 2/signal transducer and activator of transcription 3, and/or extracellular signal-activated kinase 1/2 signaling pathways. In contrast to leptin-induced levels of cyclin D1 the changes in VEGF or VEGF-R2 were more dependent on specific signaling pathways. Incubation of 4T1 cells with anti-VEGF-R2 antibody increased leptin-mediated VEGF expression suggesting an autocrine/paracrine loop. Pretreatment of syngeneic mice with LPrA2 prior to inoculation with 4T1 cells delayed the development and slowed the growth of MT (up to 90%) compared with controls. Serum VEGF levels and VEGF/VEGF-R2 expression in MT were significantly lower in mice treated with LPrA2. Interestingly, LPrA2-induced effects were more pronounced in vivo than in vitro suggesting paracrine actions in stromal, endothelial, and/or inflammatory cells that may impact the growth of MT. Although all the mechanism(s) by which leptin contributes to tumor development are unknown, it appears leptin stimulates an increase in cell numbers, and the expression of VEGF/VEGF-R2. Together, these results provide further evidence suggesting leptin is a MT growth-promoting factor. The inhibition of leptin signaling could serve as a potential adjuvant therapy for treatment of breast cancer and/or provide a new target for the designing strategies to prevent MT development.
Subject(s)
Leptin/metabolism , Mammary Neoplasms, Animal/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Cell Line, Tumor , Cyclin D1/metabolism , Female , Humans , Leptin/genetics , Mice , Mice, Inbred BALB C , Peptides/genetics , Peptides/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/genetics , Receptors, Leptin , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
Prolactin (PRL) is a 23 kDa protein hormone that is produced and secreted by the pituitary lactotrophs. Although PRL was initially regarded as an exclusive pituitary hormone, many nonpituitary tissues were later found to contain and produce this hormone. The most established extrapituitary sites that produce PRL are the decidua, the immune system, brain and endometrium. In the immune system, PRL acts as a cytokine where it plays an important role in human immune responses, including in autoimmune diseases. Here, we will discuss the regulation of PRL gene expression in human lymphocytes and review the functions of PRL made by the immune cells, including its involvement in autoimmunity.
Subject(s)
Immune System/physiology , Prolactin/physiology , Animals , Autocrine Communication , Autoimmune Diseases/metabolism , Autoimmunity/physiology , Cell Differentiation/physiology , Disease Models, Animal , Female , Gene Expression Regulation , Humans , Leukocytes/metabolism , Lupus Erythematosus, Systemic/metabolism , Lymphocytes/metabolism , Male , Mice , Mice, Inbred NZB , Paracrine Communication , Pituitary Gland, Anterior/metabolism , Prolactin/genetics , Promoter Regions, Genetic/genetics , Receptors, Cytokine/physiology , Receptors, Prolactin/metabolism , Transcription, GeneticABSTRACT
Prolactin (PRL) Is a 23 κDa protein hormone that is produced and secreted by the pituitary lactotrophs. Although PRL was initially regarded as an exclusive pituitary hormone, many nonpituitary tissues were later found to contain and produce this hormone. The most established extrapituitary sites that produce PRL are the decidua, the immune system, brain and endometrium. In the immune system, PRL acts as a cytokine where it plays an important role in human immune responses, including in autoimmune diseases. Here, we will discuss the regulation of PRL gene expression in human lymphocytes and review the functions of PRL made by the immune cells, including its involvement in autoimmunity.
La prolactina es una hormona que fue considerada durante mucho tiempo de origen exclusivamente hipofisario, y cuya función más importante era la promoción de la lactancia. Sin embargo, la prolactina no sólo se sintetiza en diversos sitios del organismo, sino que también participa en una amplia variedad de procesos biológicos. Dentro de los sitios de síntesis extrahipofisarios de esta hormona se encuentran diversas células del sistema inmunológico. A este nivel, la prolactina actúa afectando desde la proliferación celular hasta el estado inmune del individuo. En esta revisión presentamos algunos aspectos relativos a la prolactina de origen linfocitario tales como su síntesis, su participación en el sistema inmunológico y su relación con estados de autoinmunidad.
Subject(s)
Animals , Female , Humans , Male , Mice , Immune System/physiology , Prolactin/physiology , Autocrine Communication , Autoimmune Diseases/metabolism , Autoimmunity/physiology , Cell Differentiation/physiology , Disease Models, Animal , Gene Expression Regulation , Leukocytes/metabolism , Lupus Erythematosus, Systemic/metabolism , Lymphocytes/metabolism , Mice, Inbred NZB , Paracrine Communication , Pituitary Gland, Anterior/metabolism , Pituitary Gland, Anterior , Prolactin/genetics , Promoter Regions, Genetic/genetics , Receptors, Cytokine/physiology , Receptors, Prolactin/metabolism , Transcription, GeneticABSTRACT
The mammalian zona pellucida (ZP) consists of three glycoproteins (ZP1, ZP2 and ZP3), which are variably conserved among species at the genomic and amino acid levels. In order to evaluate the expression of ZP during ovarian development, a population of antibodies was selected that recognize species conserved antigenic domains of the three ZP proteins. Domain specific antibodies were selected from sera of rabbits immunized with all three native pig ZP proteins by elution of antibodies bound to each of the three human ZP recombinant proteins expressed from cDNAs, using the baculovirus expression system in insect cells. Immunoblot analysis was used to characterize the specificity of the antibodies and immunohistochemistry was used to evaluate the stage specific expression of ZP proteins during ovarian follicular development of the mouse, baboon and human. This study demonstrates that the conserved domains of all three ZP proteins are localized in the oocyte extracellular ZP matrix as well as in a subset of granulosa cells. However, this expression does vary among species with respect to the stage and cell type during early stages of ovarian follicular development. These antibodies should serve as excellent markers for evaluating early stages of human ovarian follicular development and in the development of contraceptive agents.
Subject(s)
Antigens/analysis , Egg Proteins/immunology , Membrane Glycoproteins/immunology , Ovary/chemistry , Receptors, Cell Surface , Animals , Antibody Specificity , Antigens/immunology , Baculoviridae/genetics , DNA, Complementary/genetics , Egg Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix/chemistry , Female , Granulosa Cells/chemistry , Humans , Immunoblotting , Immunohistochemistry , Membrane Glycoproteins/genetics , Ovarian Follicle/physiology , Recombinant Proteins/immunology , Species Specificity , Spodoptera/metabolism , Swine , Transfection , Zona Pellucida/chemistry , Zona Pellucida/ultrastructure , Zona Pellucida GlycoproteinsABSTRACT
La zona pelúcida (ZP) es una capa de glicoproteínas que rodea al ovocito de los mamíferos. Esta cubierta está formada por tre familias de glicoproteínas denominadas ZP1, ZP2 y ZP3, que difieren en sus propiedades inmunológicas y funcionales debido a modificaciones postraduccionales. Estudios llevados a cabo en el ratón sugieren que la función de estas proteínas se encuentra relacionada con el reconocimiento del espermatozoide por la ZP, confiriéndole muy probablemente la connotación de receptores. Esta observación ha permitido que varios laboratorios hayan iniciado la producción y obtención de las proteínas de la ZP, permitiendo la exploración de su papel en procesos fisiológicos y clínicos, y ha abierto la posibilidad de utilizarlas en el desarrollo de un método inmunológico anticonceptivo. En la actualidad la posibilidad de obtener anticuerpos específicos contra los constituyentes proteínicos de la ZP representa una estrategia novedosa para el control de la fertilidad en el humano.
Subject(s)
Contraception, Immunologic/methods , Oocytes , Zona Pellucida/immunology , Fertility , Sperm-Ovum Interactions/immunologyABSTRACT
La vitamina D cobró importancia desde que se descubrió la naturaleza esteroidea y carácter hormonal de uno de sus metabolitos: el calcitriol. Su participación en el mantenimiento de la homeostasis mineral, así como su capacidad para regular la transcripción génica y fomentar la diferenciación celular, han hecho de su estudio tema de gran interés. Los recientes avances en el estudio de la enzima encargada de convertir la 25-hidroxivitamina D en 1.25-dihidroxivitamina D3 (calcitriol), así como la descripción de su mecanismo de acción, han permitido ampliar el conocimiento de los factores reguladores que controlan el equilibrio del sistema endocrino de la vitamina D, y de las implicaciones del calcitriol en la salud en general y en el embarazo en particular.
Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase , Pregnancy , Vitamin D , Calcitriol , Cholecalciferol , Ergocalciferols , PlacentaABSTRACT
In this study, the influence of steroid and thyroid hormones and epidermal growth factor on the productin of SHBG by placenta tissue explants was investigated. Explants of trophoblastic tissue obtained from normal term placentas were cultured for 48 h in serum free culture medium, and then for an additional 24 h period in the presence or absence of various concentrations of either estradiol (0.25 - 5 nM), testoterone (0.5 - 500 nM), triiodothyronine (0.01 - 100 nM) or EGF (2-40 µM), respectively. Human SHBG concentration in culture media was estimated on each day by specific two-site time-resolved fluoroimmunometric assay and the results expressed as pmol/mg tissue protein. Binding characteristics and molecular structure of secreted SHBG were determined by [3H]5a-DHT binding assays and Western blot analysis, espectively. Estradiol and triidothyronine but not testoterone increased significantly (p<0.05 vs. control) the secretion of SHBG into the culture media. Addition of EGF did not significantly change the production of SHBG at the various concentrations studied. [3H]5a-DHT binding assays and Western blot analysis of placental SHBG resulted in identical binding affinities (Kd 2.0 ñ 0.16 x 10-9M= and molecular structure to those obtained in serum from normal pregnant women. These findings support and extend previous observations by our laboratory indicating that SHBG gene is expressed in the placenta and provide further evidence on the hormonal regulatory characteristics of this steroid-binding protein in cultured placenta
