RESUMO
The mammalian oviductal lumen is a specialized chamber that provides an environment that strictly regulates fertilization and early embryogenesis, but the regulatory mechanisms to gametes and zygotes are unclear. We evaluated the oviductal regulation of early embryonic development using Ovgp1 (encoding an oviductal humoral factor, OVGP1)-knockout golden hamsters. The experimental results revealed the following: (1) female Ovgp1-knockout hamsters failed to produce litters; (2) in the oviducts of Ovgp1-knockout animals, fertilized eggs were sometimes identified, but their morphology showed abnormal features; (3) the number of implantations in the Ovgp1-knockout females was low; (4) even if implantations occurred, the embryos developed abnormally and eventually died; and (5) Ovgp1-knockout female ovaries transferred to wild-type females resulted in the production of Ovgp1-knockout egg-derived OVGP1-null litters, but the reverse experiment did not. These results suggest that OVGP1-mediated physiological events are crucial for reproductive process in vivo, from fertilization to early embryonic development. This animal model shows that the fate of the zygote is determined not only genetically, but also by the surrounding oviductal microenvironment.
Assuntos
Tubas Uterinas , Oviductos , Humanos , Gravidez , Animais , Cricetinae , Feminino , Mesocricetus , Células Germinativas , Ovário , Mamíferos , GlicoproteínasRESUMO
Diverse lines of evidence indicate that the mammalian oviduct makes important contributions to the complex process of reproduction other than being simply a conduit for the transport of gametes and embryos. The cumulative synthesis and transport of proteins secreted by oviductal secretory cells into the oviductal lumen create a microenvironment supporting important reproductive events, including sperm capacitation, fertilization, and early embryo development. Among the components that have been identified in the oviductal fluid is a family of glycosylated proteins known collectively as oviduct-specific glycoprotein (OVGP1) or oviductin. OVGP1 has been identified in several mammalian species, including humans. The present review summarizes the work carried out, in various mammalian species, by many research groups revealing the synthesis and secretion of OVGP1, its fate in the female reproductive tract upon secretion by the oviductal epithelium, and its role in modulating biological functions of gametes and embryos. The production and functions of recombinant human OVGP1 and recombinant OVGP1 of other mammalian species are also discussed. Some of the findings obtained with immunocytochemistry will be highlighted in the present review. It is hoped that the findings obtained from recent studies carried out with recombinant OVGP1 from various species will rekindle researchers' interest in pursuing further the role of the oviductal microenvironment, of which OVGP1 is a major component, in contributing to the successful occurrence of early reproductive events, and the potential use of OVGP1 in improving the current assisted reproductive technology in alleviating infertility.
Assuntos
Tubas Uterinas , Oviductos , Animais , Desenvolvimento Embrionário , Feminino , Células Germinativas , Glicoproteínas/metabolismo , Humanos , Masculino , MamíferosRESUMO
PURPOSE: To investigate the effects of recombinant human oviduct-specific glycoprotein (rHuOVGP1) alone and in combination with progesterone (P4) on intracellular Ca2+ concentration [Ca2+]i and to investigate if rHuOVGP1 in combination with P4 can further enhance tyrosine phosphorylation (pY) of sperm proteins during human sperm capacitation. METHODS: Fluorometric flow cytometry was performed to examine the effects of rHuOVGP1 on [Ca2+]i in human sperm during capacitation. Confocal microscopy was used in conjunction with live cell imaging to analyze the influence of rHuOVGP1 and P4 on [Ca2+]i in the sperm tail and to examine the involvement of CatSper channels in their effect on [Ca2+]i. Western blot analysis was performed to assess the protein levels of p105, a major tyrosine-phosphorylated sperm protein. RESULTS: rHuOVGP1 increases [Ca2+]i in human sperm at the beginning of capacitation and further increases and sustains the level of [Ca2+]i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca2+]i in the sperm tail. P4 alone can increase pY of a major human sperm protein, p105, yet yields a further increase when used in combination with rHuOVGP1. CONCLUSION: The present study revealed that rHuOVGP1 may work with P4 to upregulate [Ca2+]i at the beginning of capacitation in part through CatSper channels which, in turn, leads to the downstream event of pY of sperm proteins and enhancement of sperm capacitation.
Assuntos
Cálcio , Progesterona , Humanos , Masculino , Cálcio/metabolismo , Cálcio/farmacologia , Progesterona/farmacologia , Progesterona/metabolismo , Motilidade dos Espermatozoides , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio/farmacologia , Sêmen/metabolismo , Capacitação Espermática , Espermatozoides/metabolismo , Tirosina/metabolismo , Glicoproteínas/metabolismoRESUMO
Placentation is one of the most important determinants for a successful pregnancy, and this is dependent on the process of trophoblast migration and invasion. Progesterone receptors (PGR) are critical effectors of progesterone (P4) signaling that is required for trophoblast migration and invasion conducive to a successful gestation. In immune complicated pregnancies, evidence has shown that abnormal placentation occurs because of aberrant expression of PGR. Therapeutic intervention with tacrolimus (FK506) was able to restore PGR expression and improve pregnancy outcomes in immune-complicated gestations; however, the exact mode of action of tacrolimus in assisting placentation is not clear. Here, we attempt to uncover the mode of action of tacrolimus by examining its effects on trophoblast invasion and migration in the human-derived extravillous trophoblast (EVT) cell line, the HTR-8/SVneo cells. Using a variety of functional assays, we demonstrated that low-dose tacrolimus (10 ng/mL) was sufficient to significantly (p < 0.001) stimulate the migration and invasion of the HTR-8/SVneo cells, inducing their cytosolic/nuclear progesterone receptor expression and activation, and modulating their Nitric Oxide (NO) production. Moreover, tacrolimus abrogated the suppressive effect of the NOS inhibitor Nω- Nitro-L-Arginine Methyl Ester (L-NAME) on these vital processes critically involved in the establishment of human pregnancy. Collectively, our data suggest an immune-independent mode of action of tacrolimus in positively influencing placentation in complicated gestations, at least in part, through promoting the migration and invasion of the first trimester extravillous trophoblast cells by modulating their NO production and activating their cytosolic/nuclear progesterone-receptors. To our knowledge, this is the first report to show that the mode of action of tacrolimus as a monotherapy for implantation failure is plausibly PGR-dependent.
Assuntos
Tacrolimo , Trofoblastos , Movimento Celular , Feminino , Humanos , Óxido Nítrico/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Tacrolimo/farmacologia , Trofoblastos/metabolismoRESUMO
Polycystic ovary syndrome (PCOS) is a major anovulatory infertility affecting a great proportion of women of childbearing age and is associated with obesity, insulin resistance and chronic inflammation. Poor endometrial receptivity and recurrent implantation failure are major hurdles to the establishment of pregnancy in women with PCOS. The accumulating body of evidence obtained from experimental and clinical studies suggests a link between inherent adaptive and innate immune irregularities and aberrant endometrial features in PCOS. The use of conventional therapeutic interventions such as lifestyle modification, metformin and ovarian stimulation has achieved limited clinical success in restoring ovulation and endometrial receptivity in women with PCOS. Unlike other immunosuppressive drugs prescribed in the clinical management of autoimmune and inflammatory disorders that may have deleterious effects on fertility and fetal development, preclinical studies in mice and in women without PCOS but with repeated implantation failure revealed potential therapeutic benefits for the use of low-dose tacrolimus in treating female infertility. Improved systemic and ovarian immune functions, endometrial progesterone receptor and coreceptor expressions and uterine vascular adaptation to pregnancy were among features of enhanced progesterone-receptor sensitivity in the low-dose tacrolimus-treated mouse model of the disease. In this review, we have compiled available experimental and clinical data in literature on endometrial progesterone resistance and current therapeutic options, as well as mechanisms of actions and reported outcomes relevant to the potential therapeutic benefits for the use of low-dose tacrolimus in treating PCOS-associated female infertility.
Assuntos
Endométrio/efeitos dos fármacos , Infertilidade Feminina/tratamento farmacológico , Síndrome do Ovário Policístico/tratamento farmacológico , Tacrolimo/uso terapêutico , Relação Dose-Resposta a Droga , Endométrio/anormalidades , Endométrio/patologia , Feminino , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Resistência à Insulina/genética , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/patologia , Gravidez , Doenças Uterinas/genéticaRESUMO
PURPOSE: To investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)-enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding. METHODS: Immunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay. RESULTS: pY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding. CONCLUSIONS: The present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Glicoproteínas/genética , Capacitação Espermática/genética , Espermatozoides/metabolismo , Animais , Feminino , Fertilização in vitro , Humanos , Masculino , Camundongos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oviductos/metabolismo , Fosforilação , Reprodução/genética , Sêmen/metabolismo , Cauda do Espermatozoide/metabolismo , Interações Espermatozoide-Óvulo/genética , Tirosina/metabolismo , Zona Pelúcida/metabolismoRESUMO
BACKGROUND: T2DM is a high-risk pregnancy with adverse fetal and maternal outcomes including repeated miscarriages and fetal malformations. Despite the established association between placental insufficiency and poor maternal Th1-adaptability to the development of pregnancy complications in T2DM, there have been no established data to assess benefits of pre-pregnancy immunosuppression relative to gestational outcomes in T2DM. We hypothesized that pre-pregnancy macrolide immune suppression can re-establish normal placental development and uterine vascular adaptation in a mouse model of obesity-associated T2DM. METHODS: Fetal live birth rate, postnatal variability, mid-gestational uterine and umbilical flow dynamics and certain morphological features of spiral artery modification were examined in the New Zealand Obese (NONcNZO10/Ltj) female mice (n = 56) weaned to ages of 32 weeks on a 60% calories/g high-fat diet (also referred to as HFD-dNONcNZO), and which received either tacrolimus (0.1 mg/kg s.c. q2d) , its vehicle (castor oil and ethanol) or metformin (in drinking water 200 mg/dL p.o. ad libitum). HFD-BALBc-Rag2/IL2-gc female mice (n = 24) were used as HFD-immunodeficient controls. RESULTS: Treatment of the HFD-dNONcNZO female mice with tacrolimus improved live birth rates and postnatal viability scores (p < 0.01), normalized OGTT (p < 0.001), inhibited fetal malformation rates, restored morphology of spiral arterial modification; and improved uterine arterial and umbilical blood flow (p < 0.01). Placental production of TNFαand IL16 in the tacrolimus-treated HFD-dNONcNZO dams were restored to non-diabetic levels and the treatment resulted in the inhibition of aberrant monocyte/macrophage activation during pregnancy in the HFD-dNONcNZO dams. CONCLUSIONS: Our present data suggest a casual association between chronic maternal overnutrition and aberrancy in the maternal Th1-immune maladaptation to pregnancy and defective spiral artery modification, placental insufficiency and adverse fetal outcomes in the T2DM subjects. Further safety studies into the use of tacrolimus in the pre-pregnancy glycemic control may be beneficial.
Assuntos
Diabetes Mellitus Experimental/complicações , Doenças Fetais/etiologia , Doenças Fetais/prevenção & controle , Resultado da Gravidez , Tacrolimo/uso terapêutico , Animais , Osso e Ossos/anormalidades , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/embriologia , Osso e Ossos/patologia , Diabetes Mellitus Experimental/tratamento farmacológico , Dieta Hiperlipídica , Feminino , Doenças Fetais/tratamento farmacológico , Teste de Tolerância a Glucose , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Obesos , Gravidez , Coluna Vertebral/irrigação sanguínea , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/fisiopatologia , Tacrolimo/farmacologia , Cordão Umbilical/irrigação sanguínea , Artéria Uterina/fisiopatologia , Remodelação Vascular/efeitos dos fármacosRESUMO
The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1), which has been shown to interact with gametes and early embryos. Here we report the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with a purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVGP1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium further enhanced tyrosine phosphorylation of sperm proteins in a time-dependent manner. After 4-h incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.
RESUMO
Oviductin or OVGP1, also known as oviduct-specific glycoprotein, has been shown to enhance sperm capacitation in addition to its other beneficial effects on fertilization and early embryo development. We hypothesized that estrus stage-specific hamster oviductin (eHamOVGP1) can potentiate the enhancement of tyrosine phosphorylation of sperm proteins during capacitation. Immunofluorescent staining and confocal microscopy as well as immunocytochemistry and surface replica technique localized tyrosine-phosphorylated proteins to the equatorial segment and midpiece after incubation of hamster sperm in capacitation medium in the presence or absence of eHamOVGP1. Increase of tyrosine phosphorylation level in the equatorial segment occurred as early as 5 min after incubation in the presence of eHamOVGP1. Immunostaining for eHamOVGP1 further increased upon prolonged incubation of sperm in medium containing the glycoprotein. Regardless of the presence or absence of eHamOVGP1, phosphotyrosine expression was observed along the tail, particularly at the midpiece. Western blotting of NP40-extracted sperm proteins (25, 37, and 44 kDa) and NP40-non-extractable sperm proteins (70, 83, 90 kDa) showed increased immunolabeling intensity after 5, 60, 120, and 180 min of capacitation in the presence of eHamOVGP1. Mass spectrometric analysis identified several proteins of functions known to be involved in metabolic pathways responsible for enhancement of tyrosine phosphorylation in its presence. The present investigation provides evidence that eHamOVGP1 regulates the expression of protein tyrosine phosphorylation in sperm capacitated in vitro, further supporting an important role of the presence of OVGP1 in the oviductal milieu during the process of fertilization.
Assuntos
Serina Endopeptidases/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/metabolismo , Animais , Cricetinae , Feminino , Masculino , Fosforilação , Fosfotirosina/metabolismoRESUMO
PURPOSE: The aim was to culture primordial follicles in vitro to reach preantral stage in vitrified human ovarian tissue. METHODS: Ovarian tissue samples were obtained from six women. Tissue strips were vitrified by infiltration with a cryoprotectant followed by mounting on a stainless steel carrier. After culturing for 7 days the morphology and developmental stages of follicles enclosed in fresh and vitrified groups were analyzed. RESULTS: High proportion of viable follicles in vitrified ovarian strips was obtained. After culturing for 7 days the percentage of secondary and preantral follicles increased significantly (P < 0.05) whereas primordial and transitory follicles showed a significant decrease (P < 0.05) compared to their respective counterparts at day 0 of culture. CONCLUSIONS: Vitrification of ovarian strips with an improved carrier device and culturing of follicles in ovarian strips after warming yielded developed follicles with high viability and morphological integrity that may be suitable for use in fertility preservation among cancer patients.
Assuntos
Criopreservação/métodos , Folículo Ovariano/crescimento & desenvolvimento , Vitrificação , Adulto , Sobrevivência Celular , Feminino , Humanos , Células Estromais/citologia , Técnicas de Cultura de TecidosRESUMO
Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.
Assuntos
Aborto Espontâneo/etiologia , Diabetes Mellitus Tipo 1/complicações , Implantação do Embrião , Endométrio/metabolismo , Mucina-1/metabolismo , Aborto Espontâneo/metabolismo , Animais , Concanavalina A , Diabetes Mellitus Tipo 1/metabolismo , Endométrio/ultraestrutura , Feminino , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Gravidez , Proteínas Inibidoras de STAT Ativados/metabolismo , Receptores de Progesterona/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
PURPOSE: To investigate the effects of cryo-storage duration in liquid nitrogen on oocyte cryo-survival, fertilization and embryonic development following vitrification and warming. METHODS: Mature mouse oocytes were vitrified with McGill Cryoleaf and stored in liquid nitrogen for a period of 8-10 days, 90-92 days and 180-182 days, respectively. After warming, the survived oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and cultured for 120 h. The rates of oocyte cryo-survival, cleavage and embryonic development were compared. RESULT(S): The oocyte cryo-survival rate declined following cryo-storage duration for 180-182 days (90.4 ± 7.9%) compared to that of the other two groups (97.4 ± 3.0% and 98.0 ± 3.3%). The fertilization rate in the group of 180-182 days (66.6 ± 22.0%) was also significantly reduced (P < 0.05) compared with the groups of 8-10 days (92.2 ± 10.8%) and 90-92 days (94.7 ± 9.1%). In addition, the number of embryos developed to the blastocyst stage declined significantly (P < 0.05) following long cryo-storage duration (72.1 ± 8.2%, 25.2 ± 3.8% and 5.5 ± 13.6%, respectively). CONCLUSION(S): The cryo-survival, fertilization rate and embryonic development of mouse oocytes are affected significantly, in an adverse manner, by the cryo-storage duration in liquid nitrogen.
Assuntos
Blastocisto/citologia , Criopreservação , Desenvolvimento Embrionário/fisiologia , Oócitos/crescimento & desenvolvimento , Animais , Técnicas de Cultura Embrionária/métodos , Feminino , Fertilização in vitro/estatística & dados numéricos , Masculino , Camundongos , Oócitos/citologia , Gravidez , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides/citologia , VitrificaçãoRESUMO
PURPOSE: to evaluate the effect of female reproductive age on oocyte cryo-survival, fertilization and the subsequent embryonic development following vitrification using the mouse model in order to address the question of how maternal reproductive age is related to fertility preservation. METHODS: oocytes were collected from mice of different reproductive age: (1) 8-10 weeks, (2) 16-20 weeks, (3) 32-36 weeks, and (4) 44-48 weeks. Following vitrification and warming, the oocytes in each group were assessed for cryo-survival, fertilization and embryonic development as well as for the quality of blastocysts. Fresh oocytes without undergoing vitrification were used in each age group as controls. RESULTS: the mean number of oocytes retrieved following superovulation was found to reduce significantly (P < 0.05) in mice from 32-36 weeks of age (18.1 ± 8.5) compared with 8-10 weeks of age (26.8 ± 9.8) and 16-20 weeks of age (23.9 ± 4.2) respectively. The cryo-survival rate of oocytes was reduced significantly (P < 0.05) in mice of 44-48 weeks of age (90.4% ± 7.9) compared with the other 3 groups (98.8% ± 2.1, 98.0% ± 3.3 and 98.5% ± 2.2, respectively). The cleavage rate of vitrified oocytes declined significantly following the increase in maternal age in mice of 32-36 weeks of age (69.7% ± 20.8) forward (63.6% ± 9.2). However, no significant difference in the cleavage rate was found among the control groups of different maternal ages. The rate of embryo development to the blastocyst stage in the vitrified oocytes also significantly declined following the increase in maternal age (71.8% ± 8.8, 66.4% ± 10.7, 64.2% ± 17.4 and 4.1% ± 8.3 respectively). There were no such differences in the rates of embryo development to the blastocyst stage among the control groups following the increase in maternal age (75.9% ± 12.2, 79.5% ± 28.9, 70.2% ± 17.4 and 69.3% ± 19.0 respectively). However, the quality of blastocysts produced from 32-36 weeks and 44-48 weeks of ages was significantly poor in term of total cell numbers and the ratio of inner cell mass(ICM) / trophectoderm (TE) compared to younger age in both vitrified and control groups CONCLUSIONS: cryo-survival of oocytes following vitrification and warming procedures is associated with female reproductive age. There is a more negative impact on the oocytes following vitrification and warming with the increase of maternal age.
Assuntos
Criopreservação , Desenvolvimento Embrionário , Oócitos , Fatores Etários , Animais , Blastocisto/citologia , Técnicas de Cultura Embrionária , Fertilização in vitro , Camundongos , Camundongos Endogâmicos , Indução da OvulaçãoRESUMO
Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), leukotriene C(4) synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high-affinity CysLT receptors: CysLT(1)R, CysLT(2)R, and GPR 17. We sought to investigate vascular sites of CysLT(2)R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT(2)R was investigated by reporter gene expression in a novel CysLT(2)R deficient-LacZ mouse model. CysLT(2)R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FITC-labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT-mediated permeability, which was blocked by application of BAY-u9773, a dual CysLT(1)R/CysLT(2)R antagonist or by CysLT(2)R deficiency. Endothelial human CysLT(2)R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT(2)R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca(2+) signaling. Our results reveal that CysLT(2)R can mediate inflammatory reactions in a vascular bed-specific manner by altering transendothelial vesicle transport-based vascular permeability.
Assuntos
Transporte Biológico/fisiologia , Permeabilidade Capilar/fisiologia , Receptores de Leucotrienos/fisiologia , Animais , Transporte Biológico/genética , Sinalização do Cálcio/fisiologia , Permeabilidade Capilar/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Expressão Gênica , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Receptores de Leucotrienos/biossíntese , SRS-A/análogos & derivados , SRS-A/farmacologia , Albumina SéricaRESUMO
Polycystic ovary syndrome (PCOS) is characterized by failure of ovulation and is associated with obesity and chronic inflammation. Recent evidence suggests that anomalous activation of ovarian macrophages and numerical and functional deficits in the Th17 (CD4+IL17A+) and the CD4+CD25+CD127low Tregs plays crucial role in PCOS. We have shown that the pre-pregnancy use of tacrolimus prevents adverse reproductive outcomes in a mouse model of PCOS. Here we used the HFD-NONcNZO mice to test a hypothesized beneficial use of tacrolimus relative to metformin in favorably influencing the ovarian and systemic immune milieux conducive to gestational success in subjects with PCOS. Compared to normative controls, our data revealed an aberrant peri-conceptional suppression of the CD4+CD25+CD127low Tregs together with an overexpression of the Th17 T cells and lack of coordinated activation of ovarian macrophages in untreated HFD-dNONcNZO mice. Significant variances in treatment outcomes favoured the use of tacrolimus over metformin in treated mice. Consistent with the human fertility studies, this investigation reveals a beneficial systemic use of tacrolimus (0.1 mg/kg) in promoting early pregnancy in individuals with PCOS and suggests the need for further research into the selective inhibition of IL17A as a plausibly alternative immunotherapeutic approach in the clinical management of infertile individuals with PCOS.
Assuntos
Homeostase , Ovário/imunologia , Ovário/patologia , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/imunologia , Tacrolimo/uso terapêutico , Animais , Citocinas/metabolismo , Dieta Hiperlipídica , Relação Dose-Resposta a Droga , Feminino , Homeostase/efeitos dos fármacos , Humanos , Terapia de Imunossupressão , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Ovário/efeitos dos fármacos , Fenótipo , Gravidez , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Tacrolimo/farmacologiaRESUMO
Diabesity is often associated with subfertility and recurrent miscarriages. Evidence links systemic and local uterine cytotoxicity to the pathogenesis of implantation failure (IF) in diabetes. Immunosuppression with tacrolimus improved pregnancy outcomes in obese and diabetic mice and repeated IF in women with elevated Th1/Th2 blood cell ratios. However the mode of action of tacrolimus in protecting against IF and the molecular mechanisms associated with recurrent miscarriages in the obese and diabetic subjects are yet to be elucidated. Here we administered tacrolimus (FK506) (0.1 mg/kg) for four consecutive weeks to the NONcNZO10/LtJ mice, a model of human PCOS, chronically fed with 60% kCal fat for 16 consecutive weeks to simulate human obesity-associated T2DM. Compared to those immunosuppressed with tacrolimus and their normative controls, high-fat fed (HFD) diabetic NONcNZO mice exhibited higher rates of peri- and post-implantation resorption and had aberrant expression of uterine IFNγ and progesterone receptor (PGR) and its immunophilin co-chaperone FKBP52 at nidation. Immature uterodomes and lack of activation of uterine STAT3 and NFκB at implantation were characteristics of IF in the HFD-dNONcNZO dams also low in the deciduogenic factors IL11 and GM-CSF. Therapeutic interventions with tacrolimus or metformin normalized the expression of decidual IFNγ, PGR and FKBP52, increased co-localization of protein inhibitor of activated STATy (PIASy) to PGR and resulted in the upregulation of uterine IL11and LIF. Rescued phosphorylation of STAT3 and NFκBp65 and uterodome maturation at nidation defined implantation success in treated dams. To our knowledge this is the first report to show that the impact of HFD on the hemochorial implantation is at least in part mediated through disruption of PGR signaling at nidation and that immunosuppression with tacrolimus or treatment with metformin restores PGR-mediated influences during implantation in the obese and diabetic subjects.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Implantação do Embrião/efeitos dos fármacos , Terapia de Imunossupressão , Metformina/farmacologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Tacrolimo/farmacologia , Útero/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interferon gama/metabolismo , Interleucina-11/metabolismo , Fator Inibidor de Leucemia/metabolismo , Camundongos , Camundongos Obesos , NF-kappa B/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismoRESUMO
Exosomes and microvesicles are extracellular vesicles released from cells and can contain lipids, miRNAs and proteins that affect cells at distant sites. Recently, microvesicles containing miRNA have been implicated in uterine microenvironment of pigs, a species with unique epitheliochorial (non-invasive) placentation. Here we report a novel role of conceptus-derived exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) in embryo-endometrial cross-talk. We also demonstrate the stimulatory effects of EVs (PTr2-Exo) derived from porcine trophectoderm-cells on various biological processes including the proliferation of maternal endothelial cells (PAOEC), potentially promoting angiogenesis. Transmission immuno-electron microscopy confirmed the presence of EVs in tissue biopsies, PTr2-Exo and PAOEC-derived EVs (PAOEC-Exo). RT-PCR detected 14 select miRNAs in CD63 positive EVs in which miR-126-5P, miR-296-5P, miR-16, and miR-17-5P were the most abundant angiogenic miRNAs. Proteomic analysis revealed EV proteins that play a role in angiogenesis. In-vitro experiments, using two representative cell lines of maternal-fetal interface, demonstrated bidirectional EVs shuttling between PTr2 and PAOEC cells. Importantly, these studies support the idea that PTr2-Exo and PAOEC-Exo containing select miRNAs and proteins can be successfully delivered to recipient cells and that they may have a biological role in conceptus-endometrial cross-talk crucial for the pregnancy success.
Assuntos
Comunicação Celular , Endométrio/citologia , Vesículas Extracelulares/metabolismo , Feto/citologia , Troca Materno-Fetal , Animais , Biomarcadores/metabolismo , Proliferação de Células , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/ultraestrutura , Endométrio/metabolismo , Células Endoteliais/citologia , Exossomos/metabolismo , Vesículas Extracelulares/ultraestrutura , Feminino , MicroRNAs/metabolismo , Modelos Biológicos , Gravidez , Proteômica , Sus scrofa , Tetraspanina 30/metabolismo , Trofoblastos/citologiaRESUMO
Recently, we provided evidence that the glycosylation of hamster oviductin, a member of the mucin family of glycoproteins, is regulated during the estrous cycle. In order to further elucidate the glycosylation process of oviductal glycoproteins, we identified biosynthetic pathways involved in the assembly of mucin-type O-linked oligosaccharide (O-glycan) chains in the hamster oviduct. Our results demonstrated that the hamster oviduct has high activities of glycosyltransferases that synthesize O-glycans with core 1, 2, 3 and 4 structures as well as elongated structures. Oviduct therefore represents a typical mucin-secreting tissue. Our results also showed that specific glycosyltransferase activities are regulated during the estrous cycle. Mucin-type core 2 beta6-GlcNAc-transferase (C2GnT2) is responsible for synthesizing core 2 and core 4 structures in the oviduct. Specific assays for C2GnT2 revealed a cyclical pattern throughout the estrous cycle with high activity at the stages of proestrus and estrus and low activity at diestrus 1. Using semiquantitative RT-PCR, the mRNA levels for C2GnT2 in the estrous cycle stages could be correlated with the enzyme activities. An increase in glycosyltransferase activity in the hamster oviduct at the time of ovulation suggests that glycosylation of oviductal glycoproteins may be necessary for these proteins to exert their functions during the process of fertilization.
Assuntos
Estro/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Oviductos/metabolismo , Polissacarídeos/biossíntese , Animais , Cricetinae , Feminino , Mesocricetus , N-Acetilglucosaminiltransferases/análise , N-Acetilglucosaminiltransferases/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/fisiologiaRESUMO
The zona pellucida (ZP) is an extracellular coat that encloses growing oocytes, ovulated eggs, and preimplantation embryos in mammals. The ZP contains receptors that mediate initial interactions between the sperm and egg and the relative species-specificity during gamete interaction. It also prevents polyspermy and protects the developing embryo prior to implantation. The current model of the organization of mouse ZP depicts this extracellular matrix as an extensive three-dimensional array of long interconnected filaments of a structural repeat. Using high-resolution colloidal-gold labeling methods in combination with specific lectins and monoclonal antibodies against three major ZP glycoproteins, we have characterized the outer and inner mouse ZP during folliculogenesis and shown the modifications of the ZP after ovulation and fertilization. Our immunogold labeling results also indicate the involvement of a vesicular aggregate, a specialized subcellular compartment in the oocyte, in the synthesis and secretion of ZP glycoproteins.
Assuntos
Glicoproteínas/análise , Microscopia Eletrônica/métodos , Zona Pelúcida/metabolismo , Animais , Cricetinae , Proteínas do Ovo/análise , Proteínas do Ovo/metabolismo , Feminino , Glicoproteínas/metabolismo , Coloide de Ouro/química , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Mesocricetus , Camundongos , Camundongos Endogâmicos , Folículo Ovariano/fisiologia , Ovulação , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/metabolismo , Zona Pelúcida/química , Glicoproteínas da Zona PelúcidaRESUMO
In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.