RESUMO
PURPOSE OF REVIEW: Premature ovarian failure (POF) is diagnosed by amenorrhea before 40 years of age. Owing to exhaustion of follicles in POF ovaries, egg donation is the only option. Although menstrual cycles cease in POF patients, some of them still contain residual dormant follicles in ovaries. Recently, we developed a new infertility treatment and named it as in-vitro activation (IVA), which enables POF patients to conceive using their own eggs by activation of residual dormant follicles. Here, we summarize data showing the potential of IVA as a new infertility treatment for POF patients. RECENT FINDINGS: Transgenic mouse studies revealed that the stimulation of phosphatidylinositol-3-kinase-AKT-forkhead box O3 pathway activated dormant primordial follicles. In murine and human ovaries, the phosphatase and tensin homolog inhibitors and phosphatidylinositol-3-kinase activators were demonstrated to activate dormant primordial follicles in in-vitro cultures. Subsequent studies showed that ovarian fragmentation suppressed Hippo signaling pathway, leading to ovarian follicle growth. Combining these two methods in an IVA approach followed by ovarian tissue autotransplantation, successful follicle growth, and pregnancies were reported in POF patients. Currently, two healthy babies were delivered, together with two additional pregnancies. SUMMARY: IVA treatment is a potential infertility therapy for POF patients who have residual follicles.
Assuntos
Infertilidade Feminina/terapia , Menopausa Precoce , Oócitos/fisiologia , Folículo Ovariano/fisiopatologia , Insuficiência Ovariana Primária/terapia , Técnicas de Reprodução Assistida , Criopreservação , Feminino , Fertilização , Humanos , Transdução de SinaisRESUMO
Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.
Assuntos
Infertilidade Feminina/metabolismo , Folículo Ovariano/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Adulto , Animais , Transferência Embrionária , Feminino , Fertilização in vitro , Via de Sinalização Hippo , Humanos , Immunoblotting , Recém-Nascido , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Masculino , Camundongos , Camundongos SCID , Recuperação de Oócitos , Folículo Ovariano/transplante , Gravidez , Resultado da Gravidez , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/terapia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do TratamentoRESUMO
Functional neural circuits in the cerebral cortex are established through specific neural connections between excitatory and various inhibitory cell types. However, the molecular mechanisms underlying synaptic partner recognition remain unclear. In this study, we examined the impact of clustered protocadherin-γ (cPcdhγ) gene deletion in parvalbumin-positive (PV+) cells on intralaminar and translaminar neural circuits formed between PV+ and pyramidal (Pyr) cells in the primary visual cortex (V1) of male and female mice. First, we used whole-cell recordings and laser-scan photostimulation with caged glutamate to map excitatory inputs from layer 2/3 to layer 6. We found that cPcdhγ-deficient PV+ cells in layer 2/3 received normal translaminar inputs from Pyr cells through layers 2/3-6. Second, to further elucidate the effect on PV+-Pyr microcircuits within intralaminar layer 2/3, we conducted multiple whole-cell recordings. While the overall connection probability of PV+-Pyr cells remained largely unchanged, the connectivity of PV+-Pyr was significantly different between control and PV+-specific cPcdhγ-conditional knock-out (PV-cKO) mice. In control mice, the number of reciprocally connected PV+ cells was significantly higher than PV+ cells connected one way to Pyr cells, a difference that was not significant in PV-cKO mice. Interestingly, the proportion of highly reciprocally connected PV+ cells to Pyr cells with large unitary IPSC (uIPSC) amplitudes was reduced in PV-cKO mice. Conversely, the proportion of middle reciprocally connected PV+ cells to Pyr cells with large uIPSC amplitudes increased compared with control mice. This study demonstrated that cPcdhγ in PV+ cells modulates their reciprocity with Pyr cells in the cortex.
Assuntos
Parvalbuminas , Protocaderinas , Camundongos , Feminino , Masculino , Animais , Parvalbuminas/metabolismo , Potenciais Pós-Sinápticos Inibidores , Células Piramidais/fisiologia , Córtex Cerebral/metabolismo , Interneurônios/metabolismoRESUMO
Plant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. Here we describe a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid pathways via a fungal secondary metabolism gene cluster related to the biosynthesis of sesquiterpene metabolites, including botrydial. Cluster activation during root infection suppresses host nutrient uptake-related genes and changes mineral contents, suggesting a role in manipulating host nutrition state. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic-mutualistic continuum in fluctuating environments.
Assuntos
Arabidopsis , Genes Fúngicos , Simbiose , Ácido Abscísico , Arabidopsis/genética , Família MultigênicaRESUMO
Cross-modal plasticity is the repurposing of brain regions associated with deprived sensory inputs to improve the capacity of other sensory modalities. The functional mechanisms of cross-modal plasticity can indicate how the brain recovers from various forms of injury and how different sensory modalities are integrated. Here, we demonstrate that rewiring of the microglia-mediated local circuit synapse is crucial for cross-modal plasticity induced by visual deprivation (monocular deprivation [MD]). MD relieves the usual inhibition of functional connectivity between the somatosensory cortex and secondary lateral visual cortex (V2L). This results in enhanced excitatory responses in V2L neurons during whisker stimulation and a greater capacity for vibrissae sensory discrimination. The enhanced cross-modal response is mediated by selective removal of inhibitory synapse terminals on pyramidal neurons by the microglia in the V2L via matrix metalloproteinase 9 signaling. Our results provide insights into how cortical circuits integrate different inputs to functionally compensate for neuronal damage.
Assuntos
Microglia , Córtex Visual , Animais , Neurônios/fisiologia , Sinapses/fisiologia , Células Piramidais , Córtex Visual/fisiologia , Plasticidade Neuronal/fisiologia , Vibrissas/fisiologia , Córtex Somatossensorial/fisiologiaRESUMO
BACKGROUND: In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. Recently, C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC) was found to suppress mouse oocyte maturation by promoting cyclic guanosine 5'-monophospate (cGMP) production in cumulus cells. However, regulation of NPPC/CNP expression during the pre-ovulatory period and their regulation by the LH surge have not been investigated. METHODS AND RESULTS: Based on genome-wide analysis of DNA microarray data sets using samples from periovulatory ovaries, we found increases in NPPC transcripts in granulosa cells during pre-ovulatory follicle growth in mice and a rapid decline induced by the pre-ovulatory LH/hCG stimulation. Treatment of pre-ovulatory animals with hCG decreased ovarian CNP content. In isolated ovarian cells, NPPC mRNA was predominantly expressed in mural granulosa cells exhibiting similar regulation following gonadotrophin treatment. In cultured mouse pre-ovulatory follicles, meiosis resumption in oocytes by hCG treatment was accompanied by decreases in NPPC transcript levels. In cultured mouse cumulus cell-oocyte complexes, CNP treatment inhibited the resumption of meiosis with increases in cGMP levels in both cumulus cells and oocytes. In human ovaries, CNP levels in ovarian follicular fluid were also decreased following treatment of patients with an ovulatory dose of hCG. CONCLUSIONS: Our findings demonstrate gonadotrophins regulation of NPPC/CNP expression in mouse and human ovaries and confirm the role of CNP as a potent paracrine oocyte maturation inhibitor.
Assuntos
Gonadotropina Coriônica/metabolismo , Células da Granulosa/citologia , Hormônio Luteinizante/metabolismo , Meiose , Peptídeo Natriurético Tipo C/metabolismo , Oócitos/citologia , Ovário/metabolismo , Ovulação , Adulto , Animais , Células do Cúmulo/citologia , Feminino , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Folículo Ovariano/metabolismoRESUMO
Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus-oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge.
Assuntos
Endotelina-1/fisiologia , Meiose , Oócitos/citologia , Comunicação Parácrina/fisiologia , Animais , Gonadotropina Coriônica/fisiologia , Células do Cúmulo , Feminino , Hormônio Luteinizante/fisiologia , Camundongos , Folículo Ovariano , Receptores de Endotelina/metabolismoRESUMO
Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus-oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Meiose/fisiologia , Oócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Oócitos/citologia , Folículo Ovariano/citologia , Ovário/citologia , Oviductos/metabolismo , Gravidez , Proto-Oncogene Mas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Estatística como Assunto , Útero/metabolismoRESUMO
BACKGROUND: Shortly after stimulation by the preovulatory surge of luteinizing hormone (LH), oocytes arrested at the late prophase I resume meiosis characterized by germinal vesicle breakdown (GVBD), chromosome condensation, and extrusion of the first polar body in preparation for fertilization and early embryonic development. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation. Thus, factors released by granulosa or theca cells expect to convey the LH stimuli to oocytes. To identify candidate ligand-receptor pairs potentially involved in the process of oocyte maturation, we performed DNA microarray analyses of ovarian transcripts in mice and identified Kit ligand (Kitl) as an ovarian factor stimulated by the LH/hCG surge. The purpose of this study is to investigate the roles of KITL in the nuclear and cytoplasmic maturation of preovulatory mouse oocytes. METHODS: The levels of Kitl and c-kit transcripts in mouse ovaries and isolated ovarian cells were determined by real-time RT-PCR, while expression of KITL protein was examined by immunohistochemistry. Follicle culture, cumulus-oocyte complexes (COC) and denuded oocytes culture were used to evaluate the effect of KITL on mouse oocyte nuclear maturation. To assess the effect of KITL treatment on the cytoplasmic maturation of preovulatory oocytes, we performed in vitro maturation of oocytes followed by in vitro fertilization. RESULTS: Major increase of Kitl transcripts in granulosa cells and mouse ovaries, and predominant expression of c-kit in preovulatory oocytes were identified by real-time RT-PCR. Predominant expression of KITL protein was found in granulosa cells of preovulatory and small antral follicles at 4 h after hCG treatment. In vitro cultures demonstrated that treatment with KITL enhanced first polar body extrusion in a dose-dependent manner. Moreover, treatment of COC with KITL enhanced first polar body extrusion with increase in cyclin B1 synthesis which is important for the progression of meiotic maturation after GVBD. In contrast, treatment of cultured preovulatory follicles with KITL did not affect GVBD and KITL has no effect on cytoplasmic maturation of preovulatory oocytes. CONCLUSION: Our findings suggest potential paracrine roles of KITL in the nuclear maturation of preovulatory oocytes by promoting first polar body extrusion.
Assuntos
Oócitos/metabolismo , Fator de Células-Tronco/fisiologia , Animais , Antígenos/análise , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Células do Cúmulo/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Feminino , Fertilização in vitro , Células da Granulosa/metabolismo , Hormônio Luteinizante/farmacologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Ovário/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologiaRESUMO
Recent studies indicate that LH stimulates production of ovarian paracrine factors that induce meiosis of the oocyte. DNA microarray analyses of ovarian transcripts were performed in mice and major increases of a short isoform of leptin receptor, ObRa, were identified by the preovulatory LH/human chorionic gonadotrophin (HCG) surge. In oocytes, the level of ObRa transcripts was increased shortly after HCG stimulation, whereas the level of ObRb transcripts was not changed. Leptin was produced by cumulus, granulosa, theca and interstitial cells of ovaries and its transcript level was not regulated during gonadotrophin treatment. Treatment with leptin promoted germinal vesicle breakdown (GVBD) in oocytes within preovulatory follicles, and enhance first polar body extrusion in both cumulus-oocyte complexes and denuded oocytes. The leptin-promoted GVBD and first polar body extrusion were blocked by a mitogen-activated protein kinase extracellular signal regulated kinase kinases (MEK)1/2 inhibitor, U0126, but not its inactive analogue U0124. Furthermore, leptin promoted fertilization of oocytes and the in-vitro development of zygotes to preimplantation embryos. These findings suggest paracrine roles of leptin in the enhancement of nuclear maturation of oocytes through MEK1/2 signalling, and in the promotion of cytoplasmic maturation essential for successful oocyte development to the preimplantation embryos.
Assuntos
Blastocisto , Leptina/metabolismo , Oócitos/citologia , Receptores para Leptina/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Gonadotropina Coriônica/sangue , Primers do DNA , Feminino , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Camundongos , Camundongos Endogâmicos ICR , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hormonal factors secreted by embryos and reproductive tracts are important for successful development of preimplantation embryos. We found expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) transcripts at its highest levels in the blastocyst stages. The transcripts for their receptor, TrkB, were detectable throughout the early embryonic stages with an increase after the early blastocyst stage. Both BDNF and TrkB are expressed in trophectoderm cells, whereas ligand-binding studies indicated specific binding of BDNF to trophectoderm cells. Furthermore, BDNF and NT-4/5 were produced in pregnant oviducts and uteri. Treatment with BDNF promoted the development of two-cell-stage embryos into blastocysts showing increased proliferation and decreased apoptosis. The effects of BDNF were blocked by the TrkB ectodomain or a Trk receptor inhibitor, K252a. Studies using specific inhibitors demonstrated the roles of the PI3K, but not the ERK, pathway in mediating BDNF actions. Under high-density embryo cultures, treatment with the TrkB ectodomain or K252a alone also inhibited embryonic development and survival, suggesting potential autocrine actions of BDNF produced by the embryo. In vivo experiments further demonstrated that K252a treatment suppressed early embryo development by inhibiting blastocyst cell numbers, and increasing blastocyst apoptosis. Our findings suggested that BDNF signaling plays important paracrine roles during blastocyst development by promoting the development of preimplantation embryos.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Embrião de Mamíferos/metabolismo , Animais , Feminino , Camundongos , Fatores de Crescimento Neural/metabolismo , Oviductos/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais , Útero/metabolismoRESUMO
Brain-derived neurotrophic factor (BDNF) acts through its cognate receptor tyrosine kinase-B (TrkB) to regulate diverse physiological functions in reproductive and other tissues. In normal and malignant trophoblastic cells, the BDNF/TrkB signaling promotes cell growth. Due to the highly malignant nature of choriocarcinoma, we investigated possible involvement of this system in choriocarcinoma cell invasion and metastasis. We demonstrated that treatment of cultured choriocarcinoma cells, known to express both BDNF and TrkB, with a soluble TrkB ectodomain or a Trk receptor inhibitor K252a suppressed cell invasion accompanied with decreased expression of matrix metalloproteinase-2, a cell invasion marker. In vivo studies using a tumor xenograft model in athymic nude mice further showed inhibition of cell invasion from tumors to surrounding tissues following the suppression of endogenous TrkB signaling. For an in vivo model of choriocarcinoma metastasis, we performed intravenous injections of JAR cells expressing firefly luciferase into severe combined immunodeficiency (SCID) mice. Treatment with K252a inhibited metastasis of tumors to distant organs. In vivo K252a treatment also suppressed metastatic tumor growth as reflected by decreased cell proliferation and increased apoptosis and caspases-3/7 activities, together with reduced tissue levels of a tumor marker, human chorionic gonadotropin-ß. In vivo suppression of TrkB signaling also led to decreased expression of angiogenic markers in metastatic tumor, including cluster of differentiation 31 and vascular endothelial growth factor A. Our findings suggested essential autocrine/paracrine roles of the BDNF/TrkB signaling system in choriocarcinoma invasion and metastasis. Inhibition of this signaling could serve as the basis to develop a novel therapy for patients with choriocarcinoma.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Carbazóis/uso terapêutico , Coriocarcinoma/tratamento farmacológico , Alcaloides Indólicos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Receptor trkB/antagonistas & inibidores , Neoplasias Uterinas/tratamento farmacológico , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carbazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Feminino , Humanos , Alcaloides Indólicos/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Camundongos SCID , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Receptor trkB/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrkB) receptor signaling promotes trophoblast growth in normal and abnormal pregnancy. It also regulates the growth of malignant trophoblastic, choriocarcinoma cells. However, possible involvement of this signaling system in hydatidiform mole, another major gestational trophoblastic disease, has not been determined. Here, we found the expression of BDNF in syncytiotrophoblasts and its receptor, TrkB, in cytotrophoblasts of hydatidiform mole using real-time RT-PCR and immunoassays. In molar explant cultures, treatment with soluble TrkB ectodomain or a Trk receptor inhibitor K252a inhibited trophoblast outgrowth as well as decreased cytotrophoblast proliferation and cellular viability based on histopathological analyses and glucose metabolism monitoring. These inhibitors also increased apoptosis and caspase-3/7 activities. In an in vivo model of hydatidiform molar growth based on xenotransplantation of molar tissues into kidney capsules of SCID mice, treatment with K252a suppressed molar growth as reflected by decreased trophoblast proliferation and their invasion into mouse kidney, reduced tissue levels of chorionic gonadotropin-ß, and increased apoptosis. Based on PCR array analyses to identify changes in expression profiles of cell cycle- and apoptosis-related genes in cultured molar explants, suppression of endogenous TrkB signaling led to decreases in key cell cycle-stimulatory and checkpoint genes together with the down-regulation of different antiapoptotic genes. Our findings demonstrate the importance of paracrine signaling by the BDNF/TrkB system in the proliferation and survival of molar trophoblasts. Inhibition of BDNF/TrkB signaling could provide a novel medical treatment for hydatidiform mole.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Mola Hidatiforme/metabolismo , Receptor trkB/metabolismo , Adulto , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Carbazóis/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Mola Hidatiforme/tratamento farmacológico , Alcaloides Indólicos/uso terapêutico , Camundongos , Camundongos SCID , Gravidez , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Uterine leiomyosarcoma is an aggressive tumor typically found at advanced stages due to difficulties with early diagnosis. Because uterine leiomyosarcoma is resistant to conventional radiation and chemotherapy, the development of more potent medical therapeutics is anticipated. Using quantitative real-time RT-PCR and immunostaining, we found the expression of brain-derived neurotrophic factor (BDNF) and neurotropin-4/5, together with their receptor, tyrosine kinase B (TrkB), in different uterine sarcoma cell lines and primary tumor samples from uterine leiomyosarcoma patients. We noted that levels of BDNF were more abundant than those of neurotropin-4/5. Moreover, the expression of TrkB and its ligands was elevated in a multidrug-resistant cell line and samples obtained from patients with leiomyosarcoma. In cultured uterine sarcoma cells, inhibition of endogenous TrkB signaling by treatment with either the soluble TrkB ectodomain or the Trk receptor inhibitor, K252a, suppressed cell proliferation and increased apoptosis based on cell viability and proliferation, in situ terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labeling and caspase-3/7 assays, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Correspondingly, treatment with exogenous BDNF increased cell proliferation. In in vivo studies in athymic nude mice bearing multidrug-resistant uterine sarcoma cell tumors, we demonstrate suppression of tumor growth by treatment with K252a, but not K252b, as reflected by decreased cell proliferation and increased levels of apoptosis and caspase-3/7 activities without obvious side effects. Our findings indicated that endogenous signaling of the TrkB pathway contributed to uterine sarcoma cell growth, and inhibition of TrkB signaling in these tumors could provide a novel medical therapy for patients with uterine sarcomas.
Assuntos
Receptor trkB/metabolismo , Sarcoma/metabolismo , Transdução de Sinais , Neoplasias Uterinas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Galectina 1/genética , Galectina 1/metabolismo , Expressão Gênica , Humanos , Leiomiossarcoma/genética , Leiomiossarcoma/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Sarcoma/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Uterinas/genéticaRESUMO
Although medical treatment of unruptured ectopic pregnancy using methotrexate has been established, development of more potent and safer medical treatment is needed due to limited indications and side effects of methotrexate. Brain-derived neurotrophic factor (BDNF) signals through its receptor tyrosine kinase B (TrkB) to regulate the growth of malignant trophoblastic, choriocarcinoma cell. We investigated possible involvement of this signaling system in nonmalignant human trophoblast growth in both ectopic and intrauterine pregnancy. Here, we demonstrated the expression of BDNF in syncytiotrophoblasts and extravillous trophoblasts (EVTs) together with TrkB in cytotrophoblasts and EVTs in human placental villi during both normal and ectopic pregnancies. Treatment of cultured villous explants with soluble TrkB ectodomain or a Trk receptor inhibitor K252a suppressed cytotrophoblast differentiation by inhibiting EVT outgrowth reflected by decreased levels of an EVT marker, human leukocyte antigen-G. These inhibitors also decreased cytotrophoblast proliferation and cellular viability based on histopathological analyses and monitoring glucose metabolism, together with increased apoptosis in cytotrophoblasts based on in situ terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labeling and caspase-3/7 assays. After xenotransplantation of human placental villi into SCID mice as an in vivo model of ectopic pregnancy, treatment with K252a suppressed transplanted villi growth as reflected by decreased cytotrophoblast differentiation and proliferation, reduced tissue levels of chorionic gonadotropin-ß, and increased apoptosis and caspase-3/7 activities. Thus, paracrine signaling by the BDNF/TrkB system is important for human cytotrophoblast differentiation, proliferation, and survival, and inhibition of BDNF/TrkB signaling in cytotrophoblasts could provide a novel medical treatment for ectopic pregnancy.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Gravidez Ectópica/metabolismo , Receptor trkB/metabolismo , Trofoblastos/metabolismo , Adolescente , Adulto , Animais , Vilosidades Coriônicas/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Comunicação Parácrina , Gravidez , Receptor trkB/genética , Transdução de Sinais , Adulto JovemRESUMO
Brain-derived neurotrophic factor (BDNF) signals through its receptor tyrosine kinase (Trk)B to regulate the development trophoblast cells during peri- and postimplantation periods. Possible involvement of this signaling system in malignant human trophoblastic cell growth has not been investigated. Here, we found the expression of BDNF and neurtropin-4/5 together with TrkB in human trophoblastic choriocarcinoma cells. Treatment of cultured choriocarcinoma cells with a soluble TrkB ectodomain or a Trk receptor inhibitor K252a suppressed cell proliferation and increased apoptosis associated by the disruption of mitochondrial functions, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using these specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase and ERK pathways in mediating BDNF actions. Based on PCR array analyses to identify changes in expression profiles of cell cycle- and apoptosis-related genes in cultured choriocarcinoma cells, we found that suppression of endogenous TrkB signaling led to decreases in key proproliferation cell cycle genes and increases in two inhibitory cell cycle genes together with the up-regulation of several proapoptotic genes. In vivo studies in athymic nude mice bearing choriocarcinoma cell tumors further demonstrated that treatment with K252a, but not K252b, suppressed tumor growth accompanied by decreased cell proliferation, reduced levels of a tumor marker, human chorionic gonadotropin-beta, and increased levels of apoptosis and caspase-3/7 activities. Thus, autocrine signaling of the BDNF/TrkB system is important for human choriocarcinoma cell growth, and inhibition of BDNF/TrkB signaling in these cells could provide a novel therapy for patients with choriocarcinoma.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Receptor trkB/metabolismo , Animais , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/genética , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Coriocarcinoma/tratamento farmacológico , Gonadotropina Coriônica Humana Subunidade beta/genética , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Alcaloides Indólicos/farmacologia , Alcaloides Indólicos/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fatores de Crescimento Neural/genética , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Successful implantation of the blastocyst and subsequent placental development is essential for reproduction. Expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5, together with their receptor, tyrosine kinase B (TrkB), in trophectoderm cells of blastocyst suggests their potential roles in implantation and placental development. Here we demonstrated that treatment with BDNF promoted blastocyst outgrowth, but not adhesion, in vitro and increased levels of the cell invasion marker matrix metalloproteinase-9 in cultured blastocysts through the phosphatidylinositol 3-kinase pathway. After implantation, BDNF and neurotrophin-4/5 proteins as well as TrkB were expressed in trophoblast cells and placentas during different stages of pregnancy. Both TrkB and its ligands were also expressed in decidual cells. Treatment of cultured trophoblast cells with the TrkB ectodomain, or a Trk receptor inhibitor K252a, suppressed cell growth as reflected by decreased proliferation and increased apoptosis, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using the specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase/Akt pathway in mediating the action of TrkB ligands. In vivo studies in pregnant mice further demonstrated that treatment with K252a, but not K252b, suppressed placental development accompanied by increases in trophoblast cell apoptosis and decreases in placental labyrinth zone at midgestation. In vivo K252a treatment also decreased fetal weight at late gestational stages. Our findings suggested important autocrine/paracrine roles of the BDNF/TrkB signaling system during implantation, subsequent placental development, and fetal growth by increasing trophoblast cell growth and survival.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Implantação do Embrião/efeitos dos fármacos , Placentação/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Fatores de Crescimento Neural/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Placenta/citologia , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Receptor trkB/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
Survival and apoptosis of cells in preimplantation embryos are fundamental for successful pregnancy. Relevant to these processes, tumor necrosis factor (TNF) and transforming growth factor alpha (TGFA) are produced by mammalian oviducts and uteri. In early embryos, TNF induces apoptosis, whereas TGFA could act as a survival factor. Here we investigated the TNF regulation of apoptosis in early mouse embryos and its antagonism by TGFA. TNF receptor superfamily, member 1a mRNA was detectable throughout early embryonic stages, with an increase after the early blastocyst stage, whereas the expression of TNF receptor superfamily, member 1b transcripts were detected only at the expanded blastocyst stage. Although pregnant uteri produced TNF, physiologic levels were low during the preimplantation period. Treatment with TNF inhibited the development of two-cell stage embryos to blastocysts showing decreased proliferation and increased apoptosis both in vitro and in vivo. These detrimental effects of TNF on early embryo development and survival were blocked by a neutralizing anti-TNF antibody. In addition to the death receptor-mediated pathway, TNF-induced apoptosis was further mediated by disruption of mitochondrial functions, characterized by release of cytochrome c and activation of caspase 9. The proapoptotic effects of TNF in blastocysts were counteracted by cotreatment with TGFA. The antagonistic effect of TGFA on TNF-induced apoptosis was blocked by phosphatidylionsitol 3-kinase (PI3K) inhibitors. The present findings demonstrate the stage-selective susceptibility to the apoptosis-inducing effect of TNF in mouse preimplantation embryos and that the TGFA/PI3K signaling system has an important role in the control of TNF-induced apoptosis in blastocysts.
Assuntos
Apoptose/efeitos dos fármacos , Blastocisto/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Fator de Crescimento Transformador alfa/farmacologia , Inibidores do Fator de Necrose Tumoral , Fatores de Necrose Tumoral/farmacologia , Animais , Blastocisto/metabolismo , Inibidores de Caspase , Citocromos c/metabolismo , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos , Mitocôndrias/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismoRESUMO
Optimal development of fertilized eggs into preimplantation embryos is essential for reproduction. Although mammalian oocytes ovulated after luteinizing hormone (LH) stimulation can be fertilized and promoted into early embryos in vitro, little is known about ovarian factors important for the conditioning of eggs for early embryo development. Because LH interacts only with ovarian somatic cells, its potential regulation of oocyte functions is presumably mediated by local paracrine factors. We performed DNA microarray analyses of ovarian transcripts and identified brain-derived neurotrophic factor (BDNF) secreted by granulosa and cumulus cells as an ovarian factor stimulated by the preovulatory LH surge. Ovarian BDNF acts on TrkB receptors expressed exclusively in oocytes to enhance first polar body extrusion of oocytes and to promote the in vitro development of zygotes into preimplantation embryos. Furthermore, in vivo treatment with a Trk receptor inhibitor suppressed first polar body extrusion and the progression of zygotes into blastocysts. Thus, ovarian BDNF is important to nuclear and cytoplasmic maturation of the oocyte, which is essential for successful oocyte development into preimplantation embryos. Treatment with BDNF could condition the cultured oocytes for optimal progression into the totipotent blastocysts.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Animais , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Fertilização , Gonadotropinas/metabolismo , Hormônio Luteinizante/metabolismo , Camundongos , Microscopia de Fluorescência , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Folículo Ovariano/metabolismo , Ovulação , Receptor de Fator de Crescimento Neural , Receptor trkB/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
We investigated the pathway of differentiation in a case of mucinous carcinoma of the skin (MCS) arising on the right temple of a 70-year-old man. Histopathologic findings of the tumor showed evidence of apocrine-type differentiation such as distinctive decapitation secretion. Additionally, by employing a panel of antibodies, the immunohistochemical staining pattern of tumor cells was shown to be compatible with that of apocrine glands. Although the differentiation of this neoplasm remains controversial, the findings in our case suggest apocrine differentiation.