RESUMEN
Embryo implantation into the uterine endometrium is required for pregnancy establishment in most mammals. By using global expression analysis, we investigated the molecules that are related to epithelial-mesenchymal transition (EMT) in noninvasive bovine trophoblasts and found that the transcription factor, ovo-like zinc finger 2 ( OVOL2), which is essential for mesenchymal-epithelial transition in various cancers, was down-regulated after trophoblast attachment to the endometrial epithelium in utero. In cultured bovine trophoblast cells, OVOL2 down-regulation occurred only when cells were allowed to attach to bovine endometrial epithelial cells via the TEAD3/YAP signaling pathway. This resulted in the up-regulation of the EMT-associated transcription factors, ZEB1 and SNAI2, and the mesenchymal cell markers, N-cadherin ( CDH2) and vimentin ( VIM), whereas epithelial cell marker, E-cadherin ( CDH1), was down-regulated. In contrast, OVOL2 overexpression in bovine trophoblast cells exhibited a decrease in ZEB1 transcripts and an increase in E-cadherin. These observations revealed that ovo-like protein (OVOL)2 down-regulation occurred concurrently with conceptus implantation into the uterine endometrium via the YAP/TEAD3 signaling pathway, and suggest that the down-regulation of OVOL2 expression contributes to the up-regulation of EMT-related transcription factor expression, which enables EMT progression in the noninvasive bovine trophectoderm postimplantation.-Bai, R., Kusama, K., Nakamura, K., Sakurai, T., Kimura, K., Ideta, A., Aoyagi, Y., Imakawa, K. Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.
Asunto(s)
Regulación hacia Abajo/fisiología , Implantación del Embrión/fisiología , Endometrio/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Transcripción/biosíntesis , Trofoblastos/metabolismo , Animales , Bovinos , Endometrio/citología , Femenino , Embarazo , Transducción de Señal/fisiología , Trofoblastos/citologíaRESUMEN
Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell-cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.
Asunto(s)
Retrovirus Endógenos/genética , Regulación del Desarrollo de la Expresión Génica , Placenta/fisiología , Transcripción Genética/fisiología , Vía de Señalización Wnt/fisiología , Animales , Bovinos , Femenino , EmbarazoRESUMEN
As placental morphology as well as trophoblast characteristics exhibit wide diversity across mammalian species, underling molecules were also thought to vary greatly. In the majority of cases, however, regardless of the mode of implantation, physiological and biochemical processes in conceptus implantation to the maternal endometrium including the kinds of gene expression and their products are now considered to share many similarities. In fact, recent progress has identified that in addition to the hormones, cytokines, proteases and cell adhesion molecules classically characterized, molecules related to lymphocyte homing and epithelial-mesenchymal transition (EMT) are all required for the progression of conceptus implantation to placentation. In this review, therefore, the newest findings are all incorporated into the molecular and cellular events related to conceptus implantation to the maternal endometrium; primarily from non-invasive bovine placentation and also from invasive human implantation.
Asunto(s)
Implantación del Embrión/fisiología , Endometrio/metabolismo , Animales , Bovinos , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
Dynamic changes in bovine conceptus and endometrium occur during early gestation, in which the conceptus undergoes epithelial to mesenchymal transition (EMT) after the conceptus attachment to endometrium. To characterize EMT inducing factors, we initially undertook iTRAQ analysis with bovine uterine flushing (UF) obtained from pregnant animals on days 17 (P17: pre-attachment) and 20 (P20: post-attachment). The iTRAQ analysis demonstrated that follistatin (FST), an inhibitor of activin A, increased in P20 UF. We then found that FST decreased in P22 conceptuses, whereas elevated activin A found in P20 UF and endometria was further increased on P22. In addition, phosphorylated SMAD2 increased in P22 conceptuses. In bovine trophoblast cells, the treatment with P22 UF or activin An up-regulated EMT marker expressions, which were inhibited by FST. These results suggest that the initiation of bovine conceptus EMT could be regulated through the spatiotemporal expression of FST or activin A during the peri-attachment period.
Asunto(s)
Activinas/metabolismo , Endometrio/embriología , Transición Epitelial-Mesenquimal , Folistatina/metabolismo , Animales , Bovinos , Células Cultivadas , Endometrio/metabolismo , Femenino , Fosforilación , Embarazo , Proteína Smad2/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismoRESUMEN
Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 (NANOS3(-/-)) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres (NANOS3(+/+)) were injected into NANOS3(-/-) Wagyu embryos. Subsequently, exogenous germ cells (NANOS3(+/+)) were identified in the NANOS3(-/-) ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies.
Asunto(s)
Diferenciación Celular , Células Germinativas/fisiología , Ovario/fisiología , Proteínas de Unión al ARN/genética , Animales , Blastómeros/fisiología , Bovinos , Femenino , Técnicas de Inactivación de GenesRESUMEN
A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.
Asunto(s)
Implantación del Embrión/fisiología , Endometrio/fisiología , Epitelio/fisiología , Feto/fisiología , Selectinas/fisiología , Útero/fisiología , Animales , Factor de Transcripción CDX2 , Bovinos , Células Cultivadas , Femenino , Proteínas de Homeodominio/genética , Interferón Tipo I/genética , Ligandos , Glicoproteínas de Membrana/genética , Embarazo , Proteínas Gestacionales/genética , ARN/genética , ARN Interferente Pequeño/genética , Transactivadores/genéticaRESUMEN
Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.
Asunto(s)
Proteínas Anticongelantes Tipo III/química , Criopreservación/métodos , Animales , Bovinos , Supervivencia Celular , Frío , Medios de Cultivo , Técnicas de Cultivo de Embriones , Proteínas de Peces/química , Microscopía Fluorescente , Perciformes , Proteínas Recombinantes/química , Factores de TiempoRESUMEN
Following bidirectional communication, the conceptus and the uterine epithelium must establish a proper cell-cell interaction, resulting in the progression of implantation processes. To clarify the mechanism of conceptus attachment to the uterine endometrium, we studied whether vascular cell adhesion molecule (VCAM1) was expressed in bovine conceptuses or endometrium during the peri-attachment period. Uterine VCAM1 expression was minimal in day 17 (day 0=day of estrus) cyclic and pregnant animals, but increased between days 20 and 22 of pregnancy. In the intercaruncular regions, VCAM1 protein was localized to the luminal and glandular epithelia, whereas in the caruncular regions, VCAM1 protein was detected in the stroma and endothelia of the uterine endometrium. In cultured endometrial epithelial cells (EECs), VCAM1 expression was up-regulated when treated with uterine flushings or growth factor and further increased when EECs were cocultured with bovine trophoblast CT1 cells. VCAM1 expression in CT1 cells was also up-regulated with the use of uterine flushings, and further increased when these cells were cocultured with EECs. Expression of VCAM1 receptor, integrin α 4 (ITGA4) mRNA, increased significantly in day 22 conceptuses. In day 22 pregnant uteri, VCAM1 protein was found in both EECs and conceptuses, but ITGA4 was localized only to trophoblasts. These observations indicate that cell-cell interactions between conceptuses and uterine epithelial cells are required for sufficient VCAM1 and ITGA4 expression in the bovine species and suggest that uterine VCAM1 and conceptus ITGA4 play a role in the establishment of conceptus adhesion to the uterine endometrium.
Asunto(s)
Blastocisto/metabolismo , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Endometrio/metabolismo , Trofoblastos/metabolismo , Útero/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Blastocisto/citología , Western Blotting , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Implantación del Embrión , Endometrio/citología , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas para Inmunoenzimas , Integrina alfa4/genética , Integrina alfa4/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/citología , Útero/citología , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0 â=â day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1, which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions -637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ.
Asunto(s)
Interferón Tipo I/genética , Proteínas Gestacionales/genética , Animales , Bovinos , Células Cultivadas , Implantación del Embrión/genética , Implantación del Embrión/fisiología , Femenino , Interferón Tipo I/clasificación , Filogenia , Mutación Puntual/genética , Embarazo , Proteínas Gestacionales/clasificación , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The recent decline in fertility is a serious problem in the dairy industry. To overcome this problem, we performed a genome-wide association study using 384 Holsteins and identified four loci associated with conception rates. Two of them contained gap junction-related genes: PKP2 and CTTNBP2NL. Further analysis confirmed that PKP2 increased connexin 43, a gap junction protein, whereas CTTNBP2NL dephosphorylated connexin 43. Knockdown of PKP2 or overexpression of CTTNBP2NL inhibited embryo implantation in mice. The other two loci contained neuroendocrine-related genes: SETD6 and CACNB2. Additional experiments indicated that SETD6 is involved in the transcriptional regulation of gonadotropin-releasing hormone, whereas CACNB2 controlled the secretion of follicle-stimulating hormone in cattle. The total allele substitution effect of these genes on conception rate was 3.5%. Our findings reveal important roles for gap junction communication and the neuroendocrine system in conception and suggest unique selection methods to improve reproductive performance in the livestock industry.
Asunto(s)
Bovinos/genética , Fertilización/genética , Hormona Folículo Estimulante/metabolismo , Uniones Comunicantes/genética , Variación Genética , Animales , Canales de Calcio Tipo L/genética , Conexina 43/metabolismo , Femenino , Hormona Folículo Estimulante/genética , Estudio de Asociación del Genoma Completo , Genotipo , Luciferasas , Ratones , Placofilinas/genética , Polimorfismo de Nucleótido Simple/genética , Proteína Metiltransferasas/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Interferon tau (IFNT), produced for a short interval during early pregnancy by the ruminant embryonic trophectoderm, is essential for the maintenance of early pregnancy, but the mechanism by which it is transcriptionally regulated has not been fully determined. To identify a transcription factor(s) involved in the down-regulation of IFNT genes, mRNAs for various known transcription factors were investigated by reverse-transcriptase and real-time PCR in conceptus tissues collected on Days 15, 17, and 21, or Days 17, 20, and 22 of ovine or bovine pregnancy, respectively. In particular, the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in Day 17 or 22 ovine or bovine conceptuses. Interaction between EOMES and the identified transcription factors was studied using transient transfection, revealing that ovine/bovine IFNT-reporter transactivation was down-regulated by EOMES. Transcription factor interactions with EOMES were further studied through immunoprecipitation, demonstrating an association between EOMES and cAMP-response element binding protein (CREB)-binding protein (CREBBP). Uterine flushing media collected from cyclic or early pregnancy animals were added to bovine trophoblast CT-1 cells cultured on type-I collagen (monoculture) or bovine uterine epithelial cells (coculture) in an attempt to regulate EOMES expression. In the coculture, but not the monoculture, addition of uterine flushing from Day 17 pregnant animals resulted in increased EOMES expression in CT-1 cells. These results suggest that as conceptuses attach to the uterine epithelium, IFNT gene transcription is down-regulated by an increase in EOMES expression and EOMES-CREBBP binding in the attached trophoblast cells.
Asunto(s)
Regulación hacia Abajo/fisiología , Implantación del Embrión/fisiología , Interferón Tipo I/biosíntesis , Proteínas Gestacionales/biosíntesis , Embarazo/metabolismo , Proteínas de Dominio T Box/metabolismo , Trofoblastos/metabolismo , Animales , Proteína de Unión a CREB/metabolismo , Bovinos , Línea Celular , Femenino , Ovinos , Transcripción Genética/fisiología , Trofoblastos/citologíaRESUMEN
Cryopreservation methods using liquid nitrogen (LN(2)) for gametes and embryos are prevalent in mammalian artificial reproduction. However, the pregnancy rate from frozen embryos has not improved over the past two decades because freeze-thawing causes significant damage. The strict regulation of transportation of LN(2) containers by airlines also limits exchange between breeders. In this article, we introduce a medium that enabled bovine embryos to be held for up to 7 days at 4°C. A pregnancy rate of 75% (24/32) was obtained for embryos held for 7 days in this medium and transferred to primed recipients. Its constituents were medium 199, foetal bovine serum, and HEPES for buffering. This technique will enable LN(2)-free storage and air transportation of embryos provided transplantation to recipients can be completed within 7 days.
Asunto(s)
Criopreservación , Embrión de Mamíferos/fisiología , Animales , Tasa de Natalidad , Bovinos , Femenino , Fertilización In Vitro , Embarazo , Índice de Embarazo , Factores de TiempoRESUMEN
UNLABELLED: The effect of an anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, against the human to bovine cellular response was investigated. METHODS: Human peripheral mononuclear cells (PBMCs) were cocultured with inactivated self-PBMCs (Self), bovine PBMCs with control antibody (Xeno), or bovine PBMCs with IMMU-114 (IMMU-114). Cellular responses were investigated by thymidine incorporation assay, CFSE (carboxyfluorescein diacetate succinimidyl ester)-mixed lymphocyte reaction, and cytokine production in culture medium. RESULTS: Thymidine incorporation rates at a 1:1 responder to stimulator ratio for Xeno + control antibody, Xeno + IMMU-114, Self + control antibody, and Self + IMMU-114 were 14201.3 ± 1968.4, 513.0 ± 49.5, 952.7 ± 128.7, and 423.3 ± 138.8 cpm, respectively (P = 0.032). Those at a 1:2 ratio were 6518.0 ± 690.1, 896.6 ± 92.9, 1051.0 ± 123.6, and 736.0 ± 35.6 cpm, respectively (P = 0.036). CFSE-mixed lymphocyte reaction demonstrated that the frequencies of CFSE-low, CD4(+), and CD25(+) activating T cells in Self, Xeno, and IMMU-114 were 0.27 ± 0.04%, 3.65 ± 0.53%, and 1.23 ± 0.15%, respectively (P = 0.027). Cytokine production in culture medium indicated that IMMU-114 decreased Th1-type cytokines, including interleukin-2, interferon-γ, and tumor necrosis factor-α. CONCLUSION: IMMU-114 effectively suppresses human to bovine cellular responses. The mechanism involves direct inhibition of the interaction between class II human leukocyte antigen-DR-positive cells and CD4(+) T cells, and indirect suppression of Th1 cytokine production.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Antígenos HLA-DR/inmunología , Leucocitos Mononucleares/inmunología , Trasplante Heterólogo/inmunología , Animales , Anticuerpos Heterófilos/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Antígenos Heterófilos/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Citocinas/inmunología , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Terapia de Inmunosupresión/métodos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Timidina/farmacocinéticaRESUMEN
Erythrocytes were recently found to improve the early development of mice embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine in vitro fertilized (IVF) embryos in medium supplemented with reactive oxygen species (ROS). IVF embryos were cultured in CR1aa medium supplemented with oxidizing agents, 0.5mmol/L hypoxanthine and 0.01U/mL xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5×10(4) , 5×10(5) , 5×10(6) and 5×10(7) erythrocytes/mL). After 8 days, blastocysts were examined with a stereomicroscope. HX/XOD blocked development to the blastocyst stage (HX/XOD: 0%, control: 33%), but in the presence of both erythrocytes and HX/XOD, blastocyst development was restored to about one-third to two-thirds the normal rate (5×10(5) to 5×10(7) erythrocytes/mL: 12 to 23%). Furthermore, adding erythrocytes or erythrocyte hemolysate to medium without HX/XOD increased the blastocyst rate. These results suggest that the addition of erythrocytes can attenuate the detrimental effects of ROS on embryo development in bovine species as well as in mice.
Asunto(s)
Antioxidantes , Blastocisto/fisiología , Medios de Cultivo , Desarrollo Embrionario/fisiología , Eritrocitos/fisiología , Fertilización In Vitro , Especies Reactivas de Oxígeno/efectos adversos , Animales , Blastocisto/efectos de los fármacos , Bovinos , Células Cultivadas , Embrión de Mamíferos , Desarrollo Embrionario/efectos de los fármacos , Hipoxantina/efectos adversos , Técnicas In Vitro , Ratones , Oxidantes/efectos adversos , Superóxido Dismutasa/metabolismo , Xantina Oxidasa/efectos adversosRESUMEN
The establishment of a successful pregnancy requires a "fine quality embryo", "maternal recognition of pregnancy", and a "receptive uterus" during the period of conceptus implantation to the uterine endometrium. In ruminants, a conceptus cytokine, interferon tau (IFNT), a major cytokine produced by the peri-implantation trophectoderm, is known as a key factor for maternal recognition of pregnancy. IFNT can be considered one of the main factors in conceptus-uterus cross-talk, resulting in the rescue of ovarian corpus luteum (CL), induction of endometrial gene expressions, activation of residual immune cells, and recruitment of immune cells. Much research on IFNT has focused on the CL life-span (pregnancy recognition) and uterine gene expression through IFNT and related genes; however, immunological acceptance of the conceptus by the mother has not been well characterized. In this review, we will discuss the progress in IFNT and implantation research made by us and others for over 10 years, and relate this progress to pregnancy in mammalian species other than ruminants.
RESUMEN
The transcription factor GATA1 is known to play an essential role in hematopoiesis, but its other roles have not been well characterized. The purpose of this study was to determine relationships between GATA1 and GATA2 and/or GATA3, and to identify their possible functions in ovine development. GATA1 mRNA was found in ovine conceptuses and endometrial epithelial regions of Day 15 (Day 0=day of estrus) cyclic and Days 15, 17, and 21 pregnant ovine uteri. GATA1 mRNA was strongly expressed in conceptuses on Day 21, when trophoblast attachment to the maternal endometrium progressed. Similarly, GATA1 protein expression was relatively high on Day 21. To localize GATA1 mRNA, ovine conceptuses and pregnant uteri were subjected to in situ hybridization on Days 15, 17, and 21, confirming that GATA1 mRNA was expressed in trophoblasts and uterine endometrial epithelial cells in these gestation days. The presence of GATA1 protein was further confirmed by immunohistochemistry. Because high GATA1 expression appeared to coincide with reduced GATA2/3 expression, a potential role of GATA1 was examined through transfection of a mouse Gata1 expression plasmid into bovine trophoblast F3 cells. This over-expression resulted in the down-regulation of endogenous GATA2 transcripts. These observations indicate that GATA1 exists in the ovine conceptus and uterus during the peri-attachment period, and suggest that GATA1 is integral to conceptus and endometrial development through the regulation of GATA2 and possibly other developmentally important genes.
Asunto(s)
Implantación del Embrión/fisiología , Embrión de Mamíferos/metabolismo , Endometrio/metabolismo , Factor de Transcripción GATA1/biosíntesis , Animales , Bovinos , Embrión de Mamíferos/química , Endometrio/química , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA3/metabolismo , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ratones , Especificidad de Órganos , Embarazo , Ovinos , Transfección , TrofoblastosRESUMEN
In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.
Asunto(s)
Blastocisto/metabolismo , Bovinos , Implantación del Embrión/genética , Transición Epitelial-Mesenquimal/genética , Preñez , Trofoblastos/metabolismo , Animales , Blastocisto/fisiología , Bovinos/embriología , Bovinos/genética , Bovinos/metabolismo , Bovinos/fisiología , Endometrio/metabolismo , Endometrio/fisiología , Epitelio/metabolismo , Femenino , Regulación de la Expresión Génica , Mesodermo/metabolismo , Embarazo , Preñez/genética , Preñez/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The success rate of bovine somatic cell nuclear transfer (SCNT) embryos to full term has been reported to be higher with G1 cells than with G0 cells. To better understand the reason for this, we analyzed the kinetics of luminescence activity in bovine SCNT embryos from G0 and G1 cells carrying a luciferase gene under the control of the ß-actin promoter during early embryonic development. At 60-h postfusion, when bovine embryonic gene activation (EGA) begins, the luminescence activity was higher in G1-SCNT embryos than G0-SCNT embryos. Moreover, half of the G1-SCNT embryos exhibited homogeneous luminescence among the blastomeres, whereas more than half of the G0-SCNT embryos exhibited mosaic luminescence. To characterize the differential luminescence pattern in SCNT embryos, the expressions of several endogenous genes and the level of DNA methylation were determined in all blastomeres of SCNT embryos with or without luminescence. The expressions of several development-related genes (H2AFZ, GJA1, and BAX) and level of DNA methylation of the SCNT embryos with luminescence were the same as those of normal embryos produced by in vitro fertilization. A higher success rate in G1-SCNT embryos is thought to contribute to homogeneous expression among all blastomeres at EGA.
Asunto(s)
Blastómeros/metabolismo , Desarrollo Embrionario/fisiología , Fibroblastos/citología , Fase G1/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Transferencia Nuclear , Actinas/metabolismo , Animales , Blastómeros/citología , Bovinos , Conexina 43/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Fertilización In Vitro , Fibroblastos/fisiología , Técnicas In Vitro , Luciferasas/metabolismo , Masculino , Modelos Animales , Fase de Descanso del Ciclo Celular/fisiología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells.
Asunto(s)
Factor de Transcripción GATA2/biosíntesis , Factor de Transcripción GATA3/biosíntesis , Regulación de la Expresión Génica , Trofoblastos/citología , Animales , Bovinos , Línea Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Interferón Tipo I/metabolismo , Proteínas Gestacionales/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Trofoblastos/metabolismoRESUMEN
Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.