RESUMEN
Heat stress (HS) reduces reproductive performance of cattle, possibly by disrupting endocrine regulation such as prostaglandin (PG) production from uterus and estradiol 17ß production from the dominant follicle. Prostaglandin F2α (PGF2α) secretion from endometrium surges during the luteal phase due to tumor necrosis factor (TNF) α stimulation and a positive-feedback loop with oxytocin (OT) from the corpus luteum, ultimately triggering luteolysis, while interferon τ (IFNT) inhibits upregulation of PGF2α production by TNFα and OT, thereby preventing luteolysis and triggering recognition of pregnancy. In the present study, we investigated the effect of OT, TNFα, and IFNT on PGF2α production in both types of endometrial cells under HS conditions. Stimulation of PGF2α production in endometrial epithelial cells by OT was unaffected by HS, while stimulation of PGF2α production in endometrial stromal cells by TNFα was enhanced by HS, and this increased PGF2α production was not significantly suppressed by IFNT. These results suggest that HS disrupted the regulation of PGF2α production by TNFα and IFNT in bovine endometrial stromal cells and it might be one of causes for low conception rate of cattle in summer.
Asunto(s)
Dinoprostona , Proteínas Gestacionales , Animales , Bovinos , Dinoprost , Endometrio , Femenino , Respuesta al Choque Térmico , Interferón Tipo I , EmbarazoRESUMEN
Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG) E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.
Asunto(s)
Dinoprost/biosíntesis , Dinoprostona/biosíntesis , Endometrio/metabolismo , Respuesta al Choque Térmico/fisiología , Calor , Células del Estroma/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Bovinos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Femenino , Hidroxiprostaglandina Deshidrogenasas/genética , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismoRESUMEN
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
Progesterone (P4) acts through different actuating pathways called genomic and non-genomic pathways. Here we investigated whether P4 regulates prostaglandin (PG) F2? (PGF) and PGE2 production in bovine endometrium through different pathways. Cultured endometrial cells were exposed to P4 for a short time (5-20min) or bovine serum albumin (BSA)-conjugated P4 (P4-BSA) for 24h. Progesterone treatment for 24h stimulated PGE2 production in epithelial cells, but suppressed both PGF and PGE2 production and the expression of PG-metabolising enzymes including phospholipase A2 (PLA2) and cyclooxygenase-2 (COX2) in stromal cells. Short-term (5-20min) P4 treatment did not affect PLA2 or COX2 transcript levels in either cell type. P4-BSA increased PGF and PGE2 production only in epithelial cells. Nuclear P4 receptor mRNA expression in endometrium was higher at the follicular phase than at the early- to mid-luteal stages, whereas membrane P4 receptor mRNA expression did not change throughout the oestrous cycle. The overall results suggest that P4 controls PG production by inhibiting enzymes via a genomic pathway and by stimulating signal transduction via a non-genomic pathway. Consequently, P4 may protect the corpus luteum by attenuating PGF production in stromal cells and by increasing PGE2 secretion from epithelial cells.
RESUMEN
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
Bovine primary uterine endometrial epithelial cells (EECs) are not ideal for long-term studies, because primary EECs lose hormone responsiveness quickly, and/or they tend to have a short life span. The aims of this study were to establish immortalized bovine EECs and to characterize these cells following long-term cultures. Immortalized bovine EECs were established by transfecting retroviral vectors encoding human papillomavirus (HPV) E6 and E7, and human telomerase reverse transcriptase (hTERT) genes. Established bovine immortalized EECs (imEECs) showed the same morphology as primary EECs, and could be grown without any apparent changes for over 60 passages. In addition, imEECs have maintained the features as EECs, exhibiting oxytocin (OT) and interferon tau (IFNT) responsiveness. Therefore, these imEECs, even after numbers of passages, could be used as an in vitro model to investigate cellular and molecular mechanisms, by which the uterine epithelium responds to IFNT stimulation, the event required for the maternal recognition of pregnancy in the bovine species.
Asunto(s)
Técnicas Citológicas/métodos , Endometrio/citología , Animales , Bovinos , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Dinoprost , Endometrio/metabolismo , Femenino , Vectores Genéticos , Interferón Tipo I/farmacología , Queratinas/metabolismo , Proteínas Oncogénicas Virales/genética , Oxitocina/farmacología , Proteínas E7 de Papillomavirus/genética , Embarazo , Proteínas Gestacionales/farmacología , Retroviridae/genética , Estimulación Química , Telomerasa/genética , Factores de Tiempo , Transfección , Vimentina/metabolismoRESUMEN
Cortisol (Cr), the most important glucocorticoid (GC), is well known to suppress uterine prostaglandin F2α (PGF) production. However, the details of the regulatory mechanisms controlling the cyclic changes in endometrial PGF production remain unclear. Here we investigated the expression of the GC receptor (GC-Rα), the actions of cortisol throughout the estrous cycle and the regulatory mechanism of GC-Rα in the bovine endometrium. The levels of GC-Rα protein were greater at the mid-luteal stage (Days 8-12) than at the other stages. Cr more strongly suppressed PGF production at the mid-luteal stage than at the follicular stage. GC-Rα expression was increased by progesterone (P4) but decreased by estradiol-17ß (E2) in cultured endometrial stromal cells. The overall results suggest that ovarian steroid hormones control the cyclic changes in endometrial PGF production by regulating GC-Rα expression in bovine endometrial stromal cells.