RESUMEN
The mammalian olfactory mucosa (OM) is continually renewed throughout life. Owing to their position in the nasal cavity, OM cells are exposed to multiple insults, including high levels of odourants that can induce their death. OM regeneration is therefore essential to maintain olfactory function, and requires the tight control of both cell death and proliferation. Apoptosis has been implicated in OM cell death. Olfaction is one of the senses involved in food intake and depends on individual nutritional status. We have previously reported the influence of hormones related to nutritional status on odour perception and have shown that the OM is a target of insulin and leptin, two hormones known for their anti-apoptotic properties. In the present study, we investigated the potential anti-apoptotic effect of these metabolic hormones on OM cells. Both Odora cells (an olfactive cell line) and OM cells treated with etoposide, a p53 activity inducer, exhibited mitochondrial-dependent apoptosis that was inhibited by the pan-caspase inhibitor zVAD-fmk. Insulin, but not leptin, impaired this apoptotic effect. Insulin addition to the culture medium reduced p53 phosphorylation, caspase-3 and caspase-9 cleavage, and caspase-3 enzymatic activity induced by etoposide. The apoptotic wave observed in the OM after interruption of the neuronal connections between the OM and the olfactory bulb by bulbectomy was impaired by intranasal insulin treatment. These findings suggest that insulin may be involved in OM cellular dynamics, through endocrine and/or paracrine-autocrine effects of circulating or local insulin, respectively.
Asunto(s)
Apoptosis/efectos de los fármacos , Insulina/farmacología , Leptina/farmacología , Mucosa Olfatoria/efectos de los fármacos , Animales , Animales Recién Nacidos , Antineoplásicos Fitogénicos/farmacología , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Citoprotección/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Etopósido/farmacología , Masculino , Mucosa Olfatoria/fisiología , Ratas , Ratas WistarRESUMEN
In mammals, the olfactory sensory neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are continuously renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory sensory neurons preferentially express ETB receptors, while ETA receptors are rather present in non neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect in the olfactory mucosa using a primary culture of olfactory sensory neurons lying on non neuronal cells. While a serum deprivation led to a massive decrease of the density of olfactory sensory neurons in the primary cultures, endothelin 1 (ET-1) rescued part of the neuronal population through both ETA and ETB receptors. This effect was mainly anti-apoptotic as it reduced cleaved caspase-3 signal and nuclear condensation. Furthermore, the olfactory epithelium of ETB-deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory sensory neurons, directly through ETB and indirectly by limiting non neuronal cells death through ETA.
Asunto(s)
Citoprotección/fisiología , Endotelina-1/fisiología , Mucosa Olfatoria/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Citoprotección/efectos de los fármacos , Citoprotección/genética , Endotelina-1/genética , Técnicas de Inactivación de Genes , Masculino , Fármacos Neuroprotectores/farmacología , Mucosa Olfatoria/citología , Mucosa Olfatoria/efectos de los fármacos , Neuronas Receptoras Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/metabolismo , Ratas , Ratas Mutantes , Ratas Wistar , Receptor de Endotelina B/deficiencia , Receptor de Endotelina B/genética , Receptor de Endotelina B/fisiologíaRESUMEN
Food odours are major determinants for food choice, and their detection depends on nutritional status. The effects of different odour stimuli on both behavioural responses (locomotor activity and sniffing) and Fos induction in olfactory bulbs (OB) were studied in satiated or 48-h fasted rats. We focused on two odour stimuli: isoamyl acetate (ISO), as a neutral stimulus either unknown or familiar, and food pellet odour, that were presented to quiet rats during the light phase of the day. We found significant effects of nutritional status and odour stimulus on both behavioural and OB responses. The locomotor activity induced by odour stimuli was always more marked in fasted than in satiated rats, and food odour induced increased sniffing activity only in fasted rats. Fos expression was quantified in periglomerular, mitral and granular OB cell layers. As a new odour, ISO induced a significant increase in Fos expression in all OB layers, similar in fasted and satiated rats. Significant OB responses to familiar odours were only observed in fasted rats. Among the numerous peptides shown to vary after 48 h of fasting, we focused on orexins (for which immunoreactive fibres are present in the OB) and leptin, as a peripheral hormone linked to adiposity, and tested their effects of food odour. The administration of orexin A in satiated animals partially mimicked fasting, since food odour increased OB Fos responses, but did not induce sniffing. The treatment of fasted animals with either an orexin receptors antagonist (ACT-078573) or leptin significantly decreased both locomotor activity, time spent sniffing food odour and OB Fos induction in all cell layers, thus mimicking a satiated status. We conclude that orexins and leptin are some of the factors that can modify behavioural and OB Fos responses to a familiar food odour.
Asunto(s)
Conducta Animal , Alimentos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leptina/fisiología , Neuropéptidos/fisiología , Odorantes , Bulbo Olfatorio/metabolismo , Pentanoles , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Animales , Ayuno , Péptidos y Proteínas de Señalización Intracelular/farmacología , Leptina/farmacología , Masculino , Actividad Motora , Neuropéptidos/farmacología , Receptores de Orexina , Orexinas , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leptina/metabolismo , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores de Neuropéptido/metabolismo , SaciedadRESUMEN
Food odours are major determinants for food choice; their detection is influenced by nutritional status. Among different metabolic signals, insulin plays a major role in food intake regulation. The aim of the present study was to investigate a potential role of insulin in the olfactory mucosa (OM), using ex vivo tissues and in vitro primary cultures. We first established the expression of insulin receptor (IR) in rat olfactory mucosa. Transcripts of IR-A and IR-B isoforms, as well as IRS-1 and IRS-2, were detected in OM extracts. Using immunocytochemistry, IR protein was located in olfactory receptor neurones, sustentacular and basal cells and in endothelium of the lamina propria vessels. Moreover, the insulin binding capacity of OM was quite high compared to that of olfactory bulb or liver. Besides the main pancreatic insulin source, we demonstrated insulin synthesis at a low level in the OM. Interestingly 48 h of fasting, leading to a decreased plasmatic insulin, increased the number of IR in the OM. Local insulin concentration was also enhanced. These data suggest a control of OM insulin system by nutritional status. Finally, an application of insulin on OM, aiming to mimic postprandial insulin increase, reversibly decreased the amplitude of electro-olfactogramme responses to odorants by approximately 30%. These data provide the first evidence that insulin modulates the most peripheral step of odour detection at the olfactory mucosa level.
Asunto(s)
Insulina/metabolismo , Mucosa Olfatoria/metabolismo , Receptor de Insulina/metabolismo , Animales , Células Cultivadas , Ingestión de Alimentos , Electrofisiología , Ayuno , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Estado Nutricional , Odorantes , Mucosa Olfatoria/citología , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , Isoformas de Proteínas/metabolismo , Radioinmunoensayo , Ratas , Ratas Wistar , Receptor de Insulina/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Placental growth hormone (PGH) is the product of the GH-V gene, predominantly expressed in the syncytiotrophoblast layer of the human placenta. PGH differs from pituitary growth hormone by 13 amino acids and possesses one glycosylation site. It has high somatogenic and low lactogenic activities. In the maternal circulation from 12-20 weeks up to term, PGH gradually replaces pituitary growth hormone, which becomes undetectable. PGH is secreted by the placenta in a non-pulsatile manner. This continuous secretion appears to have important implications for physiological adjustment to gestation and especially in the control of maternal IGF1 levels. PGH secretion is regulated in vitro and in vivo by glucose. Lower maternal levels of PGH are observed in pregnancies with fetal growth retardation. PGH is one example of a trophoblast hormone, which allows maternal metabolic adaptation to pregnancy. In addition, our recent data on its expression in invasive extravillous trophoblasts suggest that the physiological role of PGH might also include a direct influence of this hormone on placental development via an autocrine or paracrine mechanism.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hormonas Placentarias/metabolismo , Embarazo/fisiología , Trofoblastos/metabolismo , Adulto , Femenino , Edad Gestacional , Hormona del Crecimiento/genética , Humanos , Intercambio Materno-Fetal/fisiología , Técnicas de Cultivo de Órganos , Hormonas Placentarias/genéticaRESUMEN
The factors controlling normal placental development are poorly understood. We have previously reported the presence of ovine placental growth hormone (oPGH) and growth hormone receptors in ovine placenta, and oPGH production by the trophectoderm and syncitium during the second month of pregnancy. To identify factors regulating oPGH production, we developed a perifusion system to measure oPGH and ovine placental lactogen (oPL) production by Day 45 ovine placental explants. The mRNAs for both hormones were quantitated by real-time polymerase chain reaction in explants collected after perifusion periods of up to 8 h. Ovine PGH and oPL were released into the medium at mean rates of 2.45 +/- 0.2 and 353.6 +/- 13.6 ng/g/h, respectively. Ovine placenta produces growth hormone-releasing hormone (GHRH), but addition of GHRH to the perifusion medium did not modify either oPGH or oPL production. In vivo, oPGH production occurs between Days 30 and 60 of pregnancy. Because modulation of the maternal diet during this period affects placental development, the potential regulation of oPGH and oPL production by glucose was evaluated. Glucose supplementation of the perifusion medium resulted in a concentration-dependent decrease in oPGH release after 4 h, but oPGH mRNA levels were not affected. Production of oPL was not affected by glucose. Thus, oPGH and oPL belong to the same growth hormone/prolactin family but are differentially regulated by glucose. Ovine PGH modulations should be taken into account in metabolic experiments performed during the first trimester of pregnancy in sheep.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/biosíntesis , Placenta/metabolismo , Hormonas Placentarias/biosíntesis , Lactógeno Placentario/biosíntesis , Ovinos/metabolismo , Animales , Femenino , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Técnicas In Vitro , Hormonas Placentarias/genética , Hormonas Placentarias/metabolismo , Lactógeno Placentario/genética , Lactógeno Placentario/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Embarazo , ARN Mensajero/análisis , Factores de TiempoRESUMEN
Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M(r) precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M(r) forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M(r) forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.
Asunto(s)
Líquido Amniótico/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Factor II del Crecimiento Similar a la Insulina/fisiología , Mitógenos/fisiología , Animales , Bioensayo , Técnicas de Cultivo de Célula , Femenino , Cabras/fisiología , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/química , Ratones , Mitógenos/química , Mitosis , Peso Molecular , Embarazo , Ovinos/fisiologíaRESUMEN
Nitric oxide (NO) is involved in the transmission of light information to suprachiasmatic nuclei (SCN). By immunocytochemistry, we showed that both neuronal and endothelial NO synthase isoforms (nNOS and eNOS) were present in the SCN of rats and hamsters. nNOS-immunoreactive neurons were located mainly around the SCN with only a few nNOS neurons within the nucleus. By double-label immunocytochemistry, we also found, within the population of SCN glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes, a subpopulation of eNOS-immunoreactive astrocytes. Using Western blot analysis, we detected in SCN protein extracts eNOS and nNOS proteins having the expected 140 and 150 kDa molecular weights, respectively. By in situ hybridization of a 2.4-kb murine eNOS probe, mRNA for eNOS was located in the SCN of rats and hamsters. The transcript was further identified by detection of a RT-PCR product of the predicted size, after amplification of total RNA with primers specific for eNOS. In the SCN and cerebellum, the size of the mRNA for nNOS, detected with a rat probe on Northern blot, was approximately 10.5 kb, corresponding to that previously published. In the same tissues, we found two transcripts, one weakly expressed at approximately 4.0 kb and another more strongly expressed at approximately 2.6 kb, both hybridizing with two non-overlapping murine and rat eNOS probes. These results suggested the existence in the SCN of alternate transcripts for eNOS. We propose that two pathways could link light stimuli and NO release in the SCN: one involving N-methyl-D-aspartate (NMDA) receptors and nNOS in neurons; the other linking alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and eNOS in astrocytes.
Asunto(s)
Mesocricetus/metabolismo , Proteínas del Tejido Nervioso/análisis , Neuronas/enzimología , Óxido Nítrico Sintasa/análisis , Ratas/metabolismo , Núcleo Supraquiasmático/enzimología , Empalme Alternativo , Animales , Astrocitos/enzimología , Biomarcadores , Western Blotting , Ritmo Circadiano/fisiología , Cricetinae , Inducción Enzimática/efectos de la radiación , Proteína Ácida Fibrilar de la Glía/análisis , Hibridación in Situ , Luz , Masculino , Mesocricetus/anatomía & histología , Ratones , NADPH Deshidrogenasa/análisis , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , ARN Mensajero/análisis , Ratas/anatomía & histología , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Núcleo Supraquiasmático/efectos de la radiaciónRESUMEN
In a previous study we showed the existence of GH in the ovine placenta. We now supplement the information available on placental GH and describe the presence and distribution of GH receptor (GH-R) messenger RNA (mRNA) in uterine, fetal, and placental tissues during early pregnancy. GH mRNA was not detected in the placenta before day 27 (d27). Its expression peaked between d40 and d45 and fell after d55. GH mRNA was localized in the trophectoderm and syncytium. During the d35-d50 period, concentrations of GH in the maternal circulation were not increased. In umbilical blood, however, GH was detected from d35 and was presumed to be of placental origin, because GH mRNA was not detected in the fetal pituitary gland on d40. We report on GH-R mRNA expression in the placenta between d20-d120. The relative abundance of GH-R transcripts increased significantly between d25-d43. In the endometrium, GH-R mRNA was detected from d8-d120 of pregnancy and from d4-d16 of the cycle. GH-R mRNA was localized in the trophectoderm, fetal mesoderm, and maternal uterine stroma. In the fetal liver, GH-R mRNA was first detectable on d35. The results of this study indicate that between d35-d50 of pregnancy, the endometrium, placenta, and fetus are all potential targets for the placental GH.
Asunto(s)
Feto/metabolismo , Expresión Génica , Hormona del Crecimiento/genética , Placenta/metabolismo , Receptores de Somatotropina/genética , Alantoides/metabolismo , Líquido Amniótico/química , Animales , Endometrio/química , Femenino , Sangre Fetal/química , Edad Gestacional , Hormona del Crecimiento/análisis , Hormona del Crecimiento/sangre , Hígado/química , Hígado/embriología , Hipófisis/química , Hipófisis/embriología , Placenta/química , Embarazo , ARN Mensajero/análisis , Ovinos , Trofoblastos/química , Útero/químicaRESUMEN
We report the ability of sheep placental cotyledonary cells, isolated at different periods of pregnancy (40 to 90 days) to produce ovine chorionic somatomammotrophin (oCS) in in vitro culture conditions. This oCS production increased gradually with stage of pregnancy. Endogenous oCS net production by isolated placental cells was increased, in a dose-dependent manner, by addition of recombinant oCS (roCS). This effect was not observed after addition of recombinant ovine growth hormone. The roCS effect was more potent on cells collected during early pregnancy. Specific immunoprecipitation of oCS revealed that roCS treatment was associated with an increased dose-dependent incorporation of [35S]methionine-[35S]cysteine. These findings provide evidence that oCS may act in a paracrine/autocrine manner to up-regulate its own production during early gestation. We suggest that this autoregulation may be associated with morphological and functional differentiation of the trophoblast during the growth of the placenta.
Asunto(s)
Homeostasis/fisiología , Placenta/metabolismo , Lactógeno Placentario/biosíntesis , Preñez/metabolismo , Ovinos/metabolismo , Animales , Northern Blotting , Técnicas de Cultivo de Célula , Cisteína/metabolismo , Femenino , Expresión Génica , Hormona del Crecimiento/farmacología , Metionina/metabolismo , Placenta/citología , Placenta/efectos de los fármacos , Lactógeno Placentario/genética , Lactógeno Placentario/farmacología , Embarazo , ARN Mensajero/genética , Proteínas Recombinantes/farmacologíaRESUMEN
In several species, placenta has been found to express GH-related proteins. In the ovine placenta, such a protein, ovine chorionic somatommamotropin, has been described, but its involvement in the fetal/placental growth process is not clearly established. The aim of this study was to investigate the occurrence of another GH-related peptide in the ovine placenta. Placental extracts (days 30-140 of pregnancy) showed GH immunoreactivity between days 35-70. SDS-PAGE analysis of these extracts indicated that this immunoreactivity corresponded to 22- and 28-kDa proteins. GH-like immunoreactivity was localized on cotyledonary frozen sections in the syncytium and the trophectoderm. Northern blot analysis of placental RNA showed the expression of GH-hybridizing transcripts migrating to the same position as that of GH pituitary messenger RNA (mRNA). Those transcripts were highly expressed between days 40 and 50. Their sequence analysis showed the existence of three GH mRNA (GHP1, GHP2, and GHP3). GHP1 is identical to pituitary GH mRNA and probably codes for the 22-kDa protein. GHP2 and GHP3 encode the same protein, which differs from GHP1 by four amino acids. This study establishes the expression of GH gene and GH-immunoreactive proteins in the ovine placenta.
Asunto(s)
Hormona del Crecimiento/biosíntesis , Placenta/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Cartilla de ADN , Femenino , Hormona del Crecimiento/análisis , Datos de Secuencia Molecular , Placenta/citología , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Ovinos , Factores de TiempoRESUMEN
Growth hormone releasing factor (GHRH) has been described in the rat, mouse and human placentae. This study reports the presence of an immunoreactive GHRH activity (IR-GHRH) in the ovine placenta. This activity was detected by radioimmunoassay from day 50 (D50) until the end of pregnancy. Higher IR-GHRH concentration in placental tissue was observed on days 100 (543 +/- 123 pg/g) and 140 (550 +/- 62 pg/g) and, when compared with the GHRH content of the ovine hypothalamus (1.2 ng/hypothalamus), represents a considerable amount of GHRH per placenta (a mean of 200 ng). Perifused placenta explants released IR-GHRH in vitro at a mean rate of 200 pg/g/h. Depolarization by 55 mM KCl increased the IR-GHRH concentration of the perifusion media 1.7 times over basal values. The elution position of GHRH immunoreactivity in the gel filtration chromatography profiles was the same for placenta and hypothalamus extracts and lay very near to the molecular weight of bovine GHRH. Northern blot hybridization analysis revealed the existence of a placental transcript whose size (0.75 kb) was comparable to the size of the ovine hypothalamus and rat placenta GHRH transcripts. Hybridization signal was observed at each stage studied from D50 until D120 of pregnancy. This study demonstrated the existence of a IR-GHRH peptide in the ovine placenta.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/genética , Placenta/química , Proteínas Gestacionales/análisis , ARN Mensajero/análisis , Animales , Northern Blotting , Cromatografía en Gel , Femenino , Perfusión , Embarazo , Radioinmunoensayo , OvinosRESUMEN
The role of IGFs in placental growth is poorly understood. IGF-II receptors have been characterised in the ovine placenta and used extensively for radioreceptor assay, but their evolution during placental development has not been considered. In this study, binding sites for IGF-I were characterised in the ovine cotyledon by binding and cross-linking studies and the evolution of the number of IGF-I and IGF-II receptors on placentae collected on days 50, 75, 100 and 140 of pregnancy were compared. IGF-I bound onto placental membranes with a mean association constant of 1.7 nM-1 except on day 50 when a lower association constant was observed (0.8 nM-1). Scatchard analysis of the displacement curves led to a single binding site model. IGF-II was as potent as IGF-I at displacing the binding of 125I-labelled IGF-I on those membranes, whereas insulin cross-reaction was only 1%. IGF-II bound on our placental membrane preparations with the characteristics described previously and neither IGF-I nor insulin was able to displace this binding. Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that IGF-I was linked to a protein with a molecular weight of about 135,000 Da and IGF-II to a protein of 250,000 Da. The mean +/- S.E.M. number of IGF-I receptors was significantly higher on days 50 and 75 than on days 100 and 140 (154 +/- 12, 105 +/- 11 vs 65 +/- 4, 48 +/- 3 fmol/mg, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Placenta/metabolismo , Preñez/metabolismo , Receptores de Somatomedina/metabolismo , Ovinos/metabolismo , Animales , Autorradiografía , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Femenino , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Embarazo , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Temperatura , Factores de TiempoRESUMEN
Studies of the binding of 125I-labelled epidermal growth factor (EGF) to the ovine placenta were carried out on days 50, 90-100 and 140 of pregnancy. Membrane fractions were purified from the fetal area of the cotyledon. Two classes of binding sites were found. Their dissociation constants (Kd) were not significantly different for the three stages of pregnancy considered (high-affinity Kd 54-70.2 pmol/l; low-affinity Kd 12.2 to 19 nmol/l). However, the number of high-affinity binding sites on days 90-100 was significantly (P < 0.01) greater (146 +/- 19 fmol/mg protein) than on either day 50 or day 140 (respectively 74.2 +/- 1.26 and 56.3 +/- 5.6 fmol/mg protein). Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that the major part of the EGF was specifically cross-linked to a protein of molecular weight of 150 kDa and to lesser extent to 180 kDa and 130 kDa proteins. Membranes prepared from unfrozen tissues, in the presence of sodium iodoacetate to reduce endogenous enzymatic conversion of the 180 kDa form to the 150 and 130 kDa forms, still exhibited a major EGF-binding protein of 150 kDa. The occurrence of an increased number of EGF receptors at the period of rapid cotyledonary growth which coincides with the increase in placental hormonal secretions suggests that EGF has a role in the development of the ovine placenta.
Asunto(s)
Receptores ErbB/metabolismo , Placenta/metabolismo , Preñez/metabolismo , Ovinos/metabolismo , Animales , Autorradiografía , Electroforesis en Gel de Poliacrilamida , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/aislamiento & purificación , Femenino , Embarazo , Unión ProteicaRESUMEN
This study examined differences in selected components of uterine secretions from Large White and prolific Chinese Meishan gilts during the oestrous cycle or early pregnancy. Total recoverable protein, uteroferrin (measured as acid phosphatase activity), acyl aminopeptidase, calcium, sodium, potassium, immunoglobulins A and G, glucose, fructose, oestradiol-17 beta, and prostaglandins F2 alpha (PGF2 alpha) and E2 (PGE2) in uterine flushings were measured. During the oestrous cycle, breed effects were detected only for total protein (P = 0.07), which tended to be higher for Large White gilts. However, for pregnant gilts, total recoverable glucose (P less than 0.05), fructose (P less than 0.05) sodium (P less than 0.05), immunoglobulin A (P less than 0.01), PGF (P less than 0.01), PGE (P less than 0.01), and acyl aminopeptidase (P less than 0.05) were greater in uterine flushings from Meishan gilts. Only uteroferrin was higher (P = 0.06) in uterine flushings from Large White gilts. Concentrations of prolactin were higher (P less than 0.05) in plasma from cyclic or pregnant Meishan gilts, but concentrations of total oestrogens and progesterone were not affected by pregnancy status or breed. These results suggest that Meishan conceptuses, individually or collectively, are more stimulatory to endometrial secretion and/or transport of the components of histotroph into the uterine lumen, or that the endometrium of Meishan gilts is more sensitive to conceptus signals responsible for the accumulation of histotroph in the uterine lumen.
Asunto(s)
Útero/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Estro/fisiología , Femenino , Tamaño de la Camada/fisiología , Embarazo , Proteínas/metabolismo , Especificidad de la Especie , PorcinosRESUMEN
Seven lactating Lacaune ewes underwent either a total luteectomy on day 19 of pregnancy (D19) (compensated from that stage by a daily progesterone supplementation of 25 mg to ensure embryonic survival; group 1:4 animals) or a control laparotomy (group 2: 3 animals). Intra-jugular injection of 200 micrograms of a synthetic PGF2 alpha analogue (Dinolytic, Upjohn) caused an increase in the intramammary pressure (IMP) and a concomitant rise in oxytocinaemia only in the presence of a corpus luteum, ie in all ewes of groups 1 and 2 before D19 and only in those of group 2 after that stage. These experiments confirm that the corpus luteum, and not the other ovarian compartments, releases oxytocin when prostaglandin F2 alpha is administered.
Asunto(s)
Cuerpo Lúteo/fisiología , Dinoprost/farmacología , Glándulas Mamarias Animales/fisiología , Oxitocina/sangre , Animales , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/cirugía , Dinoprost/administración & dosificación , Femenino , Inyecciones Intravenosas , Venas Yugulares , Glándulas Mamarias Animales/efectos de los fármacos , Oxitocina/metabolismo , Embarazo , Presión , Progesterona/sangre , Tasa de Secreción/efectos de los fármacos , Ovinos/fisiologíaRESUMEN
Brown hares were made pseudopregnant by sterile matings or PMSG-hCG treatment (day of mating or hCG injection = Day 0 of pseudopregnancy). Progesterone secretion by the CL began 3-4 days after the ovulatory stimuli, reached maximum on Days 8 to 11 and decreased thereafter to reach low levels from Day 9 to 18, depending on the female. Cauterization of all large ovarian follicles on Day 7 resulted in an immediate luteolysis in young females, but had no effect in older ones. Oestradiol capsules implanted from Day 7 to Day 46 were able to maintain progesterone secretion until at least Day 30, in intact females as well as in females with all large follicles cauterized. Hysterectomy on Day 7 or 8 was followed by an immediate drop in progesterone concentrations; oestradiol capsules implanted at the time of hysterectomy prevented the drop in progesterone values, which remained elevated until Day 38. The induction of ovulation in females hysterectomized 2 months before resulted in CL of slightly shortened life-span. The injection of PGF-2 alpha on Day 7 of pseudopregnancy was followed by an immediate luteolysis. These results suggest that oestradiol secreted by the large ovarian follicles is the main luteotrophic factor in the brown hare. In old hares, the large amount of interstitial tissue could secrete oestrogens, and thus maintain pseudopregnancy. On Day 7 of pseudopregnancy, the uterus secretes a luteotrophic substance acting either directly on the ovary, or via the pituitary, to maintain oestradiol secretion by the follicles. In long-term hysterectomized females, the CL would be able to develop independently of any trophic substance, but for a reduced duration.
Asunto(s)
Mantenimiento del Cuerpo Lúteo/fisiología , Estradiol/fisiología , Lagomorpha/fisiología , Mamíferos/fisiología , Seudoembarazo , Útero/fisiología , Animales , Dinoprost/fisiología , Femenino , Folículo Ovárico/fisiología , Embarazo , Progesterona/sangreRESUMEN
This study describes the presence in and production by the ovine conceptus of an oxytocin-like peptide during the early stages of development. Oxytocin was measured by radioimmunoassay in ovine conceptuses from days 14 to 30 of pregnancy. Tissue concentrations of oxytocin increased from day 14 (24.8 +/- 5 pg/100 mg) until day 19 (122.9 +/- 52 pg/100 mg) and then decreased (3 +/- 1 pg/100 mg). Oxytocin was released into culture medium by day-15 ovine conceptuses at a rate of 262 +/- 55 pg/24 h. Reverse-phase high-performance liquid chromatography (HPLC) analysis of peptides extracted from a pool of ovine conceptuses was conducted using chromatographic conditions developed to separate oxytocin from other nonapeptides. Radioimmunoassay of HPLC fractions for oxytocin revealed an immunoactive conceptus peptide in a single fraction at the same retention time as chromatographed authentic oxytocin. Radioimmunoassay and chromatographic data therefore suggest that this oxytocin-like peptide is similar, if not identical, to authentic oxytocin. Concentrations of oxytocin in conceptus tissue were maximal during the period of inhibition of luteal regression (days 14-19). It is proposed that conceptus oxytocin is involved in the maintenance of luteal function in early pregnancy.
Asunto(s)
Mantenimiento del Cuerpo Lúteo , Embrión de Mamíferos/fisiología , Oxitocina/fisiología , Preñez/fisiología , Ovinos/fisiología , Animales , Cromatografía Líquida de Alta Presión , Embrión de Mamíferos/análisis , Femenino , Oxitocina/aislamiento & purificación , Embarazo , Factores de TiempoRESUMEN
Bilateral perifusion devices were utilized for measurement of prostaglandin secretion by luminal and myometrial surfaces of porcine endometrium. Tissues were collected from Days 10, 12 and 14 pregnant, Day 14 cyclic and Day 14 estrogen-induced pseudopregnant gilts. Each tissue was placed into duplicate perifusion devices and perifused with Krebs-Ringer Bicarbonate solution at 3 ml/10 min for 2 h, fractions collected every 10 min and oxytocin (1 IU/ml) perifused during fractions 6-10 to the luminal side of one chamber and to the myometrial side of the other chamber. Secretion rates of PGF were higher (P less than 0.05) than PGE2 for each status. Secretion rates of PGF and PGE2 were higher (P less than 0.01) from the luminal side for Day 12 pregnant, Day 14 pregnant and Day 14 pseudo-pregnant gilts, whereas secretion was higher from the myometrial side for Day 10 pregnant and Day 14 cyclic gilts. Oxytocin increased (P less than 0.01) prostaglandin secretion from the luminal side regardless of reproductive status. Pregnancy at Day 12 and Day 14, as well as estrogen treatment, were associated with prostaglandin secretion in a luminal (exocrine) orientation versus a myometrial (endocrine) orientation for Day 14 cyclic and Day 10 pregnant gilts. These data indicate an estrogen associated switch between Days 10 and 12 of pregnancy from an endocrine to an exocrine secretion of prostaglandins.
Asunto(s)
Endometrio/metabolismo , Prostaglandinas E/metabolismo , Prostaglandinas F/metabolismo , Animales , Dinoprostona , Glándulas Endocrinas/fisiología , Endometrio/efectos de los fármacos , Glándulas Exocrinas/fisiología , Femenino , Cinética , Miometrio/metabolismo , Oxitocina/farmacología , Embarazo , Seudoembarazo/fisiopatología , Porcinos , Factores de TiempoRESUMEN
Bilateral perifusion devices were utilized to measure prostaglandin secretion towards luminal and myometrial sides of bovine endometria. Tissues were collected at Day 17 post-estrus from cyclic (n = 4), pregnant (n = 5) and bred but subsequently non-pregnant (n = 6) cows. Tissue from each cow was placed into two perifusion devices, perifused with Krebs-Ringer Bicarbonate solution (3 ml/10 min) for 2.5 h and fractions collected every 10 min. Oxytocin (1 IU/ml) was perifused during fractions 7-12 to the luminal side of one device and to the myometrial side of the other device. Regardless of status, prostaglandin secretion rates (PGF and PGE2) were higher (P less than 0.01) from the luminal side than the myometrial side. Secretion rates of PGF were lower (P less than 0.01) for endometria from pregnant cows than for endometria from cyclic or bred/non-pregnant cows, whereas secretion rates of PGE2 were not affected by pregnancy status. Regardless of the side of perifusion, secretion rates of PGF and PGE2 from endometria of cyclic and bred/non-pregnant cows were elevated (P less than 0.01) throughout the period of oxytocin treatment, whereas prostaglandin secretion by endometria from pregnant cows was not stimulated by oxytocin. Decreased secretion of PGF from endometria of pregnant cows suggests that the corpus luteum and pregnancy are maintained because of an inhibition of endometrial prostaglandin synthesis or an inability to respond to stimulators of prostaglandin synthesis (i.e. oxytocin).