RESUMEN
Olfactomedin-like (OLFML) proteins are members of the olfactomedin domain-containing secreted glycoprotein (OLF) family. OLFML2A and OLFML2B are representative molecules of these glycoproteins. Olfactomedins are critical for the development and functional organization of the nervous system and retina, which is a highly conserved structure in vertebrates, having almost identical anatomical and physiological characteristics in multiple taxa. Spotted gar, a member of the Lepisosteidae family, is a freshwater fish that inhabits rivers, bayous, swamps, and brackish waters. Recently, the complete genome has been sequenced, providing a unique bridge between fish medical models to human biology, making it an excellent animal model. This study was aimed to understanding the evolution OLFML2A and OLFML2B in the retina of spotted gar through looking for the expression of these genes. Spotted gar retina was analyzed with hematoxylin-eosin staining assays to provide an overall view of the retina structure and an immunofluorescence assay to identify OLFML2A and OLFML2B protein expression. A phylogenetic tree was created using the neighbor-joining method. Forces that direct the evolution of the fish genes were tested. Spotted gar retina, as in other vertebrates, is made of several layers. OLFML2A and OLFML2B proteins were detected in the rod and cone photoreceptor layer (PRL), outer nuclear layer (ONL), and inner nuclear layer (INL). Phylogenetic tree analysis confirms the orthology within the OLFML2A gene. Purifying selection is the evolutionary force that directs the OLFML2A genes. OLFML2A genes have a well-conserved function over time and species.
Asunto(s)
Biología Computacional/métodos , Minería de Datos/métodos , Proteínas de la Matriz Extracelular/metabolismo , Peces/metabolismo , Glicoproteínas/metabolismo , Retina/metabolismo , Animales , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Glicoproteínas/genética , Proteínas de la Membrana , TranscriptomaRESUMEN
In humans, the polygenic growth hormone (GH) locus is located on chromosome 17 and contributes with three types of proteins: pituitary GH which consists of at least two isoforms one of 22â¯kDa and the other of 20â¯kDa, placental GH, which also exhibits isoforms, and chorionic somatomammotropin hormone (CSH). While pituitary GH results from the expression of the GH-1 (GH-N) gene, placental GH is produced by the expression of the GH-2 (GH-V) gene and CSH is contributed by expression of the CSH-1 and CSH-2 genes. The location where GH-1 is expressed is the anterior pituitary and the rest of the genes in the locus are expressed in placenta. On the other hand, expression and synthesis of GH in extra-pituitary tissues, including the eye, has been recently described. However, the physiological role of GH in the eye has not yet been elucidated, although a possible neuroprotective role has been hypothesized. Thus, we analyzed GH-1, GH-2, CSH1/2, Pit-1, GHR, GHRH, GHRHR, SST, SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 to elucidate the expression and regulation of the GH locus in the human eye. Through qPCR analysis, we only found evidence of GH-1 expression in retina, choroid and trabecular meshwork; its transcript turned out to be the same as pituitary GH mRNA found in major species, and no splicing variants were detected. PIT1 was absent in all the ocular tissues implying an independent GH-1 expression mechanism. We found evidence of GHR in the cornea, choroid coat and retina. These results suggest autocrine and/or paracrine regulation, possibly exerted by GHRH and SSTs (since their mRNAs and receptors were found predominantly in retinal, choroidal and corneal tissues) since expression of both molecules was detected in different ocular tissues, as well as in the same tissues where GH-1 expression was confirmed. Our results add solid evidence about the existence of a regulatory local system for GH expression and release in the human eye.
Asunto(s)
Ojo/metabolismo , Hormona del Crecimiento/metabolismo , Receptores de Somatotropina/metabolismo , Somatostatina/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Hormonas Placentarias , Adulto JovenRESUMEN
BACKGROUND: The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. OBJECTIVE: The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. METHODS: OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. RESULTS: OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. CONCLUSIONS: Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Ojo/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Código de Barras del ADN Taxonómico , Evolución Molecular , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/genética , Ojo/química , Técnica del Anticuerpo Fluorescente/métodos , Glicoproteínas/análisis , Glicoproteínas/genética , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Fenómenos Fisiológicos Oculares , Papio , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Reversa , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. OBJECTIVE: The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. METHODS: OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. RESULTS: OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. CONCLUSIONS: Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.
Asunto(s)
Humanos , Animales , Glicoproteínas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Ojo/metabolismo , Proteínas de la Membrana/metabolismo , Papio , Valores de Referencia , Glicoproteínas/análisis , Glicoproteínas/genética , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/genética , Técnica del Anticuerpo Fluorescente/métodos , Evolución Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de Proteína , Transcripción Reversa , Ojo/química , Código de Barras del ADN Taxonómico , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Fenómenos Fisiológicos OcularesRESUMEN
In order to analyze the temporal relationship between cortisol levels and glucocorticoid receptors (GR) in placenta and the possible influence in noninfectious abortion in goats, changes in GR in livers of the foetuses and mothers livers and in placenta together with foetal and maternal cortisol levels under stress conditions in non-pregnant (n = 5) and pregnant goats (n = 24) were recorded. The pregnant goats were five on days 40-50 of gestation (40-50 d), six on days 51-75, four on days 76-100, three on days 101-125, and six in more than 125 days of gestation. GR values in the placenta and maternal serum cortisol levels, measured by competitive binding methods, decreased from 10.4 ± 2.7 and 174.3 ± 59.1, in the group 40-50 d to 6.1 ± 2.3 fmol/mg of protein and 79.1 ± 66.1 nmol/l in the 76-100 d group, respectively. Values then increased, reaching the highest values (P ≤ 0.05) detected in this study (18.3 ± 2.7 and 659.6 ± 76.3) in the 101-125 d group followed by a decrease of 11.6 ± 2.1 fmol/mg protein and 231.6 ± 54.0 nmol/l in the +125 d group. Progesterone was bound competitively to placental GR. However, its binding values decreased in the 101-125 d group. Reciprocal profiles were found in maternal and fetal liver GR. These data suggest that goats have innate mechanisms for abortion that occurs in case of life-threatening conditions saving mother's life and giving opportunity for new pregnancies, allowing thus the survival of this species.
Para analizar la relación temporal entre los niveles de cortisol y los receptores a glucocorticoides (GR) en la placenta y su posible influencia en el aborto no infeccioso de las cabras, se registraron los cambios en el cortisol sanguíneo y en los GR del hígado de cabras adultas no gestantes (n = 5) y, además de los anteriores, en el hígado fetal y la placenta junto con los niveles de cortisol fetal de cabras gestantes (n = 24) bajo condiciones de estrés. Las cabras gestantes fueron: cinco de 40 a 50 días de gestación, seis de 51 a 75, cuatro de 76 a 100, tres de 101 a 125 y seis con más de 125 días de gestación. Los valores de GR en la placenta y los niveles de cortisol materno medidos por unión competitiva, decrecieron desde 10.4 ± 2.7 y 174.3 ± 59.1 en el grupo 40-50 d a 6.1 ± 2.3 fmol/mg proteína y 79.1 ± 66.1 nmol/l en el grupo 76-100 d, respectivamente. Ambos valores se incrementaron en el grupo 101-125 d, hasta alcanzar los más altos (P ≤ 0.05) detectados en este estudio (18.3 ± 2.7 y 659.6 ± 76.3) seguidos por una disminución a 11.6 ± 2.1 fmol/mg proteína y 231.6 ± 54.0 nmol/l en el grupo +125 d. Además, la progesterona se unió competitivamente a los GR placentarios; sin embargo, su unión disminuyó en el grupo 101-125 d. Los GR de los hígados materno y fetal presentaron un perfil inverso. Estos resultados permiten sugerir que las cabras cuando se encuentran en situaciones que amenazan su vida y la del feto tienen mecanismos innatos para abortar, salvando la vida de la madre y dando la oportunidad de una nueva gestación que permita la sobrevivencia de la especie.
RESUMEN
Para obtener anticuerpos específicos contra progesterona (P4) y 17ß-estradiol (E2) y la estandarización de sus radioinmunoensayos (RIAs) correspondientes, se inmunizaron conejos Nueva Zelanda y California con 100 o 160 µg de Progesterona-CMO:BSA o 17ß-Estradiol-GMO:BSA respectivamente. Las reinoculaciones se realizaron con intervalo de una semana en seis ocasiones, titulándose en cada una el suero cosechado. Con base en el mejor título, se seleccionaron los antisueros, a los que se les determinó la especificidad mediante reacciones cruzadas con varios esteroides. La reacción cruzada de los antisueros obtenidos contra P4 fue de 4.4 y 4.4 por ciento para pregnenolona, de 4.0 y 13.3 por ciento para epipregnenolona y menos del 1.0 por ciento para alopregnenolona, testosterona, E2 y cortisol para AcQKP5 y AcQKP6 respectivamente. La reacción cruzada del antisuero obtenido contra E2 fue de 3.6 por ciento para estrona y menos de 0.01 por ciento para estriol, andreostendiona, 17Ó-estradiol, P4 y testosterona. La sensibilidad de los ensayos permite destacar desde 6.25 pg/tubo de P4 y 0.5 pg/tubo de E2. Los coeficientes de variación intra e interensayo en el RIA fueron para P4 de 10.9 por ciento y 12.8 por ciento y para E2 de 5.0 y 10.0 por ciento respectivamente. Para vaslidar los RIAs se determinó la concentración de P4 y E2 en cabras y vacas en diferentes estados reproductivos. Así la concentración de p4 en cabras durante el último tercio de gestación fue de 8.3 ñ 0.4 ng/ml y durante el periodo preparto disminuyó a 5.4 ñ 0.8 ng/ml; en esta última etapa el E2 mostró valores de 55.1 ñ 8.1 pg/ml. En la vaca, los valores de P4 y E2 fueron en el estro de 0.9 ñ 0.08 ng/ml y 10.6 ñ 2.4 pg/ml, en la fase lútea de 8.6 ñ 2.7 ng/ml y 3.3 ñ 0.6 pg/ml y en la gestación de 6.4 ñ 0.6 ng/ml y 17.3 ñ 1.6 pg/ml respectivamente. La sensibilidad de los RIAs con los anticuerpos obtenidos, en capaz de identificar y diferenciar los valores característicos de P4 y E2 de los diferentes estados reproductivos en rumiantes domésticos