Estudo proteômico do desenvolvimento folicular de vacas zebuinas não gestantes
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Data
2016-03-30
Autores
Lourenço, Tarcísio Torre [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O ciclo estral da vaca é composto por 2-3 ondas de crescimento folicular, no qual vários folículos são recrutados e iniciam um novo crescimento. Durante o período denominado desvio folicular, um folículo se torna dominante e os outros entram em atresia. Este processo envolve um mecanismo ainda não completamente compreendido, incluindo proteínas específico, como já estabelecido pela expressão gênica. O objetivo do presente estudo foi caracterizar as proteínas do fluído folicular a fim de identificar macromoléculas relacionadas ao desenvolvimento dos folículos de vacas zebuínas nã-gestantes. Foram colhidos os ovários de 25 vacas mestiças não-gestantes em um abatedouro. A presença do corpo lúteo foi anotada para cada ovário. O líquido folicular foi colhido utilizando-se a imersão do ovário em meio líquido e ultrassonografia. De acordo com a mensuração do diâmetro folicular, foram formados 3grupos, folículos pequenos (≤6,5mm, n=25), médios (>6,5mm a ≤9mm, n=9) e grandes (>9,0mm, n=11). Após 2 centrifugações (600xg/10 minutos e 15.000xg/30 minutos, 4ºC) o sobrenadante foi separado e utilizado para determinação da concentração de proteína total (método de Bradford). A eletroforese foi conduzida sob condições desnaturantes e redutoras, em gel de separação de poliacrilamidaà 12%. A concentração de progesterona e estradiol do líquido folicular foi determinada a fim de identificar os folículos saudáveis. As proteínas diferenciais identificadas pela eletroforese foram submetidas à espectrometria de massas e a ontologia gênica foi investigada nos bancos de dados disponíveis. Foram encontradas 45 bandas proteicas em 45 amostras de líquido folicular. A média da concentração ± desvio padrão da progesterona foi de 129,91 ± 186,43, considerando todos os folículos. Os resultados mostram que houve uma expressão diferenciada de proteínas nas diferentes categorias de folículos.
The estrous cycle of the cow consists of 2-3 follicular waves, in which several follicles are recruited and initiate growth. During the period called follicular deviation, one follicle becomes dominant and the other come into atresia.This process involves mechanisms not yet fully understood, including specific proteins as already determined by gene expression. As a result, the objective of this study was to characterize the proteins of the follicular fluid to identify macromolecules related to the development of follicles from Zebu cow. The ovaries of nonpregnant cows 25 were harvested. The presence of luteum body was noted for each ovary. Follicular fluid was collected using ultrasound. According to the measurement of follicular diameter was 3 separate groups, small follicles (≤6,5mm, n = 28), medium (> 6.5mm to ≤9mm, n = 7) and large (> 9,0mm, n = 11). After 2 centrifugations (600xg / 10 minutes, 15.000xg / 30 minutes, 4 ° C) the supernatant was separated and used for determination of total protein concentration (Bradford method). Electrophoresis was conducted under denaturing and reducing conditions on polyacrylamide separating gel at 12%. The concentration of progesterone and follicular fluid estradiol was determined to identify healthy follicles. The differential proteins identified by electrophoresis will be submitted for MALDI TOF MS / MS approach and gene ontology will be investigated in the databases available. They found 45 protein bands in electrophoresis in 45 follicular fluid samples. The mean ± standard deviation of progesterone concentration was 129.91 ± 186.43 considering all follicles. The results show that there was a differential expression of proteins in different categories of follicles.
The estrous cycle of the cow consists of 2-3 follicular waves, in which several follicles are recruited and initiate growth. During the period called follicular deviation, one follicle becomes dominant and the other come into atresia.This process involves mechanisms not yet fully understood, including specific proteins as already determined by gene expression. As a result, the objective of this study was to characterize the proteins of the follicular fluid to identify macromolecules related to the development of follicles from Zebu cow. The ovaries of nonpregnant cows 25 were harvested. The presence of luteum body was noted for each ovary. Follicular fluid was collected using ultrasound. According to the measurement of follicular diameter was 3 separate groups, small follicles (≤6,5mm, n = 28), medium (> 6.5mm to ≤9mm, n = 7) and large (> 9,0mm, n = 11). After 2 centrifugations (600xg / 10 minutes, 15.000xg / 30 minutes, 4 ° C) the supernatant was separated and used for determination of total protein concentration (Bradford method). Electrophoresis was conducted under denaturing and reducing conditions on polyacrylamide separating gel at 12%. The concentration of progesterone and follicular fluid estradiol was determined to identify healthy follicles. The differential proteins identified by electrophoresis will be submitted for MALDI TOF MS / MS approach and gene ontology will be investigated in the databases available. They found 45 protein bands in electrophoresis in 45 follicular fluid samples. The mean ± standard deviation of progesterone concentration was 129.91 ± 186.43 considering all follicles. The results show that there was a differential expression of proteins in different categories of follicles.
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Palavras-chave
Eletroforese, Folículo, MALDI-TOF, Vacas, Cows, Electrophoresis folicule