Avaliação de fatores envolvidos na foliculogênese de búfalas com a utilização de primers delineados para bovinos
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Data
2021-10-06
Autores
Silva Junior, Edjalma Rodrigues da
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Universidade Estadual Paulista (Unesp)
Resumo
O conhecimento da fisiologia ovariana é importante durante a aplicação de biotécnicas assistidas de reprodução, tanto animal quanto humana. Durante a foliculogênese, os folículos apresentam aumento em número e diferenciação morfológica das células que os circundam e, com a progressão do desenvolvimento, se tornam unidades funcionais no parênquima ovariano. Esses folículos podem ser considerados como pool de reserva e, na dependência do estímulo, podem permanecer quiescentes, sofrerem atresia ou chegar até a ovulação. Durante esse desenvolvimento, podemos ter a presença de fatores de crescimento produzidos no parênquima ovariano, que são modulados pela presença dos hormônios folículo estimulante (FSH) e luteinizante (LH). Além disso, o FSH e o LH também apresentam ação direta durante todo o curso da foliculogênese. Dentre os fatores de crescimento mais comuns, podemos citar o fibroblástico (FGF), insulínico (IGF), epidermal (EGF) e seus respectivos receptores que atuam na diferenciação e progressão do desenvolvimento folicular, desde as fases iniciais até a ovulação. Sendo assim, os objetivos do presente trabalho foram revisar a regulação parácrina dos fatores de crescimento durante a foliculogênese, atresia folicular, descrever e quantificar a expressão gênica desses fatores (FGF2, IGF1 e EGF), seus receptores (FSHR, FGFR2C e FGFR3C) e agentes envolvidos na apoptose (CASP3) presentes no ovário de búfalas vazias e gestantes. Em abatedouros, coletaram-se ovários de búfalas vazias (n=12) e gestantes (n=12), que se encontravam no terço final da gestação. Para a padronização das amostras, todas as búfalas vazias se encontravam cíclicas, com a presença de folículos em desenvolvimento e corpo lúteo evidente no parênquima. As amostras foram encaminhadas ao laboratório em tempo máximo de uma hora após a coleta. Todos os ovários foram dissecados, os folículos antrais aspirados e as amostras de tecido ovariano coletadas e armazenadas em nitrogênio líquido (-196°C) para posterior análise. No laboratório, foram utilizados primers delineados para bovinos na utilização do RT-qPCR na quantificação gênica das amostras. Após a padronização técnica, normalização das amostras e transformações dos valores em bases logarítmicas foi realizado a quantificação por RT-qPCR das amostras dos ovários de búfalas. Foram consideradas com diferença estatística, as amostras que apresentaram significância em 5% no pós-teste de Wilcoxon. Todos os primers foram expressos no RT-qPCR, entretanto, apenas o FSHR apresentou diferença significativa entre os grupos experimentais. O FSH atua na estimulação e regulação dos fatores parácrinos na regulação da foliculogênese, uma vez que houve variação da expressão de seu receptor (FSHR) entre os grupos experimentais. No grupo experimental de búfalas vazias, houve oscilação na expressão do FSHR decorrente das diferentes fases do ciclo estral que as fêmeas se encontravam, pois a modulação da secreção de esteroides pode afetar o desenvolvimento e progressão do desenvolvimento folicular. Sendo assim, conclui-se que houve expressão gênica dos fatores de crescimento testados presentes no tecido ovariano que são responsáveis pela modulação e progressão da foliculogênese em búfalas gestantes e vazias. Não foi possível visualizar a diferença na expressão do fator apoptótico pesquisado (CASP3) entre os grupos experimentais e, em adição, é viável a utilização de primers desenhados para bovinos na pesquisa de fatores de crescimento em ovário de búfalas.
Knowledge of ovarian physiology is important when applying assisted reproductive biotechniques, both animal and human. During folliculogenesis, the follicles show an increase in number and morphological differentiation of the cells that surround the oocyte and, with the progression of development, they become functional units in the ovarian parenchyma. These follicles can be considered as a reserve pool and, depending on the stimulus, can remain quiescent, suffer atresia or reach ovulation. During this development, we may have the presence of growth factors produced in the ovarian parenchyma, which are modulated by the presence of follicle-stimulating (FSH) and luteinizing (LH) hormones. Furthermore, FSH and LH also have a direct action throughout the course of folliculogenesis. Among the most common growth factors, we can mention fibroblastic (FGF), insulin (IGF), epidermal (EGF) and their respective receptors that act in the differentiation and progression of follicular development, from the initial stages to ovulation. Therefore, the objectives of this study were to review the paracrine regulation of growth factors during folliculogenesis, follicular atresia, describe and quantify the gene expression of these factors (FGF2, IGF1 and EGF), their receptors (FSHR, FGFR2C and FGFR3C) and agents involved in apoptosis (CASP3) present in the ovary of empty and pregnant buffaloes. In abattoirs, ovaries from empty buffaloes (n=12) and pregnant women (n=12), which were in the final third of gestation, were collected. For sample standardization, all empty buffaloes were cyclic, with the presence of developing follicles and corpus luteum evident in the parenchyma. The samples were sent to the laboratory within a maximum of one hour after collection. All ovaries were dissected, antral follicles aspirated and ovarian tissue samples collected and stored in liquid nitrogen (-196°C) for further analysis. In the laboratory, primers designed for cattle were used in the use of RT-qPCR for gene quantification of the samples. After technical standardization, normalization of samples and transformation of values into logarithmic bases, quantification by RT-qPCR of samples from buffalo ovaries was performed. The samples that presented significance in 5% in the Wilcoxon post-test were considered with statistical difference. All primers were expressed in RT-qPCR, however, only FSHR showed a significant difference between experimental groups. FSH acts in the stimulation and regulation of paracrine factors in the regulation of folliculogenesis, since there was variation in its receptor (FSHR) expression between the experimental groups. In the experimental group of empty buffaloes, there was an oscillation in the expression of the FSHR due to the different phases of the estrous cycle that the females were in, as the modulation of steroid secretion can affect the development and progression of follicular development. The presence of the FSHR receptor in the ovarian parenchyma is one of the starting points for the beginning of folliculogenesis and progression of its development, through the modulation of other growth factors. In pregnant buffaloes, however, there was no such variation, due to the hormonal action of progesterone. Thus, it is concluded that there was gene expression of the growth factors tested are present in ovarian 12 tissue and are responsible for the modulation and progression of folliculogenesis in pregnant and empty buffaloes. It was not possible to visualize the difference in the expression of the investigated apoptotic factor (CASP3) between the experimental groups and, in addition, it is feasible to use primers designed for cattle in the research of growth factors in buffalo ovary.
Knowledge of ovarian physiology is important when applying assisted reproductive biotechniques, both animal and human. During folliculogenesis, the follicles show an increase in number and morphological differentiation of the cells that surround the oocyte and, with the progression of development, they become functional units in the ovarian parenchyma. These follicles can be considered as a reserve pool and, depending on the stimulus, can remain quiescent, suffer atresia or reach ovulation. During this development, we may have the presence of growth factors produced in the ovarian parenchyma, which are modulated by the presence of follicle-stimulating (FSH) and luteinizing (LH) hormones. Furthermore, FSH and LH also have a direct action throughout the course of folliculogenesis. Among the most common growth factors, we can mention fibroblastic (FGF), insulin (IGF), epidermal (EGF) and their respective receptors that act in the differentiation and progression of follicular development, from the initial stages to ovulation. Therefore, the objectives of this study were to review the paracrine regulation of growth factors during folliculogenesis, follicular atresia, describe and quantify the gene expression of these factors (FGF2, IGF1 and EGF), their receptors (FSHR, FGFR2C and FGFR3C) and agents involved in apoptosis (CASP3) present in the ovary of empty and pregnant buffaloes. In abattoirs, ovaries from empty buffaloes (n=12) and pregnant women (n=12), which were in the final third of gestation, were collected. For sample standardization, all empty buffaloes were cyclic, with the presence of developing follicles and corpus luteum evident in the parenchyma. The samples were sent to the laboratory within a maximum of one hour after collection. All ovaries were dissected, antral follicles aspirated and ovarian tissue samples collected and stored in liquid nitrogen (-196°C) for further analysis. In the laboratory, primers designed for cattle were used in the use of RT-qPCR for gene quantification of the samples. After technical standardization, normalization of samples and transformation of values into logarithmic bases, quantification by RT-qPCR of samples from buffalo ovaries was performed. The samples that presented significance in 5% in the Wilcoxon post-test were considered with statistical difference. All primers were expressed in RT-qPCR, however, only FSHR showed a significant difference between experimental groups. FSH acts in the stimulation and regulation of paracrine factors in the regulation of folliculogenesis, since there was variation in its receptor (FSHR) expression between the experimental groups. In the experimental group of empty buffaloes, there was an oscillation in the expression of the FSHR due to the different phases of the estrous cycle that the females were in, as the modulation of steroid secretion can affect the development and progression of follicular development. The presence of the FSHR receptor in the ovarian parenchyma is one of the starting points for the beginning of folliculogenesis and progression of its development, through the modulation of other growth factors. In pregnant buffaloes, however, there was no such variation, due to the hormonal action of progesterone. Thus, it is concluded that there was gene expression of the growth factors tested are present in ovarian 12 tissue and are responsible for the modulation and progression of folliculogenesis in pregnant and empty buffaloes. It was not possible to visualize the difference in the expression of the investigated apoptotic factor (CASP3) between the experimental groups and, in addition, it is feasible to use primers designed for cattle in the research of growth factors in buffalo ovary.
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Palavras-chave
FGF, FSHR, IGF, Ovário, PCR, Ovary