Efeitos da suplementação com ácido oleico e linoleico conjugado na secreção de prostaglandina E2 e F2α em células trofoblásticas bovinas
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Data
2022-04-14
Autores
Lourenço, Valeska de Castro
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Universidade Estadual Paulista (Unesp)
Resumo
A mortalidade embrionária precoce, ocasionada por falhas no reconhecimento materno-fetal (RMF) nas primeiras três semanas após a fertilização, representa a maior causa de ineficiência reprodutiva em fêmeas bovinas de corte. Estratégias que favoreçam tal reconhecimento baseiam-se em reduzir a capacidade de síntese da prostaglandina F2α (PGF2α) pelo endométrio materno e/ou maximizar o estímulo anti-luteolítico induzido pelo concepto, incluindo o aumento da síntese de prostaglandina E2 (PGE2). A PGE2, sintetizada pelo endométrio materno e células trofoblásticas do concepto, determina efeitos anti-luteolíticos e luteotróficos que favorecem o RMF. O ácido oleico (OA) e o ácido linoleico conjugado (CLA), reconhecidamente determinam modificações na via metabólica do ácido graxo poliinsaturado n-6 na biossíntese de eicosanóides, incluindo as prostaglandinas. Tal efeito, não foi avaliado em células trofoblásticas bovinas (CT-1). Hipotetizou-se que a suplementação com OA ou CLA, no meio de cultura de CT-1 aumenta a síntese de PGE2, diminui a síntese de PGF2α e aumenta a relação PGE2/PGF2α, sendo tal efeito dose-dependente. Objetivou-se determinar os efeitos da suplementação com OA (50, 100, 200, 500, 1000 μM e grupo controle) ou CLA (10, 20, 50, 100 μM e grupo controle) por até 72 horas, no cultivo in vitro de CT-1, na síntese de PGE2 e PGF2α, mensuradas por ensaio de imunoabsorção enzimática (ELISA). Assim, células CT-1 foram cultivadas em garrafas de cultivo celular por 22 dias em incubadora umidificada a 38,5°C e 5% CO2 até atingirem uma confluência de 100%. No 23º dia as CT-1 foram transferidas para placas de 6 poços com meio DMEM (1X) + GlutaMAX suplementado com 10% de soro fetal bovino (SFB), 1% de aminoácidos não essenciais, 1% de antibiótico e antimicótico e 0,001% B-mercaptoetanol, onde foram cultivadas por 5 dias, até atingirem uma confluência de 50%. No 6º dia de cultivo, 24 horas antes da suplementação, o meio de cultivo foi substituído por meio sem SFB. No 7º dia, ao meio sem SFB foram adicionados os tratamentos com CLA ou OA. Amostras de meio de cultivo foram coletadas 48 e 72 horas após a adição de OA e 0, 24, 48 e 72 horas após a adição de CLA, acondicionadas em microtubos e armazenadas a -80oC para posterior análise das prostaglandinas por ELISA. Para cada ácido graxo foram realizadas 5 repetições, onde em cada repetição foi utilizado 1 poço/tratamento/para cada tempo avaliado. A análise estatística foi feita pelo PROC MIXED do SAS considerando efeito de concentração (tratamento) e o efeito randômico da repetição cultivada. Meios de cultivo de CT-1 suplementados com OA na concentração de 1000 μM por 72 horas apresentaram redução da síntese de PGF2α e aumento da síntese de PGE2 não tendo alterado a razão PGE2/PGF2α, determinando um efeito dose-resposta. Meios de cultivo de CT-1 suplementados com CLA por 72 horas, em todas as doses testadas, apresentaram redução da síntese de PGF2α e PGE2 e aumento da razão PGE2/PGF2α. Conclui-se que as suplementações utilizadas confirmam parcialmente a hipótese, enquanto o OA na concentração de 1000 μM aumenta a síntese de PGE2 e diminui a síntese de PGF2α, o CLA aumenta a razão PGE2/PGF2α em todas as concentrações testadas.
Early embryonic mortality, caused by failures in maternal-fetal recognition three weeks after the fertilization is the leading cause of reproductive inefficiency in beef cattle. Strategies that favor such recognition are based on reducing the capacity of prostaglandin F2α (PGF2α) synthesis by the maternal endometrium and/or maximizing the anti-luteolytic stimulus induced by the conceptus, including the increase in prostaglandin E2 (PGE2) synthesis. The PGE2 synthesized by the maternal endometrium and trophoblastic cells of the conceptus, determines the effects anti-luteolytic and luteotrophics that favor the RMF. Oleic acid (OA) and conjugated linoleic acid (CLA) are known to determine changes in the metabolic pathway of n-6 polyunsaturated fatty acid in the biosynthesis of eicosanoids, including prostaglandins. This effect was not evaluated in bovine trophoblastic cells (CT-1). The hypothesis is that supplementation with OA or CLA in the CT-1 culture medium increases PGE2 synthesis, decreases PGF2α synthesis and increases the PGE2/PGF2α ratio, which is a dose-dependent effect. The objective was to determine the effects of supplementation with OA (0, 50, 100, 200, 500, 1000 μM) or CLA (0, 10, 20, 50 and 100 μM) for up to 72 hours, in cultivation in vitro of CT-1, in the synthesis of PGE2 and PGF2α, measured by enzyme immunosorbent assay (ELISA). So, CT-1 cells were cultured in Cell cultivation bottles for 22 days in a humidified incubator at 38.5°C and 5% CO2 until reaching 100% confluence. On the 23rd day, CT-1 were transferred to 6-well plates with DMEM (1X) + GlutaMAX medium supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% antibiotic and antimycotic and 0.001% B-mercaptoethanol, where they were grown for 5 days, until reaching 50% confluence. On the 6th day of cultivation, 24 hours before supplementation, the culture medium was replaced by medium without SFB. On the 7th day, in the medium without SFB were added to the treatments with CLA or OA. Samples of culture medium were collected 48 and 72 hours after the addition of OA and 0, 24, 48 e 72 hours after the addition of CLA, placed in microtubes and stored at -80ºC for further analysis of prostaglandins by ELISA. For each fatty acid, 5 replicates were performed, where 1 well/treatment/for each time evaluated was used in each replicate. Statistical analysis was performed by PROC MIXED of SAS considering concentration effect (treatment) and random effect of cultured replicate. CT-1 culture media supplemented with OA at a concentration of 1000 μM for 72 hours showed a reduction in PGF2α synthesis and an increase in PGE2 synthesis and did not change the PGE2/PGF2α ratio, determining a dose-response effect. CT-1 culture media supplemented with CLA for 72 hours, at all doses tested, showed a reduction in the synthesis of PGF2α and PGE2 and an increase in the PGE2/PGF2α ratio. It is concluded that the supplementations used partially confirm the hypothesis, while OA at a concentration of 1000 μM increases the synthesis of PGE2 and decreases the synthesis of PGF2α, CLA increases the PGE2/PGF2α ratio at all concentrations tested.
Early embryonic mortality, caused by failures in maternal-fetal recognition three weeks after the fertilization is the leading cause of reproductive inefficiency in beef cattle. Strategies that favor such recognition are based on reducing the capacity of prostaglandin F2α (PGF2α) synthesis by the maternal endometrium and/or maximizing the anti-luteolytic stimulus induced by the conceptus, including the increase in prostaglandin E2 (PGE2) synthesis. The PGE2 synthesized by the maternal endometrium and trophoblastic cells of the conceptus, determines the effects anti-luteolytic and luteotrophics that favor the RMF. Oleic acid (OA) and conjugated linoleic acid (CLA) are known to determine changes in the metabolic pathway of n-6 polyunsaturated fatty acid in the biosynthesis of eicosanoids, including prostaglandins. This effect was not evaluated in bovine trophoblastic cells (CT-1). The hypothesis is that supplementation with OA or CLA in the CT-1 culture medium increases PGE2 synthesis, decreases PGF2α synthesis and increases the PGE2/PGF2α ratio, which is a dose-dependent effect. The objective was to determine the effects of supplementation with OA (0, 50, 100, 200, 500, 1000 μM) or CLA (0, 10, 20, 50 and 100 μM) for up to 72 hours, in cultivation in vitro of CT-1, in the synthesis of PGE2 and PGF2α, measured by enzyme immunosorbent assay (ELISA). So, CT-1 cells were cultured in Cell cultivation bottles for 22 days in a humidified incubator at 38.5°C and 5% CO2 until reaching 100% confluence. On the 23rd day, CT-1 were transferred to 6-well plates with DMEM (1X) + GlutaMAX medium supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% antibiotic and antimycotic and 0.001% B-mercaptoethanol, where they were grown for 5 days, until reaching 50% confluence. On the 6th day of cultivation, 24 hours before supplementation, the culture medium was replaced by medium without SFB. On the 7th day, in the medium without SFB were added to the treatments with CLA or OA. Samples of culture medium were collected 48 and 72 hours after the addition of OA and 0, 24, 48 e 72 hours after the addition of CLA, placed in microtubes and stored at -80ºC for further analysis of prostaglandins by ELISA. For each fatty acid, 5 replicates were performed, where 1 well/treatment/for each time evaluated was used in each replicate. Statistical analysis was performed by PROC MIXED of SAS considering concentration effect (treatment) and random effect of cultured replicate. CT-1 culture media supplemented with OA at a concentration of 1000 μM for 72 hours showed a reduction in PGF2α synthesis and an increase in PGE2 synthesis and did not change the PGE2/PGF2α ratio, determining a dose-response effect. CT-1 culture media supplemented with CLA for 72 hours, at all doses tested, showed a reduction in the synthesis of PGF2α and PGE2 and an increase in the PGE2/PGF2α ratio. It is concluded that the supplementations used partially confirm the hypothesis, while OA at a concentration of 1000 μM increases the synthesis of PGE2 and decreases the synthesis of PGF2α, CLA increases the PGE2/PGF2α ratio at all concentrations tested.
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Palavras-chave
Ácidos graxos, Reprodução, In vitro, Bovino, Fatty acid, Reproduction, Bovine