Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Análise do resultado clínico-funcional do tratamento das fraturas-luxações da articulação de Lisfranc / Analysis of clinical and functional results from the treatment of Lisfranc joint fracture-dislocations

Objetivo: Analisar o resultado clínico-funcional do tratamento das fraturas-Iuxações agudas da articulação tarsometatarsal (Lisfranc) e estabelecer os fatores prognósticos, a médio prazo, destas lesões. Material e métodos: Vinte e um pacientes (21 pés), todos vítimas de traumatismos no médio pé com fratura-Iuxação de Lisfranc, foram reavaliados após tempo médio de seguimento de 71 meses. Doze pacientes eram do sexo masculino (57 por cento) e nove do feminino (43 por cento). A média de idade, no momento do trauma, foi de 26 anos (variando de cinco a 48 anos). Os traumas causados por alta energia foram os mais freqüentes, ocorrendo em cerca de 86 por cento dos casos. A redução aberta, seguida da fixação interna, foi empregada em 17 pés (81 por cento) e o tratamento incruento, em quatro (19 por cento). Imobilização com bota gessada foi utilizada durante 12 semanas e, em seguida, iniciou-se fisioterapia. Resultados: A pontuação média, segundo a escala AOFAS para médio pé, foi de 83 pontos (variando de 53 a 100). A dor residual foi o sintoma mais freqüente, presente em 12 dos 21 pés (58 por cento), com intensidade leve em seis dos pés (29 por cento) e moderada ou grave nos outros seis pés (29 por cento). Os resultados considerados insatisfatórios foram associados às fraturas expostas, à fratura das duas colunas do pé, às fraturas desviadas do cubóide e a seqüela de síndrome compartimental. A influência negativa da lesão de Lisfranc nas demais articulações foi evidenciada pela redução significativa na amplitude de movimento (perda de 20 por cento no tornozelo, 22 por cento na subtalar, 8 por cento na tarsometatarsal, 37 por cento na metatarsofalangiana do hálux e 18 por cento na interfalângica do hálux). Conclusões: O resultado clínico-funcional do tratamento das fraturasluxações de Lisfranc, a médio prazo, foi satisfatório em 72 por cento dos casos avaliados. Os fatores de mau prognóstico, associados aos resultados insatisfatórios, foram: fraturas expostas, síndrome compartimental, esmagamento do osso cubóide e as fraturas envolvendo as duas colunas do pé. Após fratura-Iuxação de Lisfranc, espera-se significativa redução da amplitude de movimento nas demais articulações do tornozelo e nas demais articulações do pé