In vitro studies on the control of human myometrial gap junctions

In this study human myometrial tissues were examined for the presence of gap junctions by quantitative electron microscopy before and after incubation in tissue culture media with and without indomethacin. The area of gap junctions was very low in tissues from pregnant women at term but not labor, before incubation. After 24 and 48 h incubation without any treatment, segments of some of the same tissues developed many gap junctions and other tissues contained few junctions. Prostaglandin E (PGE), prostaglandin F (PGF) and prostaglandin F metabolite (PGF metabolite) levels in the media at various times were measured by radioimmunoassay. The prostaglandins increased progressively during the incubation period. Treatment of tissues with indomethacin decreased prostaglandin levels in the media and increased the numbers of gap junctions in those control tissues that developed few junctions over the same incubation interval. We conclude that the capacity of human myometrial tissues to develop gap junctions in vitro may depend upon a maturational stage in preparation for labor. Furthermore, our results suggest that products of the cyclo-oxygenase or lipoxygenase pathways may control the presence of gap junctions in the human myometrium and that changes in synthesis in these patterns may occur as part of the maturational process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26692/1/0000239.pd

Effect of indomethacin on the pregnant rat

The objective of this study was to evaluate the reproductive performance, liver morphological study and post mortem characteristics of the pregnant Wistar rats treated with indomethacin, a general COX inhibitor. Indomethacin at doses of 0 (control), 0.32, 1.68 and 8.40 mg/kg/day were orally given once daily to each group (n=10) on days 3 and 4 of pregnancy (day 0 = first day of pregnancy = positive vaginal sperm). The animals were euthanized under anesthesia on day 11 of pregnancy, and were carried out necropsy and microorganism culture study. The results showed that the doses of 0.32 and 1.68 mg/kg body weight (the therapeutic dose for humans) of indomethacin caused no embryotoxic or lethal effects. The highest dose (8.40 mg/kg) of indomethacin disturbed implantation process and, thus, interrupted major development in some fetuses. The peritonitis was detected in the necropsy and in the bacteriological study of the animals treated with 8.4 mg/kg. It was considered death cause of these animals. Thus, this study analyzed a pharmacological agent on pregnancy in rodents and it provided some evidences that indomethacin presented embryotoxic and lethal effects at a high dose, but it was safe in the therapeutic dose used for humans.<br>O objetivo deste trabalho foi avaliar a performance reprodutiva, estudo morfológico do fígado e características " post mortem" de ratas Wistar prenhes tratadas com indometacina, um inibidor geral de COX. Indometacina foi administrada oralmente, nas doses de 0 (controle), 0,32, 1,68 e 8,40 mg/kg/dia (n=10/grupo), nos dias 3 e 4 de prenhez (dia 0 = primeiro dia de prenhez = esperma positivo). Os animais foram eutanasiados sob anestesia no 11º dia de prenhez, e foram realizadas necropsia e cultura de microorganismos. Os resultados mostraram que as doses de 0,32 e 1,68 mg/kg de peso corpóreo (dose terapêutica para humanos) de indometacina não causaram efeitos embriotóxicos ou letais. A maior dose (8,40 mg/kg) de indometacina prejudicou o processo de implantação e, portanto, interferiu no desenvolvimento fetal. A peritonite foi detectada na necropsia e nos estudos bacteriológicos dos animais tratados com 8,4 mg/kg e considerada a causa-morte destes animais. Portanto, este estudo analisou um agente farmacológico na prenhez de roedores e evidenciou que a indometacina apresentou efeitos embriotóxicos e letais na maior dose empregada, mas foi segura na dose terapêutica usada pelo homem

Hepes na produção de embriões bovinos in vitro Hepes on in vitro production of bovine embryos

O objetivo do presente estudo foi de avaliar a amplitude da variação de pH em meios de maturação e de cultivo embrionário, com diferentes concentrações do tampão HEPES. Inicialmente, foi determinado o efeito de diferentes concentrações de HEPES (0, 12,5 e 25,0mM) na variação do pH dos meios de maturação (TCM-199 modificado) e desenvolvimento embrionário (KSOM modificado) em meio ambiente, à temperatura de 25ºC, e na estufa, em uma atmosfera de 5% de CO2 em ar a 39ºC. Em um segundo experimento, os oócitos foram maturados em TCM-199 modificado sem HEPES (142 oócitos) ou com 25,0mM de HEPES (137 oócitos) e foi avaliado índice de blastocisto. O meio Fert-TALP foi utilizado para a fecundação, sendo que os embriões foram co-cultivados com células epiteliais de oviduto (CEO) em KSOM modificado com 10% de SFB. Um terceiro experimento foi delineado para determinar a importância da presença do HEPES no meio de cultivo embrionário sobre o desenvolvimento de embriões bovinos in vitro. Para isso, após a retirada do cumulus, os zigotos foram divididos ao acaso e co-cultivados com CEO em KSOM modificado (com 10% de SFB) sem HEPES (grupo controle; n = 95) ou com 25,0mM de HEPES (grupo HEPES; n = 92). Foram mantidos em cultivo somente os embriões com duas ou mais células, sendo considerados desenvolvidos os que atingiram o estádio de blastocisto expandido (Bx), 7 e 9 dias após inseminação. O cultivo dos oócitos e embriões, em ambos os experimentos, foi efetuado em estufa a 39ºC, com uma atmosfera contendo 5% de CO2 e umidade saturada. Os resultados mostram que os meios contendo 25,0mM de HEPES foram mais eficientes em minimizar a variação do pH que os meios com 12,5mM ou sem HEPES. Além disso, a adição de HEPES ao meio de maturação aumentou os índices de Bl sobre o total de oócitos e sobre o total de clivados (21,9% e 42,86%) com relação ao controle (10,56 e 16,67%; p<0,05). Na determinação da importância do HEPES no desenvolvimento embrionário, o grupo HEPES apresentou índices superiores de Bx (45,65%) em relação ao controle (11,58%; p<0,01). A uniformidade dos resultados foi um dos aspectos positivos observados quando o HEPES estava presente no meio de cultivo embrionário. Portanto, o uso do HEPES, durante a maturação e o cultivo embrionário, é recomendável para o aumento da produção de embriões in vitro com maior repetibilidade.<br>The aim of the present study was to evaluate the range of pH changes in maturation and embryo development media, buffered with different HEPES concentrations. Initially, the effect of different concentrations of HEPES (0, 12.5 and 25.0mM) on the variation of pH in the maturation (modified TCM-199) and embryonic development (modified KSOM) media was evaluated at room temperature (25ºC) and in an atmosphere of 5% CO2 in air at 39ºC. In another experiment, the effect of HEPES on in vitro oocyte maturation was determined. Oocytes were maturated in TCM-199 modified either with 25.0mM of HEPES (HEPES group; n = 137) or without HEPES (control group; n = 142), performing 7 replicates and evaluating the rate of blastocyst. In this study, the medium used for fertilization was Fert-TALP while for embryo development was KSOM with 10% of fetal bovine serum with monolayer of oviduct epithelial cells. A third experiment was designed to determine the effect of HEPES on embryo development. The zygotes were divided in two groups and co-incubated with oviduct epithelial cells in modified KSOM with 10% of fetal bovine serum without HEPES (n = 95) or with 25.0mM of HEPES (n = 92). For this experiment, it was used embryos with two or more cells and the embryo development was considered from cleavage to expanded blastocyst (Bx), 7 and 9 days after insemination. The oocytes and embryos were incubated at temperature of 39ºC, an atmosphere containing 5% CO2 in air and saturated humidity. The media with 25.0mM of HEPES were more efficient in minimizing the range of pH than those with 12.5mM or without HEPES. To determine the effect of HEPES during in vitro oocyte maturation, the percentage of Bl considered either the total number of oocytes or the total number of cleavages was higher in the HEPES group (21.9% or 42.9%, respectively) than those obtained in the control group (10.56% or 16.67%, respectively). When HEPES was added to embryo culture medium, the percentage of Bx (45.65%) was higher than that obtained in medium without HEPES (11.58%; p<0.01). The variability between replicates was lower when HEPES was present in embryo development medium, comparing to the medium without HEPES. Therefore, HEPES is an important compound to be present in media for oocyte maturation and embryo development, increasing the in vitro production of bovine embryos