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首页> 外文期刊>Molecular reproduction and development >Preimplantation bovine embryos express mRNA of growth hormone receptor andrespond to growth hormone addition during in vitro development
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Preimplantation bovine embryos express mRNA of growth hormone receptor andrespond to growth hormone addition during in vitro development

机译：植入前的牛胚胎表达生长激素受体的mRNA，并在体外发育过程中响应生长激素的添加

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In previous studies we demonstrated that bovine cumulus oocyte complexes (COCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in vitro maturation. The aim of this study was to investigate whether bovine zygotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether supplementation of GH during embryo culture affects embryo development. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro fertilization the embryos were cultured: (a) on a monolayer of buffalo rat liver (BRL) cells in M199 supplemented with 10% FCS and 100 ng/ml bovine GH (NIH-GH-B18); (b) in droplets of serum-free BRL-conditioned medium supplemented with 100 ng/ml GH; (c) in droplets of synthetic oviductal fluid (SOF) supplemented with 100 ng/ml GH. Cultures without GH sewed as controls. Embryos were scored morphologically and the efficiency of the culture system was evaluated (a) as the percentage of cleaved embryos 4 days after IVF, (b) the percentage of blastocysts on Day 9 expressed on the basis of the number of oocytes at the onset of culture, and (c) the percentage of hatched blastocysts on Day 11 expressed on the basis of the total number of blastocysts present at Day 9. For gene expression, immature (GV) and mature (MII) oocytes (as positive control), embryos with less than 8 cells, 16-32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GHR. Messenger RNA for GHR was found in GV and MII oocytes and in all stages of embryo development. No mRNA for GH could be detected in early and expanded blastocysts produced in SOF medium. Immunoreactive GHR was found both in trophoblastic and embryonic cells of hatched blastocysts. Addition of 100 ng/ml GH during embryo culture on a monolayer of BRL cells in M199 supplemented with 10% FCS did not affect embryo development. However, GH (100 ng/ml) supplementation during embryo culture in droplets of serum-free BRL conditioned medium significantly (P < 0.05) enhanced the proportion of > 8-cell embryos. Similarly, culture of embryos in droplets of SOF medium in the presence of GH (100 ng/ml) significantly (P < 0.05) enhanced the number of > 8-cell embryos from 53.8% in control to 70.6% in GH-treated group. Day 9 blastocyst formation in SOF medium was also significantly (P < 0.01) increased in the presence of GH (33.9%) compared to the control (20.2%). Embryos cultured in SOF without GH rarely resulted in hatched blastocysts (0.7%). However, GH supplementation remarkably enhanced the proportion of the hatched blastocysts (13%). in conclusion, expression of GHR gene in preimplantation bovine embryos, presence of the receptor, and the beneficial effect of GH on cleavage, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development. Mel. Reprod. Dev. 57:247-255, 2000.
机译：在以前的研究中，我们证明了从中小型卵泡获得的牛卵丘卵母细胞复合物（COC）在体外成熟过程中表达生长激素受体（GHR）mRNA并响应生长激素（GH）的添加。这项研究的目的是调查牛受精卵和植入前胚胎在体外受精后和早期胚胎发育过程中是否继续表达GHR基因，以及在胚胎培养过程中补充GH是否影响胚胎发育。因此，将从中小型卵泡获得的COC在补充有10％FCS和促性腺激素的M199中培养24小时。体外受精后，将胚胎培养：（a）在单层水牛大鼠肝脏（BRL）细胞中的M199中添加10％FCS和100 ng / ml牛GH（NIH-GH-B18）; （b）补充100 ng / ml GH的无血清BRL条件培养基的液滴; （c）补充100 ng / ml GH的合成输卵管液滴（SOF）。没有生长激素的文化作为对照。对胚胎进行形态学评分，并评估培养系统的效率（a）IVF后4天的卵裂卵百分比，（b）第9天胚泡的百分比，根据卵母细胞开始时卵母细胞的数量表示。 （c）第11天孵出的胚泡的百分比，以第9天出现的胚泡总数为基础表示。对于基因表达，未成熟（GV）和成熟（MII）卵母细胞（作为阳性对照），胚胎用少于8个细胞，16-32个细胞和带阴影的胚泡制备逆转录酶聚合酶链反应（RT-PCR），以评估GHR mRNA的表达。在GV和MII卵母细胞以及胚胎发育的所有阶段都发现了GHR的Messenger RNA。在SOF培养基中产生的早期和扩大的胚泡中未检测到GH的mRNA。在孵化的胚泡的滋养细胞和胚胎细胞中均发现了免疫反应性GHR。在补充了10％FCS的M199中，在单层BRL细胞的胚胎培养过程中添加100 ng / ml GH不会影响胚胎发育。但是，在胚胎培养过程中在无血清BRL条件培养基液滴中添加GH（100 ng / ml）显着（P <0.05）会增加> 8细胞胚胎的比例。类似地，在存在GH（100 ng / ml）的情况下，在SOF培养基液滴中培养胚胎会显着（P <0.05）将> 8细胞胚胎的数量从对照组的53.8％提高到GH治疗组的70.6％。与对照组（20.2％）相比，在存在GH（33.9％）的情况下，SOF培养基中第9天的胚泡形成也显着增加（P <0.01）。在没有GH的SOF中培养的胚胎很少导致孵化的胚泡（0.7％）。但是，GH的添加显着增加了孵出的胚泡的比例（13％）。总之，GHR基因在植入前牛胚胎中的表达，受体的存在以及GH对卵裂，胚泡形成和孵化率的有益作用表明GH参与了早期胚胎发育。梅尔责备。开发人员57：247-255，2000。

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