Preimplantation bovine embryos express mRNA of growth hormone receptor andrespond to growth hormone addition during in vitro development

In previous studies we demonstrated that bovine cumulus oocyte complexes (C OCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in v itro maturation. The aim of this study was to investigate whether bovine zy gotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether sup plementation of GH during embryo culture affects embryo development. Theref ore, COCs obtained from small and medium sized follicles were cultured in M 199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro f ertilization the embryos were cultured: (a) on a monolayer of buffalo rat l iver (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 suppleme nted 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. Embr yos were scored morphologically and the efficiency of the culture system wa s 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 blas tocysts 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) oocy tes (as positive control), embryos with less than 8 cells, 16-32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase cha in 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 develo pment. No mRNA for GH could be detected in early and expanded blastocysts p roduced in SOF medium. Immunoreactive GHR was found both in trophoblastic a nd 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% FC S did not affect embryo development. However, GH (100 ng/ml) supplementatio n during embryo culture in droplets of serum-free BRL conditioned medium si gnificantly (P < 0.05) enhanced the proportion of > 8-cell embryos. Similar ly, 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 fr om 53.8% in control to 70.6% in GH-treated group. Day 9 blastocyst formatio n 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 wit hout GH rarely resulted in hatched blastocysts (0.7%). However, GH suppleme ntation 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, blas tocyst formation and hatchability of the embryos point to the involvement o f GH in early embryonic development. Mel. Reprod. Dev. 57:247-255, 2000. (C ) 2000 Wiley-Liss, Inc.