Messenger RNA expression and protein localization of growth hormone in bovine ovarian tissue and in cumulus oocyte complexes (COCs) during in vitro maturation

The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) obtained from 2 to 8 mm follicles synthesize growth hormone (GH) during in vitro maturation. In addition the expression of growth hormone releasing hormone receptor (GHRH-r) in the COCs before and after in vitro maturation was investigated. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed and were prepared for immunohistochemical staining to detect the presence of GH. In addition, sections of ovary were stained to study the differential localization of GH in the ovary. At 0 and 24 hr COCs were removed and together with samples from granulosa cells and theca cells were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GH and GHRH-r. Within COCs, cumulus cells and oocytes showed GH immunoreactivity, while expression of GH mRNA was only found in the oocyte. At the onset of culture, oocytes and cumulus cells in the majority of COCs generally showed moderate and strong staining intensity for GH, respectively. While GH staining in the cumulus cells did hardly change during 24 hr of culture, GH staining in the oocyte was absent after 24 hr of culture in 70% of COCs. Within the ovary, GH was localized in antral follicles larger than 2 mm and no staining was found in primordial, primary and secondary follicles or in the stroma. The intensity of the staining increased with the size of the follicles. Within the follicular wall the GH was persistently observed in granulosa cells, while theca cells were occasionally negative. GH mRNA in follicular compartments was only found in the oocyte and mural granulosa cells. No GHRH-r mRNA was found in the COCs nor in the granulosa or the stroma. In conclusion, the gradual increase of GH staining during follicular development and the consistent synthesis of GH in oocytes and granulosa cells, suggest a paracrine and/or autocrine action for GH in bovine follicular growth and oocyte maturation. The absence of mRNA for GHRH receptor in the COCs indicates that ovarian production of GH is not regulated by GHRH.