Expression Of Ghrelin In Sheep Ovary And Pre-implantation Embryos

Ghrelin has been identified by Kojima as the first endogenous ligand for the GH secretagogue receptor (GHS-R) in 1999. Ghrelin as the new one of the neuroendocrine system plays an important role in energy homeostasis and elicits GH release; there are close relationships between energy homeostasis and reproduction. This makes us recognize the function of neuroendocrine system in the reproduction system.Follicular growth, development, marution and atresia, development of the corpora lutea (CL) or embryonic development highly related with energy homeostasis. In this sense, the activity role of ghrelin stimulates us naturally think about: Whether ghrelin may have a direct role in the dramatically net expression levels of a target gene and peptide. Ghrelin in the control of reproduction function remains to be elucidated. The aim of this paper was start with characterizing in detail the pattern of expression of ghrelin in the sheep ovary, with special attention to the cellular distribution of ghrelin peptide within the ovarian tissue as well as the the expression pattern of ghrelin in embryonic development.1. Expression of ghrelin in the sheep ovary(1) Initial RT-PCR analyses first revealed that ghrelin mRNA is expressed in sheep ovary.(2) In situ hybridization for ghrelin mRNA showed wider of hybridization with the ovarian follicles. Ghrelin mRNA was localised to the CC, GC and theca cells, as well as the ovarian surface epithelium (OSE). Ghrelin mRNA was also indicative of gene expression in the all developmental follicles (primary, secondary and pre-antral). Ghrelin mRNA was detected clearly in all developmental stages of the CL (oestrous cycle and throughout pregnancy).(3) Ghrelin protein also showed a somewhat wider pattern of cellular distribution within the sheep ovary. Interestingly, ghrelin protein was found in the oocytes of the antral follicles, as well as the follicular fluid, but it is sure the positive singal in CC, GC and theca cells. Also, the ghrelin signal was more intense in the OSE. Furthermore, the results show not only that ghrelin is present in the antral follicles, but that ghrelin was present in preantral follicle. In addition, ghrelin protein was detected in oocyte, with an expression profile that was roughly parallel to follicle development. Follicles showed variable ghrelin immunoreactivity. In preantral and antral follicles, ghrelin protein was detected in the oocytes, GC showed weak positive. Specific ghrelin signal was observed in both larger luteal cells (LLC) and small luteal cells (SLC) of the oestrous and pregnancy CL. The observed ghrelin immunoreactivity was predominantly in the cytoplasm of the LLC. To avoid inadequacy of the immunohistochemistry approach, we confirmed expression of ghrelin in the CC, GC and theca cells of the antral follicles by using immunofluorescence staining.In summary, there is weak ghrelin singanl in CC and GC compared with oocytes.2. Ghrelin mRNA Changes in the antral follicles and all development stages CL(1) Ghrelin mRNA expression was evaluated in antral follicles by means of relative quantitative real time RT-PCR. Relative expression levels of ghrelin in the different components of the antral follicles did not show significance before ovulation/ atresia/ degeneration.(2) The pattern of expression of ghrelin mRNA in CC was examined during in vitro maturation, the results indicated that expression of ghrelin mRNA was decreased evevy other 8h.(3) To further confirm mRNA changes at different development CL, mRNA was extracted from newly developed, mature and regressing CL, and also from very early, early, middle, and late pregnancy CL model was used to discriminate the expression of ghrelin mRNA between Relative mRNA levels of ghrelin varied depending on the stage of the cycle, with significantly decreased values in regressing CL (P<0.05) and significantly increased relative expression values to maximum levels during the luteal phase of the cycle. In the CL of pregnant sheep, ghrelin expression is low at the beginning, peaks during pregnancy, and is lowest late in pregnancy (P<0.05)The profile of ghrelin expression in the CL was roughly coincident with its peak in functional activity.3. Expression of the ghrelin in sheep oocytes and preimplantation embryos(1) The possibility of ghrelin expressed in sheep oocytes and preimplantation embryos was investigated by using real time PCR and immunofluorescence staining. Dynamic changes and the presence of the ghrelin signaling system persists within oocytes and pre-implantation embryos. Ghrelin also indicated a somewhat wider pattern of distribution. A. Relative real-time reverse-transciptase (RT)-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes and at the 2-cell stage blastocyst(BL) produced in vitro, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the morula. 16,32-cell and BL produced in vivo was significantly higher expression than MII, 2- and 8-cell stages, GV, 4- and 8-cell stages was lowest. B. Immunofluorescence staining results indicated ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane before 4-cell stages produced in vitro, fluorescence signal was gradually intense and localized in the cytoplasm after 4-cell stages, In embryos at the blastocyst stage, the inner cell mass (ICM) and trophectoderm(TE) are equally well-stained, ghrelin protein was detected mainly in the cytoplasm oocytes and preimplantation embryos produced in vivo and intense with development.In summary, oocytes and preimplantation embryos produced in vivo was more intense than those produced in vitro at mRNA and peptide levels.(2) The pattern of expression of ghrelin mRNA in oocytes was examined during in vitro maturation, the results indicated that expression of ghrelin mRNA was increased evevy other 8h. To ascertain the role of hormone on the expression of ghrelin mRNA based on different culture media, we added LH, FSH and E2 for purpose which element is responsible to the above results, the results showed the oestrous serum Ewe and E2 promote ghrelin mRNA expression.In this study identification of the expression of ghrelin was accomplished by molecular (RT-PCR, real time RT-PCR and ISH) and immunological approaches (Immunohistochemistry and Immunofluorescence staining), The results of this study provide the directly demonstration of the expression of ghrelin (at the mRNA and peptide levels) with somewhat wider pattern of cellular distribution in the in the sheep ovary and preimplantation embryos. Especially in the oocytes provide evidence for further study on the function of the Follicular growth, development, marution or embryonic development.In summary, the results make it worthy to evaluate the potential implication of this novel signal in follicle development. Evidence for the expression of of ghrelin signaling system within the sheep ovary paves the way for further studies on the role of this novle molecule in ovarian function.