An immunohistochemical study of the localization and developmental expression of ghrelin and its functional receptor in the ovine placenta

Abstract

Background: Ghrelin is an orexigenic hormone principally produced by the stomach, but also by numerous peripheral tissues including the placenta. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation, and has been suggested to play a role in the developmental growth of the fetoplacental unit. The placental expression of ghrelin and its role in ruminant species is not known. We tested the hypotheses that ghrelin and its functional receptor, GHSR-1a, are present in tissues of the ovine placenta, and that their expression is linked to the stage of development.

Methods: Antibodies raised against ghrelin and GHSR-1a were used in standard immunohistochemical protocols on placental tissues collected from pregnant ewes (n = 6 per gestational time point) at days 50, 80, 100, 128 and 135 of gestation (term approximately day 145). Immunostaining for ghrelin and GHSR-1a was quantified using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference.

Results: Positive immunostaining for ghrelin was detected in ovine placentae at all gestational time points, with staining localized to the maternal epithelium, caruncle and trophectoderm. There was a significant effect of gestational age (p < 0.001) on the placental expression of ghrelin, with maximal levels at gestational day 80. GHSR-1a immunostaining was detected in the fetal trophectoderm at all time points. In contrast to the gestational pattern of ghrelin expression, there was no effect of gestational age on placental GHSR-1a immunoexpression.

Conclusion: Ghrelin and GHSR-1a are both present in the ovine placenta, and ghrelin displays a developmentally-related pattern of expression. Therefore, these data strongly suggest that the ghrelin system may have a role in feto-placental development in sheep.

Figure 3

Effect of gestational age on placentomes mass and immunoexpression of placental ghrelin and growth hormone secretagogue receptor (GHSR)-1a. Effect of gestational age on the percentage of cells positively immunostained for ghrelin (a) and GHSR-1a (c) in sheep placentomes collected between days 50 and 135 of gestation (n = 6 at each gestational age). In Fig. 3a the change in total placentome mass is superimposed as the grey line on the graph for ghrelin, and in Fig. 3b the comparison is shown for the 2^nd order polynomial lines of best fit with line equations for the gestational change in ghrelin (black text equation, white squares, black line) and total placentomes mass (grey text equation, grey diamonds, grey line) (b). Values with different alphabetical superscripts are statistically significant compared to the peak (ghrelin) and nadir (GHSR-1a) levels at day 80: a versus b = p < 0.05; a versus c = p < 0.01; a versus d = p < 0.001. Significant changes within variables over time are annotated to each figure. Values are means ± S.E.M.

Figure 1

Immunohistochemical localization of ghrelin in the sheep placenta. Representative immunohistochemical photomicrographs showing localization of ghrelin (b – f) in sheep placentomes collected at days 50 (b), 80 (c), 100 (d), 128 (e) and 135 (f). The insert in the bottom right corner (b) represents an example of a negative control. Positive immunostaining in the placenta was abolished (a) when the antiserum was pre-incubated with the immunizing peptide (ghrelin). Placentome sections mainly show positive immunostaining for ghrelin (brown) in the maternal compartment (M) and the trophectoderm (T), with few cells being immunopositive for ghrelin in the fetal compartment (F). The scale bars represent 50 μm.

Figure 2

Immunohistochemical localization of growth hormone secretagogue receptor (GHSR)-1a in the sheep placenta. Representative immunohistochemical photomicrographs showing localization of GHSR-1a (b – f) in sheep placentomes collected at days 50 (b), 80 (c), 100 (d), 128 (e) and 135 (f). Insert (b) represents an example of a negative control. Positive immunostaining in the placenta was abolished (a) when the antiserum was pre-incubated with the immunizing peptide (GHSR-1a). Placentome sections mainly show positive immunostaining for GHSR-1a (brown) in the maternal compartment (M) and the trophectoderm (T), and in the blood vessels (BV). The scale bars represent 50 μm.