The general aim of our in

The general aim of our in vitro experiments was to analyze the patterns of ghrelin and GHSR-1a expression in the mammary glands of dairy goats during pregnancy and to examine the direct effect of ghrelin on cell proliferation in cultured MECs of pregnant dairy goats. For this purpose, we cultured goat MECs and examined the action of ghrelin on the cell proliferation, and the expression of proliferation-related peptides (PCNA, cyclin B1) and mammary PRL. This effect might induce mammary growth and development of pregnant diary goats.
Materials and methods
Results
Discussion This is the first study that has examined the expression of both ghrelin and GHSR-1a in the mammary glands of pregnant dairy goats, and revealed the stimulatory effects of ghrelin on the cell proliferation and the expression of proliferation-related peptides (PCNA and cyclin B1) and PRL in cultured MECs. A recent report showed that ghrelin is expressed in human mammary gland (Grönberg et al., 2008). Studies also showed that GHSR-1a is expressed in mammary glands of lactating rats and dairy goats (Roche et al., 2008, Zhang et al., 2013). In this study, the expression of ghrelin and GHSR-1a were detected at all stages, their abundance varied significantly depending on the phase of the pregnancy. The expression of both the hormone and the receptor was lower at early stage (days 30 of pregnancy) and higher at the later stages (days 90 and days 120 of pregnancy). Thus, the expression of ghrelin and GHSR-1a reached a peak when mammary gland entered into the stage of rapid epithelial cell proliferation. The immunohistochemical analysis showed that in mammary tissue, ghrelin and GHSR-1a are localized in the epithelial CORM-3 and the intensity of specific staining seemed higher at mid and late pregnancy. Our present findings suggest that ghrelin may play an important role in the mammary growth and development of dairy goats during pregnancy. We found that ghrelin significantly increased the cell proliferation in primary culture of mammary epithelial cells of pregnant goats. This observation was in line with earlier reports on the stimulatory effect of ghrelin on the proliferation of non-mammary cell types such as cardiomyocytes, endothelial cells, and ovarian cells (Baldanzi et al., 2002, Rak and Gregoraszczuk, 2008). Recently, it was reported that ghrelin modulates mammary β-casein expression and cell viability in lactating dairy goats (Zhang et al., 2013). However, the functions of ghrelin in pregnant mammary glands of dairy goats remained unknown. Our results provide the first evidence of a direct stimulatory effect of ghrelin on the cell proliferation in cultured goat mammary epithelial cells. These observations confirm our hypothesis on the proliferation-stimulating effect of ghrelin on pregnant goat MECs. This is the first evidence that expression of PCNA, cyclin B1 in MECs could be promoted by ghrelin. Our data suggest a stimulatory effect of ghrelin on mammary epithelial cell proliferation via promotion of cell cycle progression in MECs. This effect might induce mammary growth and development. It has been estimated that the significance of mammary growth was taken place during gestation in many species, the duct system increases in size and epithelial cells proliferate (Knight and Peaker, 1982). The control of mammary growth involves a complex hierarchy of interacting mechanisms. The mammary growth is regulated by the actions of steroid and polypeptide mammogens (Flint and Knight, 1997). Our results suggest that ghrelin is produced locally and may be involved in the autocrine/paracrine regulation of pregnant mammary growth of dairy goats. Our results showed expression of PRL mRNA in the mammary glands from days 60 to days 120 of pregnancy. The pattern of PRL expression showed similar trend as that of mammary ghrelin and GHSR-1a expression during pregnancy. The results are consistent with the hypothesis that ghrelin might regulate PRL expression (Zhang et al., 2009). Further, ghrelin increased the expression of PRL in cultured goat mammary epithelial cells. The primary source of PRL in plasma is the pituitary gland (Ben-Jonathan et al., 1996, Tucker, 2000). Reports have described the production of PRL in the mammary glands of various species, including sheep and goats (Fields et al., 1993, Le Provost et al., 1994, Steinmetz et al., 1993). Ghrelin infusion increases the concentration of circulating PRL in humans (Tassone et al., 2003). We have reported that ghrelin significantly upregulates the expression of PRL mRNA in the pituitary gland of dairy goats (Zhang et al., 2009). In contrast, study has shown that in rodents, ghrelin inhibits PRL secretion (Tena-Sempere et al., 2004). Although these results suggested that ghrelin regulates PRL secretion in human and animals, whether or not ghrelin directly regulates PRL expression in the mammary glands was not known. Our results showed that ghrelin directly regulated mammary PRL mRNA expression in dairy goats. PRL is involved in the growth and differentiation of the mammary glands (Ben-Jonathan et al., 1996, Bern and Nicoll, 1968). Since ghrelin promoted the cell proliferation and PRL expression in goat mammary glands, it is likely to think that ghrelin promotes the cell proliferation of mammary epithelial cells by up regulating the PRL expression. However, further experiments are needed to fully understand the mechanisms involved.