Immunohistochemistry analysis shown ghrelin and GHSR a immun
Immunohistochemistry analysis shown ghrelin and GHSR-1a immunostaining was located in the epithelial cells of Daptomycin and ducts throughout the lactation, strong immunoreactive cells were detected in L30, L60 and L120 stage. The distribution of ghrelin has been shown in many tissues, including mammary glands of postmenopausal and fertile age women (malin et al., 2008). However, the presence of ghrelin and GHSR-1a immunoreactive cells in normal mammary glands of animal has not previously been described. In this study, Ghrelin and GHSR-1a immunostaining was detected in mammary glands of dairy goats at all developmental stages, mainly in the epithelial cells. The results suggested that ghrelin may involved in the regulation of mammary function, although study has shown that ghrelin promoted β-casein expression and milk production in rats (Nakahara et al., 2003), however, the functions of ghrelin in mammary gland of dairy goat are unknown. In the second part of this study, we investigated whether ghrelin promote β-casein expression in the lactating goat mammary glands. Our results showed that ghrelin dose-dependently increased β-casein expression in the cultured mammary tissues and primary MECs, the greatest increase was observed at the dose of 103pg/ml ghrelin treated 18h. The regulation of casein expression is a complex process, a variety of hormones are known to play roles in lactogenesis, and growth factors are now being implicated as local mediators of hormonal function (Oka et al., 1991, Karensa et al., 2009, Choi et al., 2004, Plath et al., 1997, Robinson et al., 1993). Here results showed that mammary ghrelin may be one of these local mediators. Nakahara (2003) reported that daily injection of ghrelin caused a significant increase in milk yield, plasma GH levels and β-casein mRNA levers in mammary glands of rats. These results imply two possible mechanisms of action of ghrelin on milk synthesis, a direct and an indirect action. Our results confirmed that ghrelin could increase the expression of β-casein in the cultured mammary tissues and primary MECs, it support the view that ghrelin was able to directly modulate the expression of β-casein in mammary glands. In domestic animals, such as ruminants, GH has often been used as a galactopoietics hormone (Etherton and Bauman, 1998, Tauer and Knoblauch, 1997). These findings imply that ghrelin-induced GH could stimulate milk production in lactating mammals. Recent studies investigating the involvement of ghrelin in ruminant physiology have reported that ghrelin stimulates GH secretion (Javed et al., 2006, Itoh et al., 2005). Thus, ghrelin has specific central effects to regulate the GH axis in ruminants. A very recent study showed that ghrelin also has different regulatory effects on plasma pancreatic hormone levels, glucose concentration and on milk yield (Roche et al., 2008, Itoh et al., 2006). These studies showed that administration of ghrelin may stimulate milk production in animals. However, to our knowledge, these experiments were conducted In vivo, the present study is first time to explore the effects of ghrelin on β-casein expression in vitro. GHSR-1a, as the functional receptor of ghrelin, which implied that ghrelin may stimulate the expression of β-casein via GHSR-1a. In order to study receptor dependent mechanism of ghrelin action in mammary function, we used a ghrelin receptor antagonist, D-Lys-3-GHRP-6, in cultured mammary tissues and primary MECs. Our results showed D-Lys3-GHRP6 treatment suppressed ghrelin-induced enhancement of the β-casein expression. In cultures treated together ghrelin and D-Lys-3-GHRP-6, expression of β-casein mRNA and protein returned to control levels, suggesting GHSR-1a receptor mediated action. The effect of ghrelin on cell proliferation of MECs was also determined in this study. In MECs exposed to 102, 103 and 104pg/ml of ghrelin all significantly increased the cell viabilities compared to control group after 12, 18h and 24h incubation. This observation is in line with previous reports on the stimulatory effect of ghrelin on cell proliferation in non-mammary cell types, such as cardiomyocytes, endothelial cells and ovarian cells (Baldanzi et al., 2002, Rak and Gregoraszczuk, 2008). The result suggest that ghrelin may be involved in the autocrine/paracrine regulation of mammary development of dairy goat.