The caseins are a family of milk phosphoproteins

The caseins are a family of milk phosphoproteins whose biological function is to provide supersaturating concentrations of calcium, phosphates, and essential mps1 to the neonate (Vonderhaar and Ziska, 1989). α- and β-caseins, the most abundant of the milk caseins, are described as calcium sensitive because they precipitate in the presence of low concentrations of calcium, they are maintained in stable suspension in milk because of their interaction with κ-casein. Caseins are useful markers of milk protein synthesis and partial differentiation but in many studies casein expression can be stimulated in the absence of full lactation. It would be useful to make this distinction. The regulation of casein synthesis is a complex process multiple interactions between peptides and steroid hormones. Expression of caseins in mammary explants is also influenced by cell-cell and cell-matrix interactions (Levine and Stockdale, 1985, Bissell and Ram, 1989), and there is increasing evidence that growth factors play important roles in mammary functions (Oka et al., 1991). Northern blot analysis revealed that daily injection of ghrelin significantly increased mammary casein mRNA expression in rats (Nakahara et al., 2003). Here, we explore whether ghrelin regulates cultured mammary tissues and epithelial cells production of β-casein. The biological actions of ghrelin are mainly due to interaction with its specific cell surface receptor, GHSR-1a, which transduces the GH-releasing effect of synthetic GHS drugs as well as of ghrelin. GHSR-1a is a classic G proteincoupled receptor containing seven putative membrane spanning alphahelical segments (Howard et al., 1996). In order to study receptor dependent mechanism of ghrelin action in mammary function, we used a ghrelin receptor antagonist, D-Lys3- GHRP-6, in cultured mammary tissues and epithelial cells. D-Lys3-GHRP-6 has been reported as a synthetic selective antagonist of the ghrelin receptor, GHSR-1a, and its functional properties have been extensively studied both in vitro and in vivo in animals (Beck et al., 2004, Benso et al., 2007, Sibilia et al., 2006). Therefore, the objectives of the present study were (1) to explore the patterns of ghrelin and GHSR-1a expression in the mammary glands of Guanzhong Saanen dairy goats during the lactation, with special attention to the distribution of ghrelin and GHSR-1a in the mammary glands using immunohistochemical method; and (2) to investigate the effects of ghrelin on mammary function in cultured mammary tissues and epithelial cells from lactating dairy goat via receptor dependent mechanism.
Materials and methods
Results
Discussion The present study provides immunohistochemical and molecular (RT-PCR) evidence for the presence of ghrelin and its functional receptor (GHSR-1a) in mammary glands of dairy goat during several stages of lactation and the dry period. Although ghrelin immunoreactive cells have been identified in human mammary glands, breast cancer and cell lines (Malin et al., 2008, Jeffery et al., 2005), and expression of GHSR-1a mRNA has been detected in the mammary glands of lactating rats (Nakahara et al., 2003). However, no detailed characterization of the patterns of expression of ghrelin and GHSR-1a in mammary glands of dairy goat have been reported. We found that ghrelin and GHSR-1a mRNA and protein levels varied significantly depending on the stage of lactation. Immunohistochemical analyses showed that specific ghrelin and GHSR-1a signals were predominantly located in the epithelial cells of alveoli and ducts throughout the lactation. In addition, the study showed that ghrelin could act on cultured goat mammary explants or primary MECs to increase β-casein expression and cell proliferation. Similar patterns of ghrelin and GHSR-1a (mRNA and protein) expression were observed in the mammary glands during the lactation, with higher levels during peak milk yield (day 60–120 of lactation) and lower levels during late (day 180) of lactation, which shown a low-high-low pattern from L30 to L180 stage. Such a cyclic profile of expression, with peak level in the L60 and L120 stage, is highly suggestive of predominant expression of ghrelin and GHSR-1a in the peak lactating stage. The results suggested that ghelin may be important in milk production, appetite and body condition score. In addation, the expression of ghrelin mRNA and protein in the stomach exhibited a similar tendency with β-casein expression and milk yield during the lactation, higher levels during ascending, peak and declining stage of lactation and lower levels during late of lactation. In good agreement, considering that the stomach accounts for more than 65% of circulating ghrelin (Horvath et al., 2001), the results implied that ghrelin might operate as an endocrine regulator of mammary function. Our results showed that ghrelin was locally synthesized in mammary cells,which implied that ghrelin might also act as an autocrine or intracrine mediator in mammary glands. Study showed that plasma ghrelin levels are greastest in early lactation, declining as lactation progresses, consistent with the profile of growth hormone secretion and physiological state of the cow (Itoh et al., 2005). The result also support the view that ghrelin may operate as a regulator of mammary function. It is generally accepted that the increasing milk yield until peak lactation is due to increased proliferation and differentiation of mammary epithelial cells (Capuco et al., 2001). Many hormones and growth factors are known to play roles in normal mammary development and maintaining lactation persistency. So the results are easy for understanding that high levels of ghrelin led to lactogenesis and maintained mammary function in L30, L60 and L120 stages.