Molecular and cellular investigation of leptin hormone and its receptors in large ruminants

Abstract

The experimental data described in this thesis shows the first information about leptin hormone and its receptor in the Egyptian water buffalo and one-humped camel. Additionally our data shows the first investigation about the origin of locally produced leptin in the mammary gland of large ruminant animals. The study about the alteration of mammary leptin gene expression between dry and lactating gland also serves as new scientific data. The major achievements of the present PhD work are listed below: Using RT-PCR we demonstrated the expression of leptin hormonein adipose tissue and mammary gland in the lactating Egyptian water buffalo, dry and lactating cow, and dry one-humped female camel. Leptin expression is also visualized in the liver tissue of the one-humped camel. RT-PCR revealed the expression of long (Ob-Rb) and short (Ob-Ra) isoforms of leptin receptor in the mammary gland and adipose tissues of all the examined species, as well as in the liver of the on-humped camel. We prepared partial cDNA clons of leptinhormone and the examined receptors for the examined species (cow, Egyptian water buffalo, and one-humped camel) using PCR cloning. The result of cDNAs sequences and computer analysis using GCG program revealed that leptin hormone and the two examined isoforms of its receptor of both the buffalo and the camel show high homology to other mammalian species especially to ruminants. In situhybridization analysis revealed the localization of leptin and leptin receptor mRNA transcripts in the mammary gland epithelial cells of the of large ruminants Immunohistochemical staining of leptinprotein confirms the result of in situ hybridization analysis of leptin mRNA in the mammary gland. In camel hepatocytes and lining epithelial cells of the bile duct leptin mRNA and protein was localized by in situhybridization and immunohistochemistry procedures, respectively. Competitive PCR method was used to compare the leptin mRNA levels between dry and lactating mammary gland of non-pregnantcows. Leptin expression for the whole mammary tissues showed no difference. However, during lactation, the mammary adipose tissue is completely regressed. Thus, lactating epithelial cellsproduce the same amount of leptin as the epithelial cell and the fat cells together in the dry mammary gland. Finally, we can conclude that, the leptin hormone is highly conserved in the mammalian species especially among ruminants. Local paracrine and/or autocrine effects are attributed to leptin, which gives the possibility for auto-regulation on the tissue level. The idea is supported by the observation, which describes the expressionof leptin and its receptor in the same tissues. Alveolar epithelial cells of the mammary gland might be an additional source of milk leptin. The epithelial cells of the mammary gland express much of leptin during lactation, which might help maintaining continuous milk production. The hepatic leptin of the camel might be associated with the lipogenic activity of the liver in this species.