Somatotroph regulation in dwarf rats

Growth hormone (GH) and prolactin (PRL) are synthesized in specialized secretory cells in the anterior pituitary gland. The cells producing these hormones are thought to develop from a common precursor cell which co-expresses both GH and PRL. Somatotrophs are under stimulatory and inhibitory control from hypothalamic factors, influencing GH release, GH gene expression and somatotroph proliferation. The autosomal recessive dwarf rat (dw/dw) exhibits a 40% reduced growth rate compared to normal rats of the same (AS) strain. While the secretory function of dw/dw somatotrophs is relatively intact, somatotroph proliferation and GH gene expression is markedly reduced, resulting in a 75-80% reduction in somatotroph number and a 95% reduction in pituitary GH. This specific deficiency is a result of impaired cAMP accumulation in response to growth hormone releasing factor (GRF) due to the inability of Gs to activate adenylate cyclase (Downs and Frohman, 1991). Dw/dw anterior pituitary cultures, in my hands, showed no detectable increase in cAMP, while cultures from AS rats showed an increase in cAMP in a dose dependant manner. Both AS and dw/dw pituitary cultures responded to GRF with GH release in a dose dependent manner, illustrating the distinction between the cAMP dependant responses (GH gene expression and somatotroph proliferation) and the secretory response. I further investigated the cAMP response to GRF in dw/dw anterior pituitary cultures by manipulating the somatotroph number in the culture to control for the reduced proportion of GH cells in the dw/dw. This thesis provides the first description of fluorescence activated cells sorting (FACS) to purify somatotrophs from dw/dw pituitary cells. Dw/dw somatotrophs enriched to over 40% by fluorescence (equivalent to an AS pituitary culture) did not show cAMP accumulation in response to GRF. In addition, cultures of AS and dw/dw pituitary cells mixed to contain just 2% normal somatotrophs (about 6 fold less than a dw/dw pituitary) were still able to exhibit a significant cAMP response to GRF. These data support the existence of an inherent defect in the GRF signalling pathway in these cells. While my data support the existence of the GRF signalling defect in the dw/dw somatotrophs, I also present data that the phenotype is not restricted to this cell type. SDS-PAGE of anterior pituitary extracts suggested that the PRL content in the dw/dw pituitary was increased compared to the AS. Radioimmunoassay of pituitary extracts confirmed a 2-3 fold higher PRL content in both male and female adult dw/dw rats, despite profound pituitary hypoplasia. As somatotrophs and lactotrophs belong to the same lineage, I therefore decided to investigate the proportions of both cell types during development. This was carried out using two colour FACS analysis, which also allowed the quantification of the mammosomatotroph precursor and intermediate cells