Regulation and distribution of squirrel monkey chorionic gonadotropin and secretogranin II in the pituitary

Abstract

Secretogranin II (SgII) is a member of the granin family of proteins found in neuroendocrine and endocrine cells. The expression and storage of SgII in the pituitary gland of Old World primates and rodents have been linked with those of luteinizing hormone (LH). However, New World primates including squirrel monkeys do not express LH in the pituitary gland, but rather CG is expressed. If CG takes on the luteotropic role of LH in New World primates, SgII may be associated with the expression and storage of CG in the pituitary gland. The goal of this study was to evaluate the regulation and distribution of CG and SgII in the squirrel monkey. A DNA fragment containing approximately 750 bp of squirrel monkey SgII promoter was isolated from genomic DNA and found to contain a cyclic-AMP response element that is also present in the human SgII promoter and important for GnRH responsiveness. The squirrel monkey and human SgII promoters were similarly activated by GnRH in luciferase reporter gene assays in LβT2 cells. Double immunofluorescence microscopy demonstrated close association of SgII and CG in gonadotrophs of squirrel monkey pituitary gland. These results suggest that CG and SgII have a similar intercellular distribution and are coregulated in squirrel monkey pituitary gland.

Figure 1

The proximal squirrel monkey SgII promoter (smSgII) shares consensus sequences with the human SgII (huSgII) promoter. The CRE is conserved in the squirrel monkey SgII promoter (underlined and in bold). Dashes indicate nucleotides bases that are identical to the squirrel monkey sequences. Asterisks indicate nucleotide deletions.

Figure 2

Squirrel monkey SgII and CGβ promoters are regulated by GnRH. LβT2 cells were transiently transfected with luciferase-reporter plasmids driven by either the squirrel monkey (sm) SgII (−745/+35), human (h) SgII (−869/+1), or squirrel monkey CGβ (−1898/+9, smCGβ) promoter constructs. After 24 h, cells were incubated in the absence or presence of 100 nM GnRH for 6 h. Cells were harvested and assayed for luciferase activity, and data are shown as percent change in luciferase activity over that achieved in unstimulated cells, set at 100 percent. Each bar represents the mean ± SEM of at least three independent experiments. *, significantly different than control.

Figure 3

Distribution of CG and SgII in squirrel monkey pituitary gland. A, DAPI nuclear staining of squirrel monkey pituitary cells. B, The presence of CG in squirrel monkey pituitary gland was detected using 518B7 antibody, visualized with FITC-labeled goat anti-mouse secondary antibody. C, The presence of SgII was detected using an anti-SgII antibody, visualized with Texas Red-labeled goat anti-rabbit secondary antibody. D, Composite micrograph of squirrel monkey pituitary gland showing DAPI, CG, and SgII labeling in squirrel monkey pituitary gland. E, Composite micrograph showing DAPI, LH, and SgII labeling in human pituitary gland. F, Composite micrograph of squirrel monkey pituitary showing separate staining of CG and prolactin, visualized using 6F11 antibody and Texas Red-labeled goat anti-rabbit secondary antibody. Scale bars, 20 μm.