Effect of LH-RH on the Release of Sulfated Lutropin: A Potential In Vitro Bioassay for LH-RH.

Abstract

Sheep pituitary cells prelabelled with radioactive [35S] sulfate (35SO4(2-)) were incubated with different concentrations of LH-RH and the release of LH (lutropin) into the medium was monitored in terms of immunoprecipitable [35S] sulfated LH radioactivity and estimation of LH in the same sample by radioimmunoassay. A dose dependent response was obtained with a maximum of a 16 fold increase in immunoprecipitable 35SO4(2-) -labelled LH radioactivity in the medium which was confirmed by radioimmunoassay. Similar results were also obtained for Buserelin, a well known superactive analogue of LH-RH. However, the half maximal response for Buserelin was obtained at 3-5 nM in comparison to 80.5 nM for LH-RH. After the maximal response to LH-RH as well as Buserelin, a further increase in the concentrations caused a decrease in the release of immunoprecipitable [35S]-sulfate labelled LH into the medium. Differential labelling of stored and newly synthesized LH with radioactive [35S] sulfate and [3H]-labelled leucine revealed that there was a dose dependent increase in the [35S] sulfate labelled LH into the medium whereas the release of [3H]-leucine labelled newly synthesized LH did not show a parallel increase either at different concentrations of LH-RH or at different time intervals. The above observations strongly suggest the possibility of sulfation of LH being the potential signal indicating the storage of LH in sheep pituitary cells. Another important observation in our study was that the dose dependent response of LH-RH in the form of release of [35S]-sulfate labelled LH, which was monitored by immunoprecipitation with specific LH antiserum, can be used in an in vitro bioassay for LH-RH. We believe that a new cheap and sensitive in vitro bioassay could be developed on the basis of this observation.

Sulfation of LH does not affect intracellular trafficking

“…Sulfation of LH has been proposed to act as a sorting signal (Pierce & Parsons, 1981, Kohli & Muralidhar, 1990, Boime et al , 1999, Bousfield George R. et al , 2006). Kohli and Muralidhar (1990) compared the LH-RH response of LH by indirect double labeling experiments.…”

“…Kohli and Muralidhar (1990) compared the LH-RH response of LH by indirect double labeling experiments. From these data they suggested that sulfate could be a sorting signal for LH.…”

“…Sulfation of LH has been proposed to act as a sorting signal (Pierce & Parsons, 1981, Kohli & Muralidhar, 1990, Boime et al , 1999, Bousfield George R. et al , 2006). Analysis of the asparagine (N)-linked oligosaccharides of human, bovine and ovine gonadotropins revealed that these structures terminate with sulfate or sialic acid (Pierce & Parsons, 1981, Green & Baenziger, 1988a).…”