Geneeskundige Stichting Koningin Elisabeth ... - GSKE - FMRE

94
At this stage of our experiments, it appears that the difference of response to guinea pig
and rat GnRH probably resides in differences in the interaction of the GnRH molecules with the
GnRH-receptor molecules, rather than in differences in in vivo degradation of the peptides.
In our in vivo experiments, we have experienced that also LH of the guinea pig displays
unusual species specificity. Indeed, the well established in vitro bioassays for measurement of
LH with use of testicular Leydig cells from rat or mice testis, successfully applied to measure LH
from a variety of mammalian species, were shown to be inadequate for measurement of guinea
pig LH, as guinea pig LH fails to stimulate testosterone secretion from Leydig cells from these
two species. Therefore, we had to set up a specific bioassay for measurement of guinea pig LH,
using dispersed Leydig cells from guinea pig testes.
Establishment of new research tools
In previous years, for our studies of the neuro-endocrine regulation of reproduction, we
have used two experimental models : in vitro GnRH secretion by the isolated mediobasal hypothalamus
of the male guinea pig (either in a static incubation system or in a continuous superfusion
incubation system) and in vivo secretion of LH in the male guinea pig with use of a specific
bioassay for measurement of guinea pig LH. In the past year, we have now validated a
new experimental model, i.e. the in vitro LH secretion by an immortalised pituitary cell line of
murine gonadotrophic origine that acquired the capability to secrete intact LH after permanent
transfection with the rat gene for the bèta chain of LH (gift from P. Mellon ; Centre for Molecular
Genetics, University of California at San Diego). In the validation of this model we have demonstrated
in the cell line the presence of the estrogen-receptor (ERa), we have demonstrate the presence
of GnRH-receptor mRNA by RT-PCR, we have demonstrated cellular calcium mobilisation
in response to exogenous GnRH by fluorescence technique (pretreatment of the cells with Fluo-
3) and, finally, we have demonstrated that we can measure both spontaneous and GnRH-stimulated
LH release in the cell-culture medium and modulation of this response by estrogens.
Presently, we are also working on the establishment and validation of cultures of an immortalised
GnRH secreting cell line from murine hypothalamic origine.
Publications
•GAO CQ, VAN DEN SAFFELE J, GIRI M, KAUFMAN JM - Guinea-pig
gonadotropin releasing hormone : immunoreactivity and biological activity. Journal of
Neuroendocrinology 12:355-359,2000
•GAO C, GIRI M, VAN HOECKE MJ, MERTENS K, VAN DEN SAFFELE J, KAUFMAN JM.
Marked species specificity of guinea pig luteinizing hormone : validation of a bioassay. J
Androl 2001,22 (in press)