In situ hybridization of GnRH mRNA in the rat and the mink hypothalamus using biotinylated synthetic oligonucleotide probes.
The cellular localization of GnRH messenger RNA (mRNA) in the rat and the mink hypothalamus has been examined using a newly developed highly sensitive non-radioactive in situ hybridization procedure. Synthetic oligonucleotides labeled by addition of a biotin-21-dUTP tail at their 3' end can be used to detect GnRH mRNA in both species. Streptavidin-alkaline phosphatase revealed with nitroblue tetra-zolium-bromo-chloro-indolyl-phosphate as substrate makes possible detection of the biotinylated oligonucleotides. In the rat, our findings confirm results previously obtained using synthetic radioactive probes, and demonstrate the potency of and interest in using biotinylated oligonucleotides to identify related sequences of bases in tissues. The principle advantages include rapid signal detection, excellent spatial resolution, and low background. In the mink, the in situ hybridization method clearly confirms the characterization of GnRH-producing cells and also allows detection of GnRH cell bodies in conditions in which they are not detected by immunohistochemistry. Adaptation of the in situ hybridization to the detection of GnRH mRNA in species like the mink which shows seasonal reproductive activity is a crucial step. This method offers a new approach to problems as fundamental as changes in gene expression depending on photoperiod or under a variety of experimental conditions.
Boissin-Agasse
L
L
Laboratoire de Neurobiologie endocrinologique, URA 1197 CNRS, Université de Montpellier II, France.
de Bouard
V
V
Roch
G
G
Boissin
J
J
D000469:Alkaline Phosphatase; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D015342:DNA Probes; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis*; D007031:Hypothalamus / Q000378:metabolism*; D008297:Male; D008907:Mink / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats / Q000378:metabolism*; D011919:Rats, Inbred Strains; D012621:Seasons; D012726:Sexual Behavior, Animal; D013045:Species Specificity; D019809:Streptavidin
D001426:Bacterial Proteins; D015342:DNA Probes; D012333:RNA, Messenger; D007987:Gonadotropin-Releasing Hormone; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase