PARENT SESSION

(M511) ACCURATE IDENTIFICATION OF MONOHORMONAL GONADOTROPES WITH DUAL BRIGHT FIELD IN SITU HYBRIDIZATION AND PROBES FOR GONADOTROPIN BETA SUBUNIT mRNA OR hnRNA.

Childs, Gwen¹, ¹ University of Arkansas for Medical Sciences, Little Rock, AR

ABSTRACT- Monohormonal gonadotropes have been detected by dual-immunocytochemistry (ICC) or, by dual in situ hybridization (ISH)-immunolabeling (ISH-ICC). Their numbers may vary with the physiological state. Monohormonal LH cells are most abundant before the proestrous surge; only 40% of cells with LH mRNA contain FSH antigens, in spite of the fact that stores of LH and FSH proteins are at a peak. Monohormonal FSH cells increase 14 days after castration. This suggests independent roles for monohormonal gonadotropes. However, application of dual ISH is needed to confirm their monohormonal identity. This report describes the development and application of a new dual ISH protocol that detects biotinylated and digoxygenin-labeled oligoprobes complementary to LH and FSH-beta subunit mRNAs or hnRNAs (produced by GeneDetect.com with GreenStar technology and 10 reporter molecules/probe). After simultaneous hybridization with the two probes, their reporter molecules were detected by the simultaneous addition of 1:200 monoclonal anti-biotin and sheep anti-digoxygenin, Fab fragments (alkaline phosphatase labeled). Each reaction was then amplified by simultaneously adding horse anti-mouse IgG linked to peroxidase micropolymers (ImmPRESS, Vector Labs) and 1:200 donkey anti-sheep IgG linked to alkaline phosphatase. Peoxidase was then detected (diaminobenzidine) followed by alkaline phosphatase (BCIP/NBT). Substitution of sense for antisense probes abolished the reaction. The detection systems resolved patches of label for the two mRNAs in different or contiguous sites in bihormonal gonadotropes. The technology also successfully detected differential expression of hnRNA in the nucleus during the cycle. Most importantly, dual ISH detected significantly more monohormonal gonadotropes in many of the populations tested (male and cycling female rats). In male rats, dual ISH detected significantly more (27±1%) total gonadotropes than by dual ICC or ICC-ISH (15-16%); half were monohormonal LH or FSH. Furthermore, 49±1% of LH cells and 46±1% of FSH cells were monohormonal. The previous dual labeling protocols, which relied on ICC or ISH-ICC, may not detect all gonadotropes and, depending on the physiological state, may underestimate the proportion of gonadotropes that are monohormonal. Supported by NIH R21 HD47467

KEY WORDS: gonadotropin beta subunits, in situ hybridization, monohormonal gonadotropes, mRNA and hnRNA