Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. In addition to hypothalamic GnRH (GnRH-I), a second GnRH form (pGln-His-Trp-Ser-His-GlyTrp-Tyr-Pro-Gly; GnRH-II) with unknown function has been localized to the midbrain of many vertebrates. We show here that a gene encoding GnRH-II is expressed in humans and is located on chromosome 20p13, distinct from the GnRH-I gene that is on 8p21-p11.2. The GnRH-II genomic and mRNA structures parallel those of GnRH-I. However, in contrast to GnRH-I, GnRH-II is expressed at significantly higher levels outside the brain (up to 30؋), particularly in the kidney, bone marrow, and prostate. The widespread expression of GnRH-II suggests it may have multiple functions. Molecular phylogenetic analysis shows that this second gene is likely the result of a duplication before the appearance of vertebrates, and predicts the existence of a third GnRH form in humans and other vertebrates.Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating reproduction. Release of GnRH from the hypothalamus controls the production of pituitary gonadotropins responsible for gonadal development and growth in all vertebrates. This function for GnRH has been highly conserved during 500 million years of vertebrate evolution despite the fact that its amino acid sequence varies by 50% (1).In addition to the hypothalamic GnRH of variable sequence, many vertebrate species have been shown to express a second, invariant GnRH form (pGln-His-Trp-Ser-His-Gly-Trp-TyrPro-Gly; GnRH-II) (2). By using antibody staining, this form of GnRH has been found in the midbrain in all species where its location has been described (reviewed in ref. 1). Furthermore, nucleic acid probes have been used to identify GnRH-II expression in the midbrain of several fish species and one mammal (1).Recently, a cDNA encoding this second form of GnRH was found in a placental mammal, the tree shrew Tupaia glis (1), thus leading us to search for it in humans. Here we describe the cloning of a cDNA encoding a second form of GnRH in humans and the subsequent isolation and sequencing of the complete human GnRH-II gene, the first description of a nonhypothalamic GnRH gene form in any species. In addition, the structure and chromosomal location of the new GnRH-II gene is compared with that of the previously described form in humans. Finally, we have constructed a molecular phylogeny of GnRH evolution, incorporating new sequence data for GnRH-II cDNAs from three placental mammals: human (this paper), tree shrew (1), and musk shrew (Suncus murinus; R.B.W., T.L.K., S. White, and R.D.F., unpublished data). MATERIALS AND METHODSLibrary Screen. A 270-nt partial cDNA for the putative human GnRH-II was cloned from human thalamus poly(A) RNA (CLONTECH) by using reverse transcription-PCR (RT-PCR) and 3Ј-RACE (rapid amplification of cDNA ends) as described (1). Oligomers flanking putative...

Gonadotropin-releasing hormone (GnRH) is known and named for its essential role in vertebrate reproduction. Release of this decapeptide from neurons in the hypothalamus controls pituitary gonadotropin levels which, in turn, regulate gonadal state. The importance of GnRH is underscored by its widespread expression and conservation across vertebrate taxa: five amino acids are invariant in all nine known forms, whereas two others show only conservative changes. In most eutherian mammals, only one form, expressed in the hypothalamus, is thought to exist, although in a recent report, antibody staining in developing primates suggests an additional form. In contrast, multiple GnRH forms and expression loci have been reported in many non-mammalian vertebrates. However, evidence based on immunological discrimination does not always agree with analysis of gene expression, since GnRH forms encoded by different genes may not be reliably distinguished by antibodies. Here we report the expression of three distinct GnRH genes in a teleost fish brain, including the sequence encoding a novel GnRH preprohormone. Using in situ hybridization, we show that this form is found only in neurons that project to the pituitary and exhibit changes in soma size depending on social and reproductive state. The other two GnRH genes are expressed in other, distinct cell populations. All three genes share the motif of encoding a polypeptide consisting of GnRH and a GnRH-associated peptide. Whereas the GnRH moiety is highly conserved, the GnRH-associated peptides are not, reflecting differential selective pressure on different parts of the gene. GnRH forms expressed in nonhypothalamic regions may serve to coordinate reproductive activities of the animal.

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In vertebrates, the gonadotropin-releasing hormone (GnRH) decapeptide is secreted from hypothalamic nerve terminals to regulate reproduction via control of synthesis and release of pituitary gonadotropins. Only one GnRH peptide has been found in mammals, with one exception, although numerous other vertebrate species express more than one of the eight known decapeptide forms as shown by immunocytochemical labeling of distinct cell groups in the brain.