Six novel gonadotropin-releasing hormones are encoded as triplets on each of two genes in the protochordate, Ciona intestinalis.

GnRH is the key regulator of the reproductive axis in vertebrates, but little is known about GnRH before the origin of vertebrates. We have identified two genes encoding GnRH in a protochordate, Ciona intestinalis, thought to be related to the ancestral animal that gave rise to vertebrates. Each gene, Ci-gnrh1 and Ci-gnrh2, encodes in tandem three GnRH peptides, each of which is unique compared with known forms. Ci-gnrh1 encodes three peptides and contains no introns, whereas Ci-gnrh2 encodes three more peptides but has two introns. This is the first report in which more than one GnRH peptide is encoded on a single gene. The Ciona genes reveal consensus promoter elements that are conserved compared with human GNRH1. Both tunicate genes are expressed as mRNA early and throughout development, measured at the stages of four-cell, gastrulation, tail release, and tail resorption. In a closely related tunicate species, Ciona savignyi, we used in silico analysis to identify two similar genes encoding six peptides, only one of which is unique compared with C. intestinalis. Immunohistochemistry showed that at least one GnRH peptide was in the nerve net that surrounds the dorsal strand. Synthetic forms of the seven novel tunicate peptides induced release of gametes in adult tunicates. In contrast, the peptides did not activate the human GnRH-I receptor or cause release of LH in a rat pituitary cell assay. These data provide insight into the structural evolution of the GnRH peptides and their genes and show a functional role for GnRH in tunicate spawning.

Transcription and translation of the salmon gonadotropin-releasing hormone genes in brain and gonads of sexually maturing rainbow trout (Oncorhynchus mykiss).

Rainbow trout sexually mature at the end of Year 3. The form of GnRH that controls gonadotropin release in trout is salmon GnRH (sGnRH). In the tetraploid rainbow trout, two genes encode an identical sGnRH peptide. The sGnRH gene-1 produces one mRNA, whereas sGnRH gene-2 can produce more than one. This study asks whether the transcripts and their protein products are expressed in the brain and gonads and whether the pattern correlates with sexual maturity over the final year leading to first spawning. Brain sGnRH mRNA and protein were continuously present throughout the third year. We show for the first time that the long sGnRH-2 mRNA transcript is expressed in neural tissue and not exclusively in gonadal tissue. Expression of the long sGnRH-2 mRNA in the brain coincides with high levels of sGnRH peptide in the brain during a time of increased gonadal growth. Thus, the long sGnRH-2 mRNA in the brain may act to regulate sGnRH production in a stage-specific rather than a tissue-specific manner. In gonads, local sGnRH is thought to play an autocrine/paracrine role in regulating gonadal maturation and spawning. In the maturing gonads, sGnRH gene-1 and -2 are expressed intermittently. Strikingly, sGnRH peptide was not detected in the gonads at any time during Year 3. These results suggest that either the sGnRH transcripts in the gonads are not translated into protein or, if translated, the protein is rapidly released, resulting in gonadal content below 1 fM per fish.

Three forms of gonadotropin-releasing hormone, including a novel form, in a basal salmonid, Coregonus clupeaformis.

Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH(2). HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the alpha-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.

Guinea pig gonadotropin-releasing hormone: expression pattern, characterization and biological activity in rodents.

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.