Vertebrate Gonadotropin-releasing Hormone Research Articles

Chimeric Analogues of Vertebrate Gonadotropin-Releasing Hormones Comprising Substitutions of the Variant Amino Acids in Positions 5, 7, and 8: Characterization of requirements for receptor binding and gonadotropin release in mammalian and avian pituitary gonadotropes

Abstract Variant forms of mammalian gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) are present in chicken ([Gln8] GnRH and [His5, Trp7, Tyr8]GnRH), salmon ([Trp7, Leu8]GnRH), and lamprey ([Tyr3, Leu5, Glu6, Trp7, Lys8] GnRH). To delineate the functional importance of the variant amino acids in positions 5, 7, and 8, the natural peptides and chimeric analogues were tested for gonadotropin-releasing activity and receptor-binding activity in rat, sheep, and chicken pituitaries. The results demonstrate that (i) the mammalian receptor has a high fidelity for Arg8 while the chicken receptor is less discriminatory and accepts basic or neutral amino acids in this position. Arg8 may contribute to conformational stabilization, and conformational constraint with D-Trp6 restored activity to analogues lacking Arg8 in the mammalian systems. D-Trp6 incorporation did not generally enhance activity in the chicken pituitary. (ii) His5 accompanying Arg8 in analogues markedly diminished activity in the chicken while gonadotropin-releasing activity was retained in the sheep pituitary. Receptor-binding activity was increased in the sheep indicating an uncoupling of receptor occupancy and activation. (iii) Substitution in position 7 is tolerated by the mammalian and chicken receptor. With Trp7-substituted analogues receptor-binding activity was relatively lower than gonadotropin-releasing activity in the sheep pituitary, suggesting an enhanced receptor activation by these analogues or the existence of different GnRH receptors.

Identity of gonadotropin-releasing hormone in passerine birds: Comparison of GnRH in song sparrow ( Melospiza melodia) and starling ( Sturnus vulgaris) with five vertebrate GnRHs

Gonadotropin-releasing hormone (GnRH) was detected in the brains of passerine birds, a recently evolved and diverse avian group. The molecular forms of GnRH in two species of birds under breeding conditions were deduced using methods of HPLC and immunology. The brain extracts of song sparrows ( Melospiza melodia) contained a form of GnRH identified as chicken I GnRH-like peptide by its HPLC elution pattern and cross-reactivity with four antisera. In contrast, starling ( Sturnus vulgaris) brain extracts showed molecular heterogeneity of GnRH forms; equal amounts of chicken I and chicken II GnRH-like peptides were present. Neither bird contained GnRH that could be identified as mammalian, salmon, or lamprey GnRH. Chicken II GnRH-like peptide may not have evolved after the separation of the song sparrow and starling as both peptides are found in chicken, a more primitive bird. The possibility remains that different stages of the life cycle are associated with the expression of these GnRH-like peptides or their ratio. Only determination of the primary structure will establish whether our chromatographic and immunological evidence is correct that chicken I and II GnRH are present in passerine birds and have been conserved in representatives throughout the reptiles and birds. Starlings can be added now to the growing list of submammalian species that express multiple forms of GnRH in their brains.

Activity of position-8-substituted analogs of mammalian gonadotropin-releasing hormone (mGnRH) and chicken and lamprey gonadotropin-releasing hormones in goldfish

Several vertebrate gonadotropin-releasing hormones (GnRH) and analogs were tested for activity in vivo in goldfish. Each peptide was administered intraperitoneally of goldfish, pretreated with pimozide or vehicle for pimozide, and changes in serum levels of gonadotropin were determined. Pimozide potentiates the activity of GnRH in vivo in goldfish by blocking the endogenous gonadotropin release-inhibitory activity of dopamine; relative potencies of GnRH peptides become evident in vivo in goldfish pretreated with pimozide (R. Peter et al. (1985), Gen. Comp. Endocrinol. 58, 231–242). Mammalian GnRH (mGnRH) was used as reference standard. [Tyr 3, Leu 5, Glu 6, Trp 7, Lys 8]-GnRH (lamprey GnRH), [Gln 8]-GnRH (chicken GnRH-I), and [His 5, Trp 7, Tyr 8]-GnRH (chicken GnRH-II) caused increases in serum gonadotropin level similar in magnitude to mGnRH. [His 5, d-Arg 6, Trp 7, Tyr 8]-GnRH is superactive in the goldfish. [Asn 8]-, [Met 8]-, [Phe 8]-, and [Ser 8]-GnRH had activity similar to mGnRH in goldfish; [His 8]-, [Ile 8]-, and [Leu 8]-GnRH had a lower level of activity; [Glu 8]-GnRH had no apparent activity. The results indicate that there is no particular requirement for a hydrophobic or hydrophilic amino acid, or for a positively charged amino acid in position 8 of mGnRH for activity in vivo in the goldfish; a negatively charged amino acid in position 8 is detrimental for activity.

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake ( Merluccius capensis) and tilapia ( Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp 7, Leu 8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp 7, Leu 8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp 7, Leu 8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln 8-GnRH) and Trp 7, Leu 8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln 8-GnRH and Trp 7, Leu 8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp 7,Leu 8-GnRH, while tilapia brain contains both Trp 7,Leu 8-GnRH and Gln 8-GnRH. Additional GnRH molecular forms were detected in both species. The presence of multiple GnRH-related molecules in teleost fish brain suggests that the GnRH molecule has experienced gene duplication during evolution and the different structures co-opted for as yet undetermined physiological functions.