Susan Walker Farmer

Evolution of gonadotropin structure and function.

Publisher Summary It has long been recognized that the control of gonadal function by pituitary hormones (gonadotropins) is a general feature of vertebrate reproductive physiology. Numerous studies on the hormones of eutherian mammals have established the existence of two chemically distinct types of gonadotropin molecules in the pituitary-luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Recent biochemical studies on these molecules have revealed that each is a glycoprotein consisting of two chemically non-identical subunits, designated α and β. The physiological actions of the two gonadotropins in mammals are still subject to intensive investigation; however, it is agreed that FSH and LH have somewhat different roles in the regulation of gonadal function. This chapter describes a flow diagram of the protocol for the fractionation of pituitary hormones, with particular reference to gonadotropins and growth hormone (GH), from various nonmammalian species. The strong resemblance in chromatographic behavior among the fractions identified as FSH and LH from most nonmammalian species and their mammalian counterparts provided preliminary evidence of chemical similarity among the different species of FSH and of LH. Biochemical analyses on the six highly purified species of LH and FSH (chicken, turkey, alligator, snapping turtle, sea turtle, and bullfrog) yielded additional evidence for the homologies between mammalian and nonmammalian hormones.

Effect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and seawater adaptation of sockeye salmon (Oncorhynchus nerka)

Purified Tilapia growth hormone (GH), which has already been shown to have slight but significant activity in the rat tibia assay, was found to promote growth in two intact teleost species, Tilapia mossambica and Oncorhynchus nerka (sockeye salmon). Tilapia GH and bovine GH were approximately equipotent in stimulating an increase in both length and weight of these fish. In salmon, ovine prolactin (PRL) stimulated growth with a much lower potency than Tilapia GH, while Tilapia PRL was inactive. Furthermore, bovine GH, Tilapia GH, and ovine PRL facilitated adaptation of the salmon to sea water, while Tilapia PRL had no effect. These data, along with published results of teleost bioassays specific for Tilapia PRL, demonstrate that teleosts can readily distinguish between Tilapia GH and Tilapia PRL. This separation contrasts with previously reported similarities between the two hormones with respect to amino acid composition, immunochemistry, and activity in the rat tibia assay.

Purification and properties of teleost growth hormone

Highly purified growth hormone (GH) has been prepared from the pituitary glands of a euryhaline teleost, Tilapia mosambica. Tilapia GH was obtained in a yield of 1400 mg/kg wet weight tissue. It was found to have a molecular weight (gel filtration) of 22,200 daltons, a sedimentation coefficient (s20,w) of 2.19, and an α-helix content (circular dichroism) of 50%. Isoleucine was found to be the major amino-terminal residue; leucine was found to be COOH terminal. The amino acid composition, disc gel electrophoresis pattern, and circular dichroism spectra were similar to those of mammalian GHs. Tilapia GH was found to have a low but significant activity in the rat tibia assay and showed immunological relatedness to mammalian GH in a rat GH radioimmunoassay. Antiserum was prepared against the Tilapia GH and characterized in agar diffusion experiments and radioimmunoassay. Results from these investigations demonstrated a significant degree of cross-reaction between Tilapia GH and pituitary extract from another teleost (perch), but purified tetrapod GHs were essentially nonreactive. The data indicate a significant resemblance between Tilapia GH and mammalian GHs and suggest that the GH structure has been strongly conserved during evolution.

Isolation and properties of teleost prolactin.

Highly purified prolactin (PRL) has been isolated from the pituitary tissue of a euryhaline teleost, Tilapia mossambica. It has a molecular weight of 19,400 daltons, with a single NH2-terminal residue, valine, and a single COOH-terminal residue, half-cystine. Amino acid composition data revealed the presence of one tryptophan and four half-cystine residues, which is characteristic of all known vertebrate growth hormones (GH) but not of mammalian PRLs. The circular dichroism spectrum of Tilapia PRL was similar to that of porcine PRL and showed an α-helix content of 65%. Tilapia PRL was considerably more potent than ovine PRL in two teleost PRL bioassays: the sodium-retaining assay in Tilapia and the reduction of water permeability in the urinary bladder of Gillichthys mirabilis, but it did not stimulate mammary tissue or the pigeon crop sac. Tilapia PRL had equivocal but suggestive activity in the rat tibia assay, and showed cross reaction in two GH (rat and snapping turtle) radioimmunoassays. A specific Tilapia PRL antiserum was prepared in a rabbit which gave a precipitin reaction against Tilapia PRL in agar diffusion but showed no cross reaction with purified mammalian PRLs or pituitary extracts from other teleosts. The data show that Tilapia PRL has features common to both mammalian PRLs and GHs as well as to Tilapia GH, lending support to the hypothesis that PRL and GH originated from a common ancestral molecule.

Immunocytochemical identification of the prolactin- and growth hormone-secreting cells in the teleost pituitary with antisera to tilapia prolactin and growth hormone

Antisera raised to highly purified tilapia (Sarotherodon mossambicus) prolactin (PRL) and growth hormone (GH) were used to locate PRL and GH cells in the adenohypophysis of seven species of teleosts by immunoenzymological methods using horseradish peroxidase. The only pituitary cells in the tilapia that immunologically reacted with anti-tilapia PRL were the PRL cells located in the rostral pars distalis (RPD). In the pituitary glands of two species of salmonids and two species of marine fishes, the tilapia PRL antibodies bound to both PRL and GH cells; no prolactin-immunoreactive cells were identified in the goldfish and eel pituitaries. Antiserum to tilapia GH reacted specifically with the GH cells located in the proximal pars distalis of all teleosts examined in this study, but not with any presumed PRL cells in the RPD. These results indicate that tilapia PRL antisera are specific for tilapia PRL cells but not necessarily for these cells in other teleost species. Tilapia GH antisera showed no species specificity among the teleosts tested.

Isolation and characterization of luteinizing hormone from amphibian (Rana Catesbeiana) pituitaries

Abstract We report here the first isolation of an anterior pituitary hormone from an amphibian species, the bullfrog ( Rana catesbeiana ). Highly purified luteinizing hormone was isolated from alkaline extracts of bullfrog pituitaries by salt fractionation, chromatography on ion-exchangers and gel filtration. Characterization studies show the hormone to contain 9% carbohydrate and to possess an amino acid composition similar to ovine luteinizing hormone. Sedimentation-velocity experiments in the ultracentrifuge indicate that the bullfrog gonadotropin dissociates in acidic solution and is composed of subunits. Bullfrog luteinizing hormone is highly active in an in vitro toad ovulation assay and also ellicits testosterone production in vitro from isolated rat testis Leydig cells.

Immunochemical studies on the pituitary gonadotropins (FSH and LH) from the bullfrog, Rana catesbeiana

Separate antisera against bullfrog (Rana catesbeiana) FSH (FSH ) and LH (LH ) were generated in rabbits with partially purified gonadotropins. Ouchterlony agar diffusion techniques indicated that each antiserum was relatively specific for the homologous antigen, and there was no detectable precipitin reaction with pituitary gonadotropins from a variety of other tetrapod species, including several mammals, birds, and reptiles. Radioligand studies were performed with iodinated preparations of highly purified Rana FSH and LH. Binding studies with these labeled hormones indicated some cross-reactivity with both antisera; i.e., each antiserum bound the heterologous label to some extent. However, this cross-reaction was abolished by adsorbing the antisera with the opposite antigen; this adsorption did not affect the binding of the homologous label. Thus, the apparent cross-reactivity is attributed to separate antibodies in each serum. Electrophoretic analyses on polyacrylamide disc gels at basic pH further demonstrated the distinctiveness of each gonadotropin and its antiserum. Radioimmunoassay with adsorbed and unadsorbed sera confirmed this specificity of each serum for its homologous antigen and showed that this technique could be used as a sensitive method for quantifying gonadotropins. The degree of cross-reaction between gonadotropins was estimated at about 1% or less. Comparison with crude pituitary extract from bullfrog showed that both gonadotropins were considerably purified. Little or no cross-reaction was seen with bullfrog growth hormone or with FSH, LH, or TSH from other nonamphibian species with either antiserum. Biological studies demonstrated that the LH neutralized the activity of Rana LH in a specific bioassay (in vitro ovulation in Xenopus), whereas the FSH had no effect on the LH. In tests for FSH activity in Anolis, the activity of Rana FSH was blocked by both antisera, but the FSH did not affect the biological activity of Rana LH. Thus, the activity of both bullfrog hormones in Anolis is judged to be intrinsic to the molecules. These results demonstrate that the antisera cross-react with the biologically active gonadotropin molecules.

Pituitary gonadotropins in snakes

Conventional fractionation procedures used for separation and purification of pituitary FSH and LH in diverse tetrapods were employed to study pituitary gonadotropin (Gn) from five genera of snakes representing three families: Elaphe and Ptyas—Colubridae; Naja and Bungarus—Elapidae; Crotalus—Viperidae. The hormones purified from each of these snakes were shown to be relatively potent in a variety of Gn bioassays and radioreceptor assays (RRAs) in snakes but there was no clear evidence for two separate types of Gn molecule. These snake materials were also active in RRAs and bioassays (testis growth, androgen production, and ovulation) in lizards, but poor dose-response characteristics and relatively low potencies were observed in most cases. An even higher degree of species specificity was evident when snake hormones were tested in nonsquamate species, including other reptiles. Most snake Gn fractions were essentially inactive in all RRAs and bioassays employing amphibians, birds, mammals, and turtles; these assays included several that typically show broad species cross-reactivity (e.g., anuran ovulation and spermiation, 32P uptake by chick testis, and in vitro androgen production in birds and turtles). Radiolabeled Gn from a snake (Naja) bound specifically only to gonadal receptors from snakes and lizards. Biochemical analyses of the snake Gn confirmed their glycoprotein nature but failed to show clear structural homology to either FSH or LH. Chromatographically, the purified Gn from Naja naja tended to behave predominantly like an LH and its electrophoretic mobility on polyacrylamide gels also suggested an LH-like molecule, but several problems complicated interpretation of these results. Amino acid composition of this snake Gn revealed similarities to both FSH and LH, but it was not consistently like either. Radioimmunological studies (RIAs) with several heterologous gonadotropin antisera also failed to show a consistent relatedness between snake hormones and either FSH or LH; in fact, the snake Gns are exceptional among tetrapods in showing a lack of cross-reactivity in several FSH and LH-RIA systems. Antisera were raised against Gn from two species of snake. Antiserum to Naja Gn blocked the biological and binding activities of other species of snake Gn and it also selectively neutralized the activity of FSH (but not LH) from a turtle, alligator, and bird. However, in homologous RIA, the Naja Gn showed a slight cross-reaction only with another elapid species. In a heterologous RIA (using antiserum to Ptyas Gn and 125I-labeled Naja Gn), relatively high cross-reactivity was seen with Gn from several elapids and colubrids but not viperids or any non-snake species. Thus, the snake hormones show partial relatedness to one another but are generally distinct from FSH or LH; the only evidence of cross-reaction with heterologous (non-serpentine) gonadotropin suggests that they share common immunochemical determinants with reptilian FSH. Overall, the snake gonadotropins appear to be unique among tetrapod gonadotropins in terms of their biological cross-reactivity, biochemical composition, and immunochemical properties. Data suggest that snakes may only have a single gonadotropin that does not show a clear homology to either FSH or LH.

A teleost (Tilapiamossambica) gonadotropin that resembles luteinizing hormone

Abstract A purified gonadotropin preparation was obtained from pituitaries of a teleost fish ( Tilapia mossambica ). This gonadotropin was found to resemble LH in that it behaved identically to mammalian and non-mammalian LHs in several chromatographic systems, and stimulated testerone production in isolated rat Leydig cells. In this assay, specific for LH, the Tilapia gonadotropin was less potent than mammalian LHs but significantly more active than avian, reptilian or amphibian LHs. The Tilapia gonadotropin was found to be a glycoprotein; preliminary amino acid composition data show resemblances to both mammalian and non-mammalian LHs.

Comparison of the steroidogenic and melanotropic activities of corticotropin, α-melanotropin and analogs with their lipolytic activities in rat and rabbit adipocytes

The ability of alpha-melanotropin and a series of synthetic peptides related to adrenocorticotropin (ACTH) to stimulate steroidogenesis in isolated rat adrenal cells, melanin dispersion in frog melanophores and lipolysis in rat and rabbit fat cells have been studied. It was found that the steroidogenic activity closely paralleled the lipolytic activity of these peptides in rat fat cells, whereas the melanocyte stimulating activity paralleled the lipolytic activity in rabbit fat cells. These results indicate that the structural requirements for stimulating steroidogenesis in isolated rat adrenal cells and lipolysis in isolated rat fat cells are quite similar. The structural features required for eliciting lipolysis in rabbit fat cells appear to be very similar to those necessary for stimulating frog melanophores. The possibility that regulation of lipid metabolism in the rabbit may be a new function acquired by melanotropin is discussed.