Ted Hayashida

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.

Immunological studies with rat pituitary growth hormone (RGH). II. Comparative immunochemical investigation of GH from representatives of various vertebrate classes with monkey antiserum to RGH.

Abstract The immunochemical relatedness of GH in pituitary extracts from various vertebrate species has been studied by means of precipitin reactions in agar and by radio-immunoassay. Extracts were prepared from the pituitaries of animals representing most of the vertebrate classes. The results which were based on the use of monkey antiserum to RGH, indicated that GH from mammalian species must share a relatively large number of common antigenic determinants and that the greater the phylogenetic distance from the mammal, the fewer such determinants were shared by the GH molecule from any particular species. The agar diffusion studies based on reactions of identity and reactions of partial identity and the quantitative results of immunochemical relatedness based on radioimmunoassays, revealed a separation of GHs from the various vertebrate species into a series of immunochemical categories which closely corresponded to their respective phylogenetic classes based largely upon morphological considerations. Bioassays were also performed to determine the capacity of GH in the pituitary extracts from representativees of the various vertebrate classes to stimulate growth by means of the tibia test in hypophysectomized rats. It was concluded on the basis of the current series of investigations that theresults of the immunochemical approach have shown a general correlation with evolutionary development and was more reliable in this regard than the biological approach.

Fish growth hormone: a biological, immunochemical, and ultrastructural study of sturgeon and paddlefish pituitaries.

Abstract In contrast to pituitary growth hormone (GH) of modern bony fish (teleosts), the hormone in pituitary extracts of two primitive bony fish (chondrosteans)—the Pacific white sturgeon and the paddlefish—stimulated growth of the hypophysectomized rat as demonstrated by the tibia assay and immunochemically cross-reacted with monkey antiserum to rat GH, as shown by radioimmunoassay. In addition, electron microscopic studies revealed that these primitive fish, unlike the teleosts, possess a median eminence, with morphological features similar to those of tetrapods.

Prolactin Localization in the Primate Pituitary by Immunofluorescence

Cells which contain prolactin were clearly distinguished from those which contain growth hormone in adult monkey pituitary glands by means of histologic and fluorescent antibody techniques. The results indicate that in primates, as well as in other mammals, prolactin is immunochemically distinguishable from growth homone.

Amphibian Pituitary Growth Hormone and Prolactin: Immunochemical Relatedness to Rat Growth Hormone

Growth hormone and prolactin were electrophoretically isolated from amphibian pituitaries and then were tested in a radioimmunoassay with labeled rat growth hormone and antiserum to the same hormone. This isolation and purification of the hormones increased the steepness of the slopes of competitive inhibition in this system when compared to those of crude extracts. Both hormones from most species tested showed high immunochemical cross-reactivity, indicating that amphibian growth hormone and prolactin are structurally related to rat growth hormone.

Histologic identification and immunochemical studies of prolactin and growth hormone in the primate pituitary gland

Abstract The two acidophilic cell types were tinctorially differentiated in rhesus monkey pituitaries as well as in pituitaries of other species of monkeys and apes and were identified by immunofluorescence as the prolactin and the growth hormone (GH) cells. The number of prolactin cells was significantly greater in adult than in juvenile rhesus monkeys. During late pregnancy and lactation, these cells appeared to occupy 60–80% of the entire anterior lobe of the rhesus monkey. The concentration of prolactin and CH was measured by radioimmunoassay in pituitary extracts (PE) from pools of juvenile male, juvenile female, adult male and adult female rhesus monkey pituitaries. More prolactin was present in PEs from adult than from juvenile animals, with the highest concentration being present in adult females. A similar relationship was observed with GH. The qualitative immunologic relationship between ovine prolactin and prolactin in the rhesus monkey PEs, and between human GH and GH in the PEs, was established using agar gel diffusion. The two prolactin hormones were partially related immunochemically, while the two GHs were shown to be immunochemically identical. In intact juvenile female rhesus monkeys given estradiol benzoate, the concentrations of pituitary and serum prolactin were elevated over that of the controls, while the concentration of pituitary GH was decreased. An increase in the number of prolactin cells was also observed in the steroid-treated animals. These observations demonstrate further that prolactin and GH reside in separate cells in the primate pituitary and that the concentration of these two hormones varies depending upon age, sex, or stage of the reproductive cycle of the animal.

Biological and immunochemical studies with growth hormone in pituitary extracts of holostean and chondrostean fishes

Abstract The results of the present investigation suggest that growth hormone (GH) in extracts of pituitaries from representatives of the two existing genera of holostean fish, the bowfin (Amia) and the gar (Lepisosteus), and in an extract of pituitaries of a chondrostean fish (Polypterus), shows significant immunochemical relatedness to mammalian (rat) GH and appears to be capable of stimulating growth in the mammal on the basis of the tibia test in the rat. These findings are analogous to those previously reported for GH in pituitary extracts of two other chondrostean fish, the sturgeon and the paddlefish, but are in direct contrast to the negative results obtained with pituitary extracts of modern bony fish (teleostean). The findings to date indicate that a general correlation appears to exist between the immunochemical relatedness of fish GH with respect to mammalian (rat) GH, the ability of that fish GH to stimulate growth in the mammal (rat tibia test) and the general degree of development of the median eminence in the particular species from which the GH originates.

Pituitary growth hormone from the turtle and duck: purification and immunochemical studies.

Summary Turtle and duck pituitaries were fractionated and preparations of purified growth hormone (GH) were obtained. Phenylalanine and leucine are present as N-terminal residues in both species of GH and the amino acid composition of both is similar. Studies by agar-gel diffusion and radioimmunoassay employing antirat GH serum and antiturtle GH serum showed that the purified GHs were identical to the immunoreactive material in pituitary extracts. We are grateful to Miss Eleanor Lasky, Miss Laurie Hidekawa, and Mr. Dan Viele for their skilled technical assistance. We thank Dr. Stanley Ellis for making available the highly purified rat and rabbit GH and rat prolactin preparations; the Endocrinology Study Section of the National Institute of Arthritis and Metabolic Diseases, for supplying us with ovine prolactin. Some of the turtle pituitaries were made available to us by Dr. Paul Licht. We are indebted to Doctors Selna Kaplan and Melvin Grumbach of the Department of Pediatrics for the iodinations of rat GH.

Immunological studies with rat pituitary growth hormone: I. Basic studies with immunodiffusion and antihormone tests

Abstract Four rhesus monkeys, four rabbits, and three guinea pigs were immunized with rat pituitary growth hormone (RGH) in Freunds adjuvant. Antisera from all monkeys produced good precipitin reactions with RGH in the double-diffusion procedure in agar, while antisera from none of the rabbits showed any precipitin reaction, even after continued immunization. All guinea pigs yielded antisera that would precipitate RGH, but the reactions were much less intense than those obtained with any of the monkey antisera. These results demonstrated a significant quantitative difference in precipitin antibody response on the part of three different host species that were immunized with the same antigen, RGH. Monkey antiserum to RGH, tested by double diffusion in agar or by immunoelectrophoresis, gave a single, strong precipitin line with highly purified RGH or with a crude saline extract of the rat pituitary. Monkey antiserum to RGH was capable of demonstrating a reaction of identity between RGH and growth hormone from a wide variety of species, including the sheep, the ox, the pig, the rabbit, the guinea pig, and the humpback whale, suggesting the existence of a common antigenic relationship in the structure of growth hormone from these species. Only the monkey or human growth hormone did not show a cross-reaction with this antiserum, a phenomenon which was also observed with guinea pig antiserum to RGH. In vivo experiments utilizing the tibia assay in hypophysectomized rats showed that the monkey antiserum was capable of neutralizing the growth-promoting activity of purified RGH, a crude saline extract of rat pituitary, or purified growth hormones from other species, when these preparations were injected subcutaneously at a different site from that of antiserum injection. However, human growth hormone could not be neutralized even when three times the amount of antiserum was employed. The findings of this investigation, based on precipitin reactions in agar and neutralization of hormone activity in vivo, have clearly demonstrated the antigenicity of RGH, the detectability of RGH in crude pituitary extracts, the similarity in antigenic structure of growth hormones from a wide variety of animal species, and the apparent difference of this common antigenic structure from that of primate growth hormone.