Harold Papkoff

Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species

The present study describes the development and characterization of a monoclonal antibody (518B7) generated against bovine LH (bLH). Although 518B7 was extremely specific for LH, very low species specificity was observed. A RIA using this antibody and radioiodinated equine LH (eLH) showed good sensitivity for all mammalian LH preparations tested, with the exception of human LH (15%, relative to the eLH reference standard). Activities of most mammalian LHs ranged between approximately 50-200%. Much less activity was detected with reptilian LH (less than 1.5%). Amphibian and avian LH fractions were essentially inactive. The reactivities of LH alpha and beta subunits from a variety of mammals clearly showed that the antibody reacts with the beta subunit. Sensitive RIAs were also developed utilizing 125I-bovine and 125I-rat LH. Interestingly, all hormone preparations which showed sufficient reactivity for statistical analysis within the dose ranges used in the present study (0.01-1000 ng/tube) produced a displacement curve parallel to the reference standard. We have also validated the use of 518B7 in detecting LH in serum. Parallel dilution curves relative to purified LH reference standards were observed with equine and bovine serum samples and equine pituitary extract. High (average 94%) recoveries were also seen with bovine serum with known amounts of exogenously added bLH. Similar patterns of LH secretion were detected with a RIA based upon 125I-bLH and 518B7 and a previously described polyclonal antibody-based RIA in bovine serum samples during estrus. Thus, a monoclonal antibody for LH has been produced which can be used to develop sensitive and specific RIAs in many different mammalian species.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Preparation of ovine interstitial cell-stimulating hormone in high yield

Abstract A new procedure is described for the purification of interstitial cell-stimulating hormone (ICSH) from sheep pituitaries. Extraction of ground sheep pituitaries at pH 4.0 followed by ammonium sulfate precipitation, chromatography on sulfoethyl-Sephadex C-50, and gel-filtration on Sephadex G-200 give yields of ICSH of about 300 mg per kilogram of glands. Contamination with other pituitary activities is very low. The preparation obtained by this procedure has been characterized with respect to biological activity, molecular weight, amino acid composition, and carbohydrate content. The results show it to be identical with previously described preparations of purified ICSH.

Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris gallopavo)

Abstract Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were purified from the pituitary glands of the turkey (Meleagris gallopavo). These hormones were characterized biochemically and biologically and compared with chicken gonadotropins prepared in several independent laboratories. Amino acid and carbohydrate analyses demonstrated homology between turkey and other species of gonadotropin. The turkey LH purified here had significantly higher carbohydrate content than a previous preparation of turkey LH. Immunological studies further confirmed that the turkey FSH and LH were distinct from one another and that each was homologous to the respective gonadotropin from other vertebrates; the immunopotencies of the turkey hormones were similar to those from chickens. A variety of bioassays and radioreceptor assays (RRAs) confirmed the biological activity of the two turkey gonadotropins and revealed that the turkey LH was distinct from that of the chicken. As expected, the two types of turkey hormones were approximately equipotent in total gonadotropin bioassays (frog spermiation and 32P uptake by chick testes), and only the turkey LH was active in the rat Leydig cell assay and in RRA for LH in mammals. However, the turkey LH was also highly potent in several assays considered to be relatively FSH specific, including the Anolis lizard assay and several RRA systems using mammalian, turtle and avian gonadal receptors with 125I-labeled human FSH as tracer. Turkey and chicken FSH are similar in the RRAs, but the turkey LH was consistently more potent than either avian FSH in competing for FSH-binding sites. Chicken LH had relatively low activity by comparison. It is suggested that the evolution of the structure of active sites in turkey LH has involved convergence on those of the FSH molecule.

Granulosa cells as hormone targets : the role of biologically active follicle-stimulating hormone in reproduction

Publisher Summary This chapter describes that with recent advances in cellular and molecular biology, significant progress has been made to elucidate the effect of diverse hormones at the ovarian granulosa cells. Follicle-stimulating hormone (FSH) is the primary regulator of granulosa cell differentiation. The mechanisms by which FSH induces the expression of multiple genes associated with feedback actions, ovulation, steroidogenesis, and other differentiated functions are studied. The chapter highlights the reception, action, and local synthesis of various growth factors and further explains the paracrine and autocrine roles of these growth factors in the modulation of FSH actions at the granulosa cells. The granulosa cell aromatase bioassay (GAB) allows measurement of serum levels of bioactive FSH in diverse species. The characterization of bioactive FSH molecules secreted by eukaryotic cell lines transfected with human FSH genes provides clinically useful reagents for stimulating follicle maturation and spermatogenesis.

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.

Purification of turkey prolactin and the development of a homologous radioimmunoassay for its measurement.

Prolactin has been purified from pituitary glands of the turkey (Meleagris gallopavo) and a radioimmunoassay has been developed for its measurement. The prolactin is a molecule of about 26,000 MW. It is high in glutamic and aspartic acids and in leucine, while it contains few methionine and half-cystine residues. Turkey prolactin elutes from Sephadex G-100 with a ratio of 2.0. It displays multiple bands in disc electrophoresis, when run at pH 8.3. The most prominent prolactin band is at a Rf value of 0.83–0.85. Prolactin is active in stimulating pigeon crop-sac development, but does not give a parallel dose-response to ovine prolactin. An antiserum was developed against turkey prolactin and was used to develop a homologous RIA using 125I-labeled turkey prolactin as the radioligand. The antiserum binds about 20% of the radioligand at a dilution of 1:7500. The least detectable dose of prolactin was 0.42 ± 0.13 ng when tracer and sample were simultaneously mixed with antiserum. The 50% bound point averaged 3.8 ± 0.32 ng. Considerably greater sensitivity was achieved by incubating the sample with the antiserum for 1 or 2 days prior to addition of tracer. The within-assay coefficient of variation was 7.9% for samples in the 45–65% bound range. Between-assay coefficient of variation was 8.9%. Binding is inhibited by sera from turkeys in some, but not all, stages of growth and reproductive development. Serial dilutions of sera and pituitary extracts yield dose-response curves which do not differ in slope from the purified turkey prolactin standard. Potency estimates of pituitary preparations by cropsac assay and by the prolactin RIA are in excellent agreement. Rat pituitary extract, rat prolactin, ovine prolactin, turkey FSH, LH, and GH all show no inhibition of binding even at very high doses. The antiserum neutralizes crop sac-stimulating activity of turkey prolactin.

Purification and properties of ovine follicle-stimulating hormone

Abstract A procedure is described for the purification of follicle-stimulating hormone (FSH) from sheep pituitaries. The method utilized extraction of the glands at pH 4.0, metaphosphate, ammonium sulfate, and alcohol precipitation, and chromatography on CM-cellulose, sulfoethyl-Sephadex C-50, and Sephadex G-100. A yield of 5–6 mg per kilo of glands was obtained which had an activity of 45 times NIH-FSH-S1. The preparation has been analyzed by electrophoretic techniques and shown to be highly purified. The material has also been studied in the ultracentrifuge. Amino acid composition and carbohydrate content have been determined.

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.

Human pituitary interstitial cell stimulating hormone: Primary structure of the α subunit

Abstract The primary structure of the α subunit of human ICSH has been deduced from amino acid sequences of tryptic peptides and the compositions of cyanogen bromide fragments as well as the known structure of ovine ICSH-α. The proposed structure is shown in Figure 1. It consists of 89 amino acids with the single isoleucine residue in position 22, and the three methionine residues in positions 26, 44 and 68.