Isolation and characterization of subunits from two distinct salmon gonadotropins

Two distinct gonadotropins, GTH I and GTH II, isolated from female chum salmon pituitary glands, were separated into subunits by acid treatment and subsequent fractionation on reversed-phase high-performance liquid chromatography. GTH II was completely dissociated in 0.1% trifluoroacetic acid, while GTH I was partially dissociated. The acid-stable form of GTH I exhibited a potency identical to that of GTH I in stimulating estradiol-17 beta production in vitro. Both GTH I and GTH II consist of two dissimilar subunits. One subunit (alpha) is common to both GTHs, has Tyr as its N-terminal residue, and a molecular weight (Mr) of 22K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. The other subunit (beta) has a Mr of 17K and an N-terminal residue of Gly for GTH I, whereas GTH II beta is 18K and has an N-terminal residue of Ser, after reduction.     

Induction of spermiation in anurans by mammalian pituitary gonadotropins and their subunits

Tests with highly purified ovine FSH and LH in three species of anurans show that the spermiation response in these amphibia does not discriminate between the two pituitary gonadotropins: FSH and LH are essentially equipotent. Similar results are obtained with both hypophysectomized and intact frogs. Less purified gonadotropin preparations (NIH) give more variable results: LH is considerably more potent than FSH in Rana pipiens, but FSH is about twice as potent as LH in Hyla regilla and Eleutherodactylus coqui. There is a slight synergism between FSH and LH. However, the use of trypsin to inactivate LH and the use of antiserum to block FSH confirmed that the activity of each purified gonadotropin is an intrinsic property of the molecule. The relative potencies of the highly purified gonadotropin preparations in the anuran spermiation test differed markedly from those observed with the same preparations in mammalian and reptilian assays. In anurans, the relative potency of purified LH was about 20 times greater than in other systems, whereas, the relative potency of the highly purified FSH was only about $\text{1}\text{5}$ that observed in the other tests.The two individual subunits of LH had little activity in the frogs (ca. 2.5% of LH) and there was slight difference in their potencies. In contrast the β subunit of FSH was relatively potent (about 25% of native FSH), but the α subunit was inactive (<3%). In all three species of anuran, a crude extract of anuran (bullfrog, R. catesbeiana) pituitary glands was as potent as the most active purified mammalian hormone.     

Immunological studies with the gonadotropins and their subunits from the green sea turtle Chelonia mydas

Antisera generated in rabbits against highly purified follicle-stimulating hormone (FSH), luteinizing hormone (LH), and the β subunit of LH from the green sea turtle, Chelonia mydas, were used to obtain additional biochemical information on the chemical and biological relatedness between the two turtle gonadotropins. Evidence that the antisera were directed against the biologically active gonadotropins was obtained from neutralization studies. At concentrations of 1 ml of antiserum per 100 μg of hormone, FSH and LH could each be selectively neutralized with homologous antisera. These results provide additional evidence that both gonadotropins have intrinsic biological activities in assays related to testis growth and androgen production in lizards and in vitro androgen production by snake testes. Each antiserum ($\text{a}\text{s}$) showed some binding to the heterologous radioiodinated hormone, but binding was clearly maximal with homologous tracer, and some of this cross-reaction was shown to be due to heterogeneity in antibody populations. Highly specific radioimmunoassays (RIAs) were developed for both FSH and LH with these antisera. When FSH $\text{a}\text{s}$ was used in conjunction with ¹²⁵I-labeled FSH, Chelonia LH and thyroid-stimulating hormone (TSH) showing virtually no cross-reaction (e.g., contamination of LH with FSH was < 1%). Alternatively, RIA using either LHβ $\text{a}\text{s}$ or LH $\text{a}\text{s}$ combined with ¹²⁵I-labeled LHβ as tracer was highly specific for LH and especially the β subunit; there was no cross-reaction with either FSH, TSH, or the α subunit of LH. When LH $\text{a}\text{s}$ was used with ¹²⁵I-labeled α as tracer, the specificity of the RIA was reduced; in this case, LHβ alone showed no cross-reaction but LHα was highly potent. Thus, these systems provide a means for studying the importance of the two separate subunits in immunorelatedness among pituitary glycoprotein hormones and for measurement of circulating gonadotropin levels.     

Studies on the immunochemical relatedness among tetrapod gonadotropins and their subunits with antisera to sea turtle hormones

Immunological relatedness among gonadotropins from the four classes of tetrapods was studied with three antisera ($\text{a}\text{s}$) raised in rabbits against follicle-stimulating hormone (FSH), luteinizing hormone (LH), and LHβ subunit from the green sea turtle, Chelonia mydas. Only pituitaries or FSH from turtles showed appreciable cross-reaction in the homologous RIA for Chelonia FSH, and preparations from the heterologous suborder Pleurodira showed weaker and less complete cross-reactivity than from species of Cryptodira. However, the Chelonia FSH $\text{a}\text{s}$ was capable of neutralizing the biological activity of crocodilian FSH and showed specific binding to ¹²⁵I-labeled FSH from birds and mammals (but not amphibians). Considerable immunorelatedness was evident among the LH preparations of different species and this appeared to result primarily from common antigenic determinants on the α subunits of LH. First, all chelonian, crocodilian, and avian LH preparations showed high activity in RIA based on anti-Chelonia LH serum and ¹²⁵I-labeled Chelonia LHα as tracer; in fact, several of these heterologous species of LH were more potent than the Chelonia LH. LH from eutherian mammals (human, ovine, bovine) showed essentially no cross-reactivity, but preparations of ovine LHα and kangaroo LH showed measurable activity in LHα RIA. Second, the antiserum for LH, which contained antibodies to the LHα subunit, neutralized the biological activity of LH from other species of turtles, crocodilians, and a mammal (sheep). Third, ¹²⁵I-labeled preparations of avian (turkey) and mammalian (ovine and rat) LH showed specific binding to this antiserum. In contrast, parallel tests with an antiserum raised against Chelonia LHβ showed high cross-reactions only with LHs from other species of turtles; i.e., little or no cross-reaction with any nonchelonian species was indicated in RIA, neutralization tests, or binding studies with ¹²⁵I-labeled hormones. Gonadotropins from amphibians and snakes showed no cross-reaction in any tests with any of the three antisera used. This conspicuous lack of immunological cross-reactivity of snake gonadotropins, especially with antibodies to LHα, provides further evidence for a pronounced structural divergence between the hormones of squamates and the other reptilian orders.     

Initiation and control of ovulation in the mouse luteal phases. Effects of gonadotropins and gonadotropin releasing hormone

The aim of the present study was to examine the induction of ovulation during pregnancy, pseudopregnancy, and suckling-delayed pregnancy in mice using exogenous gonadotropins. The present results demonstrate that there are mature follicles in the ovary which can be induced to ovulate with administration of either exogenous human chorionic gonadotropin (hCG) or luteinizing hormone (LH) during pregnancy (Days 1-12) and pseudopregnancy (Days 4-8) in the mouse. hCG was relatively ineffective in initiating ovulation during suckling-delayed pregnancy, and hCG could not induce ovulation on Days 3-6 in any animals, suggesting that follicular growth is not continuous during suckling-delayed pregnancy in the mouse. Ovulation occurred in pregnant and pseudopregnant mice following injection of gonadotropin releasing hormone (GnRH) in a gelatin delay vehicle. Injection of GnRH in saline did not initiate ovulation in pregnant or pseudopregnant mice. A large release of LH was shown to occur following injection of GnRH in gelatin, but no release occurred after the same dose of GnRH in saline. In conclusion, the experiments demonstrate the existence of mature follicles during murine pregnancy and pseudopregnancy, and the lack of inductable follicles during suckling-delayed pregnancy.     

Synthesis and release of gonadotropins and their subunits by long-term organ cultures of human fetal hypophyses

From weeks 13 to 26 of fetal life human hypophyses disclosed a constant content of radioimmunoassayable FSH. Although present already before this period, LH content increased considerably at week 17, along with the appearance of free beta-LH. In long-term organ culture experiments such early differentiating pituitaries proved to be endowed with autonomous synthesis and release of FSH and of free alpha-subunit, while LH, beta-LH and TSH declined to very low levels within a few weeks. Supplementation of the medium with LH-RH (12 ng/ml) significantly increased FSH synthesis and release but was not sufficient to sustain production of beta-LH and LH. It is suggested that other factors than LH-RH are required for differentiation of beta-LH biosynthesis and thus for production of LH.     

Comparative mapping of human thyrotropin, gonadotropins, and free subunits with antipeptide antibodies.

To compare the structural topology of the human TSH to that of the structurally related gonadotropins, 10 peptides covering the entire primary sequence of the alpha- and beta-subunits of TSH were synthesized and used as antigens for the preparation of polyclonal antibodies. The alpha-subunit was synthesized as 4 nonoverlapping peptides (1-25, 26-51, 49-73, 72-92) while the beta-subunit was segmented in 6 overlapping sequences (2-18, 10-38, 31-51, 53-76, 77-96, 92-112). Most of the peptide sequences were predicted to contain a putative antigenic determinant. All antipeptide antisera were found to bind to the corresponding synthetic sequence in an enzyme-linked immunosorbent assay as well as to denatured TSH subunits after Western blotting. The N-terminal half of the alpha-subunit was found differentially accessible in TSH and gonadotropins compared to the free subunit: antipeptide-alpha 1-25 antibodies exhibited variable affinity for the four glycoprotein hormones whereas anti-alpha 26-51 displayed a remarkable recognition of free alpha-subunit. Four peptides proved to be accessible in the TSH beta-subunit: the N-terminal peptide (beta 2-18) elicited antibodies that bound to free TSH-beta and poorly to the dimer while antibodies against the C-terminal sequence (beta 92-112) recognized equally well free beta-subunit and TSH. Antipeptide-beta 31-51 antibodies proved to be specific for TSH while the beta 53-76 contiguous peptide appeared accessible in both TSH and gonadotropins. The current findings therefore demonstrate that most of the sequences predicted to contain antigenic sites in the alpha- or the beta-subunits are indeed accessible at the surface of these proteins. Additionally, both subunits appear to contain amino acid sequences that are differentially expressed in TSH and gonadotropins as well as in free and combined subunits.     

Effects of naloxone and morphine on the proestrous surge of prolactin and gonadotropins in the rat

The effects of naloxone hydrochloride and morphine sulfate on the proestrous surge of PRL and gonadotropins (LH and FSH) were investigated in normal cycling Sprague-Dawley rats. Blood samples (0.45-0.50 ml) were withdrawn without anesthesia every 20 min from 1400-2000 h through an atrial cannula implanted the same morning. RIA revealed that a single iv injection of naloxone (0.2 mg/kg) at 1400 h completely suppressed the surge of PRL, and this was reversed by a concomitant injection of morphine (10 mg/kg). Morphine itself did not alter the peak of the PRL surge. Morphine suppressed only the early phase of the LH surge, and this was reversed by naloxone. Naloxone alone did not change the peak of the LH surge but maintained higher levels than controls during the declining phase. The FSH surge was not altered by either morphine or naloxone. These results suggest that endogenous opioid peptides may have a role in regulating the PRL and LH surges during proestrus in the rat.     

Subunits of an avian (ostrich) follicle-stimulating hormone and their hybridization with subunits of mammalian gonadotropins

Follicle-stimulating hormone (FSH) from the ostrich, Struthio camelus, was dissociated by methods previously used to prepare subunits from mammalian gonadotropins. Two chemically dissimilar subunits were obtained from the ostrich FSH and these resembled the alpha- and beta-subunits of mammalian FSH in amino acid composition. The subunits were relatively inactive in radioimmunoassay, radioreceptorassay, and bioassay when tested alone, but significant activity was regenerated upon their recombination. Incubations of mixtures of one of the ostrich subunits with the opposing subunit of ovine FSH also resulted in a significant regeneration of binding and biological activity in FSH assays. These hybrid recombinants demonstrated that the species specificity of the FSH molecule is conferred entirely by the source of the beta-subunit; in fact, hybrids formed between ovine LH-alpha and either ovine or ostrich FSH-beta were more potent in FSH assays than were those containing even two homologous FSH subunits. In contrast, there was little regeneration of LH bioactivity when FSH subunits (from ostrich or sheep) were combined with the subunits of ovine LH, although some LH binding activity was obtained when the mixture contained either one of the LH subunits; i.e., even LH-alpha + FSH-beta showed significant enhancement of binding activity for LH receptors. Thus, the subunits of avian FSH show biochemical and functional homologies to those of mammalian FSH, but there is only limited interchangeability with ovine LH subunits.     

Effects of antihypertensive therapy on human alpha- and beta-adrenoceptors.

The present study was designed to investigate the role of plasma catecholamines in the regulation of adrenoceptors in human hypertension. Thirty-three patients with newly detected essential hypertension were treated for 4 weeks with either nifedipine (2 x 20 mg/day), hydergine (2 x 2 mg/day), or both (20 and 2 mg/day, respectively, twice daily each), which lowered blood pressure equally well. Plasma noradrenaline increased during nifedipine, decreased during hydergine and was unaltered during the combination therapy. Lymphocyte beta 2-adrenoceptor density decreased by similar amounts, independent of whether blood pressure was normalized by treatment with nifedipine or hydergine. Platelet alpha 2-adrenoceptor density, however, decreased during nifedipine, slightly increased during hydergine and was unchanged during the combination treatment. We conclude that lymphocyte beta 2-adrenoceptors of hypertensive patients are not regulated primarily by plasma catecholamines but rather by a distinct factor associated with the extent of blood pressure elevation. The density of platelet alpha 2-adrenoceptors, however, appears to be dynamically regulated by catecholamines.     

Effects of luteal estradiol on the secretory transformation of human endometrium and plasma gonadotropins.

To study the role of luteal estradiol (E2), we interrupted the supply of E2 during the luteal phase of E2 and progesterone (P) replacement cycles. Thirty-one women, aged 26-37 yr, with absent or inactive ovaries received three different treatment regimens: group I (n = 11) received transdermal E2 and vaginal P according to a protocol designed to approximate levels of estrone (E1), E2, and P seen during the menstrual cycle. Groups II (n = 11) and III (n = 9) received identical treatments, except that in group II no E2, and in group III no E2 or P, was administered after day 15. Endometrial biopsies were obtained on days 20 and 24 in groups I and II, and on days 14 and 20 in group III. In group I, plasma E1 and E2 reached menstrual cycle levels, whereas in groups II and III, discontinuation of the E2 supply on day 15 resulted in a prompt decrease to castrate levels of plasma E1 and E2. In groups I and II, menopausal FSH and LH levels decreased to 26 +/- 6 and 30 +/- 7 IU/L, respectively, on day 13 (mean +/- SEM). In group I, administration of E2 and P starting on day 15 further lowered plasma gonadotropin levels. In group II, administration of P only failed to induce a similar decrease in plasma FSH and LH. No uterine bleeding occurred before day 25 in women of groups I or II, while women of group III bled within 2 days of E2 withdrawal. Endometrial biopsies were similar in groups I and II. Histological features were characteristic of early and late luteal phases on days 20 and 24, respectively. Endometrial maturation assessed by estrogen and progesterone receptors identified by immunocytochemistry showed the typical distribution seen on day 24 of the menstrual cycle with no difference between groups I and II. We conclude that in women deprived of ovarian function, administration of P only after 14 days of E2 priming prevented uterine bleeding and induced normal secretory transformations of the endometrium, but failed to suppress plasma gonadotropins.     

Effects of teleost gonadotropins and their antibodies on gonadal histology in winter flounder

The gonadotropic actions of teleost vitellogenic and maturational hormones were studied in flounder hypophysectomized shortly after reinitiation of vitellogenesis and spermatogenesis in August. Maturational hormone alone was able to stimulate formation of yolky oocytes, but vitellogenic hormone required the action of estrogen before it could stimulate production of yolky oocytes. An antiserum to vitellogenic hormone induced atresia of yolky oocytes and lowered the gonadosomatic index in vitellogenic flounder whereas an antiserum to maturational hormone did not affect ovarian histology or gonadosomatic index to any great extent. Preincubation of flounder ovarian sections with vitellogenic hormone, followed by incubation with antiserum to vitellogenic hormone and fluorescein isothiocyanate-labeled goat-anti-rabbit γ-globulin in succession, resulted in immunofluorescence in ooplasm of both large immature and vitellogenic oocytes, and in follicular envelopes of vitellogenic oocytes. Preincubation of ovarian sections with maturational hormone resulted in fluorescence in the interstitial tissue, and the follicular envelopes and the perinuclear regions of yolky oocytes. Maturational hormone stimulated spermatogenesis in hypophysectomized male flounder. The significance of these results in relation to previous studies was discussed.     

Effects of intranasal administration of LHRH superanalogue on endogenous LHRH and gonadotropins in humans

Plasma LH, FSH and endogenous LHRH were determined by radioimmunoassay (RIA) when 100 micrograms of LHRH superanalogue, des-Gly10-[D-Leu6]-LHRH ethylamide (leuprolide) was intranasally administered to both 7 healthy subjects aged 22 to 63 years and 6 patients with various hypogonadism aged 22 to 43 years [2 with anorexia nervosa, 3 with hypophysial hypogonadism and 1 with isolated gonadotropin (Gn) deficiency]. The response of plasma LH after the intranasal administration of leuprolide was observed to be 814 +/- 236 (mean +/- SE)% in the healthy subjects at 2 to 10 hours, and 528 +/- 183% in the patients with hypogonadism at 2 to 4 hours. These elevations were observed until 24 hours after administration in both groups. The response of plasma FSH was observed to be 449 +/- 99% in the healthy subjects at 3 to 8 hours, and 593 +/- 238% in the patients with hypogonadism at 2 to 6 hours. These elevations were observed until 24 hours in the healthy subjects and until 12 hours in the patients with hypogonadism. Furthermore, these responses tended to be higher than those after 100 micrograms intravenous administration of LHRH; LH responded up to 386 +/- 50% in the healthy subjects and up to 390 +/- 91% in the patients with hypogonadism, and FSH up to 282 +/- 38%, 343 +/- 90%, respectively. The elevation of plasma immunoreactive LHRH which was determined using RIA having no immunocrossreactivity with leuprolide (less than 0.01%) was observed at 6 hours after the administration of leuprolide in the healthy subjects (789 +/- 497%).(ABSTRACT TRUNCATED AT 250 WORDS)