Purification and characterization of gonadotropin I and II from pituitary glands of tuna (Thunnus obesus)

The duality of teleost pituitary gonadotropins was established in an advanced marine fish, the tuna (Thunnus obesus). Two different molecular forms of gonadotropins, designated tGTH I and tGTH II, were isolated from an alcoholic extract of pituitary glands following ion-exchange chromatography and reversed-phase HPLC. Both tGTH I and tGTH II stimulated estradiol-17 beta and testosterone production in tuna ovarian follicles in vitro, although responses to tGTH II were significantly greater than those to tGTH I. Each gonadotropin consisted of alpha- and beta-subunits. tGTH I was stable in acidic conditions, whereas tGTH II dissociated into two subunits after acid treatment. Alpha subunits of tGTH I and tGTH II had identical amino acid sequences of 94 amino acid residues. The tGTH I beta and tGTH II beta consisted of 102 and 115 amino acid residues, respectively, and showed 35% sequence identity. tGTH I beta is structurally more similar to salmon GTH I beta than to salmon GTH II beta, whereas tGTH II beta is more similar to salmon GTH II beta. Thus it is evident that the tuna pituitary gland produces two chemically distinct gonadotropins.     

cDNA cloning of the beta subunit of teleost thyrotropin

cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human follitropin (42%), human lutropin (32%), and salmon gonadotropin (31-33%) beta subunits. The identification of TSH in addition to two gonadotropins (gonadotropins I and II) in the teleost fish suggests that the divergence of three kinds of glycoprotein hormones from an ancestral molecule took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods. Northern blot analysis showed that the expression of the rainbow trout TSH beta gene is specific to the pituitary gland and is significantly higher in immature fish than in mature fish, suggesting that TSH plays some role in the biological processes of immature fish.     

The value of a fabric-coated self-expanding stent in iliac arterial occlusions or aneurysms--the primary and long-term results

The present study focused on the role of catecholaminergic neurons and estrogens on the release of gonadotropins I and II in immature and early vitellogenic female rainbow trout. The ovariectomy-induced increase of GtH I blood levels (from about 10 to 15 ng/ml) was prevented in vitellogenic fish by E2 supplementation. E2 implantation of immature fish decreased blood GtH I levels (from about 6 to 1 ng/ml). Blood levels of GtH II were low (about 0.5 ng/ml) and not altered by ovariectomy and E2 treatment. These data demonstrate that estrogens exert a negative feedback on the release of GtH I in trout. A treatment with alpha-methyl-p-tyrosine (MPT), an inhibitor of catecholamine synthesis, increased blood GtH II levels of sham-operated vitellogenic fish and ovariectomized fish implanted with E2, but had no effects in ovariectomized fish. MPT did not modify blood GtH I levels in any experimental group. A treatment of E2-implanted immature or vitellogenic fish with the dopamine antagonist pimozide also increased blood GtH II levels, but did not significantly change blood GtH I levels. These data demonstrate that release of GtH II, but not of GtH I, depends on an E2-activated DA inhibitory tone.     

Atypical microtubule organization in undifferentiated human colon cancer cells

Immunocytochemical identification of GTH I and GTH II cells in the pituitary of the bluefin tuna (Thunnus thynnus) was performed using antisera specific for the common alpha-subunit and the two distinct beta-subunits of tuna (Thunnus obesus) GTH I and GTH II. Cells of the dorsal part of the proximal pars distalis (PPD), in close association with somatotrophs, displayed immunoreactivity of GTHIbeta. GTH IIbeta immunoreactivity was present in cells of the central part of the PPD and the external border of the pars intermedia. Anti-GTHalpha immunostained both GTH Ibeta- and GTH IIbeta-immunoreactive cells and also thyrotrophs. Both GTH Ibeta- and GTH IIbeta-immunoreactive cells were observed in immature bluefin tuna, although there were greater numbers of GTH IIbeta immunoreactive cells. These results suggest that GTH I and GTH II are synthesized in separate cells in the pituitary of the bluefin tuna. The localization and appearance of the two distinct gonadotropic cells of the tuna are compared with the salmonid arrangement.     

Modulation of 17-alpha-hydroxy-20 beta-dihydroprogesterone or gonadotropic extract activity on the in vitro intrafollicular maturation of rainbow trout (Salmo gairdnerii) oocytes by various steroids lacking maturing activity

The efficiency of 17 alpha-hydroxy-20 beta-dihydroprogesterone (17 alpha-20 beta Pg) or of a Trout pituitary gonadotropic extract to induce intrafollicular maturation of trout oocytes can be modulated by some steroids which do not present any direct maturing action: gonadotropic extract efficiency is lowered by estradiol and estrone, and enhanced by testosteron. As these steroids do not present a significant effect on 17 alpha-20 beta Pg induced maturation, their site of action may be located in the follicular tissues. The corticosteroids, particularly cortisol and cortisone enhance the maturing efficiency of gonadotropin and stimulate much more strongly the efficiency of 17 alpha-20 beta Pg. This suggests a direct effect on oocyte sensitivity to 17 alpha-20 beta Pg.     

In vitro reconstitution of an intermediate assembly stage of vaccinia virus

The effects of chronic exogenous testosterone treatment on the synthesis and/or secretion of two sturgeon gonadotropins (stGTH I and stGTH II) were assessed in 2-year-old juvenile white sturgeon (Acipenser transmontanus) surgically implanted with silastic capsules filled with 75 mg of testosterone and in previtellogenic female white sturgeon females implanted with 150 mg of testosterone. In groups of juvenile white sturgeon sacrificed 30, 60, 90, or 442 days postimplantation, pituitary concentrations of stGTH I were significantly greater in testosterone-treated fish (P < 0.01) when compared to those of controls. Pituitary concentrations of stGTH II were significantly higher (P < 0.01) in juvenile fish treated 60, 90, or 442 days with testosterone when compared to those of controls. Exogenous testosterone had no effect on plasma concentrations of either stGTH. Additional testosterone-treated juvenile sturgeon which were injected intraperitoneally 90 or 442 days postimplantation with 10 microg/kg of the gonadotropin releasing hormone analog d-Ala6-des-Gly10-GnRH ethylamide (GnRHa) also showed no change in plasma concentrations of stGTHs. Similar results were obtained for previtellogenic white sturgeon, as pituitary concentrations of stGTH I and stGTH II were significantly greater (P < 0.01) after 60 days of testosterone treatment compared to those of controls. A second group of 60-day testosterone-treated previtellogenic females also failed to exhibit increases of plasma stGTHs when administered 10 microg/kg of GnRHa. These results indicate that long-term testosterone treatment stimulates the accumulation of pituitary stGTHs in both juvenile and previtellogenic white sturgeon but does not affect basal or GnRHa-induced stGTH secretion.     

Gonadal steroid modulation of vasopressin secretion in response to osmotic stimulation

As deficiencies in osmotic stimulation of vasopressin (VP) messenger RNA (mRNA) content in castrated rats have been reported, experiments were performed to determine whether castration altered osmotically stimulated VP release in vitro. Perifused explants of the hypothalamo-neurohypophyseal system were obtained from sham and gonadectomized male rats. There were no significant differences in VP release stimulated by a ramp increase in the osmolality of the culture medium between the two groups. As testosterone was undetectable in the perifusion medium, the effect of addition of testosterone on osmotically stimulated VP release was evaluated. Testosterone (3 ng/ml) and its metabolites, estradiol (50 pg/ml) and dihydrotestosterone (DHT; 3 ng/ml), inhibited osmotically stimulated VP release in hypothalamo-neurohypophyseal system explants. The osmotically induced increase in VP mRNA content was also inhibited by testosterone and estradiol, but not by DHT. Neither estradiol nor DHT affected stimulus-secretion coupling of hormone secretion, because they did not inhibit KCl (25 mM)-stimulated VP release. BSA conjugates of estradiol (200 nM) and DHT (10 mM) also inhibited osmotically stimulated VP release, and VP mRNA content was inhibited by BSA-estradiol, but not by BSA-DHT, suggesting nongenomic actions of the steroids. The differential effects of estradiol and DHT on VP mRNA imply distinct actions for these steroids, and the DHT mechanism uncouples regulation of VP release from VP mRNA content.     

Interaction of activin A and gonadal steroids on FSH secretion from primary cultured rat anterior pituitary cells

To clarify the mechanism of FSH secretion from the pituitary induced by activin A, we studied the interaction between activin A and gonadal steroids in inducing FSH release from primary cultured female rat pituitary cells in serum-free medium. The basal release of FSH was stimulated by activin A, testosterone (T) and progesterone (P), and T and P also facilitated basal FSH release stimulated by activin A. The GnRH-stimulated FSH release was facilitated by activin A, P and 17 beta-estradiol (E2), but suppressed by T. The effect of activin A on GnRH-stimulated FSH release was facilitated by P, but not affected by T or E2. These findings suggested that the interaction between activin A and T or P may be involved in the regulation of FSH secretion during the estrus cycle in rats.     

Effect of human and murine interferon-alpha on steroid production by rat ovarian cells

The effect of interferon on the rat ovarian cell function was investigated. Cells from the ovary of juvenile rats were used as a model to investigate the effect of IFN-alpha on the secretion of estradiol and testosterone. In addition the effect of human IFN-alpha (hIFN-alpha) on the secretion of testosterone by the rat adult testis was studied. Present results show that leukocyte hIFN-alpha decreased the human chorionic gonadotropin (hCG) stimulated secretion of estradiol and testosterone by ovarian cells, and the production of testosterone by testis cells. Basal secretion of steroids was affected later and in less proportion than the hCG-dependent production. The IFN-alpha obtained from murine leukocytes, also inhibited the response of ovarian cells to the hCG stimulus.The nature of this effect in the secretion of the steroids is dose and time-dependent. The incubation of hIFN-alpha with an specific antibody completely blocked the effect of the cytokine on ovarian cells.     

A comprehensive evolutionary analysis based on nucleotide and amino acid sequences of the alpha- and beta-subunits of glycoprotein hormone gene family

On the basis of nucleotide sequences of the coding region and their predicted amino acid sequences, 58 glycoprotein hormone subunit genes were compared, aligned and used to construct phylogenetic trees for this family. The analysis included 17 alpha-subunits, eight TSH beta-, six FSH beta-, 17 LH beta/CG beta-, four fish gonadotropin (GTH)-I beta-, five fish GTH-II beta- and one additional fish GTH beta-subunit. The reliability of the phylogenetic trees was probed with the bootstrapping test. Our results indicated that: both the alpha- and beta-subunits of the family diverged from a common ancestral gene about 927 million years ago, the initial precursor of the beta-subunit duplicated to give rise to the LH beta and a second hormone, the latter then duplicating to FSH beta and TSH beta, so that FSH beta is related more to TSH beta than to LH beta; and bony fish GTH-I beta is highly related to mammalian FSH beta, whereas the bony fish GTH-II beta is more related to mammalian LH beta. For scientific consistency and convenience, we propose that the following nomenclature be adopted, all fish gonadotropins of type I be classified as FSH and all type II be classified as LH hormones. In addition, on the basis of results from this and other studies, we propose an evolutionary history for this glycoprotein hormone family. Reconstruction of the evolutionary history of this family would not only provide clues to understanding thyrotropin and gonadotropin functions, but would also allow further revision of the present nomenclature of the gonadotropins in fish.     

Tuft-to-capsule adhesions and their precursors: differences between the vascular and tubular poles of the human glomerulus

The hypothalamo-pituitary-adrenal (HPA) axis is modulated by sex hormones. Few data exist on the relation between acute estrogen deficit and HPA axis response to corticotropin-releasing hormone (CRH). The effects of a sudden drop in estradiol levels on basal and CRH-stimulated levels of ACTH, cortisol, testosterone, androstenedione and 17-hydroxyprogesterone (17-OHP) were assessed in nine premenopausal women (44-48 years of age), before and after ovariectomy. The CRH test was performed before and 8 days after ovariectomy. A significant reduction in ACTH and adrenal steroids but not in cortisol response to CRH was observed after ovariectomy. The ratio of deltamax androstenedione/17-OHP after CRH stimulation was substantially the same before and after ovariectomy, whereas deltamax 17-OHP/cortisol was significantly lower in ovariectomized women showing increased 21- and 11beta-hydroxylase activity. The results show that the acute estrogen deficit induces changes in the HPA axis characterized by reduced stimulated secretion of ACTH and steroids but normal stimulated cortisol production.     

Endocrine therapy of transsexualism and potential complications of long-term treatment

Physiological principles of the interrelationship of sex hormones and their regulation are the foundation of understanding appropriate treatment of the transsexual patient. While both genetic males and females have estrogens and androgens, the quantitative sex hormone production is genetically predetermined by sex hormone production both in the gonads and via peripheral conversion of hormone precursors to sex steroids. Sex hormones exert a negative feedback on the hypothalamus and pituitary gland whereby gonadotropin-releasing hormone (GnRH), pituitary luteinizing hormone (LH), and follicle-stimulating hormone (FSH) are regulated or suppressed by the endogenous levels of these hormones. Sex hormonal therapy induces attenuated GnRH stimulation of LH and FSH causing a reduction of serum sex hormone levels. It is clear that estrogen as well as androgen therapy have a dual role: (i) induction of feminization or virilization and (ii) suppression of the hypothalamic-pituitary-gonadal axis leading to a reduction of endogenous estradiol or testosterone secretion. Cross-sex hormonal treatment may have substantial medical side effects. The smallest dosage of hormonal therapy compatible with the above clinical aims should be used.     

A case report of multiple hepatic hilar cyst associated with hepatocellular carcinoma

Two types of cDNA encoding gonadotropin beta subunits (GTH beta) were isolated from a cDNA library prepared from pituitary gland of Baikal omul (Coregonus autumnalis migratorius Georgi). The nucleotide sequences of cDNA were determined. The CTHI beta and GTHII beta cDNAs code for polypeptides of 137 and 142 amino acids, respectively. Both of them include a putative signal peptide of 24 amino acids. The predicted amino acid structures of omul gonadotropins were compared with those of other vertebrate species.     

The capability of androgen aromatization by bank vole Leydig cells in vitro: the effect of aromatase inhibitor

The effect of increasing concentrations of aromatase inhibitor CGS 16949A on LH- and testosterone-supplemented estradiol secretion by Leydig cells was studied. Leydig cells were obtained from immature and mature bank voles, which were reared in a long or a short photoperiod conditions. They were either incubated for 6 hrs with LH and/or testosterone (LH/testosterone-supplemented cultures), or as the control cultures. Aromatase inhibitor was added for further 18 h. After 24-h incubation period testosterone and estradiol secretion were radioimmunologically examined. This study revealed that in bank voles, Leydig cells' aromatase activity appeared to be photoperiod- and age-dependent. However, the differences in its activity were seen only in case of mature tissue. In immature bank voles, reared in both regimes of light aromatase activity was low, while in mature animals a 50% increase was observed. Thus, aromatase activity and estradiol biosynthesis were much lower in the Leydig cell culture from a short day than in the analogus culture fom a long one. The present study also showed different sensitivity to the stimulation of LH between young and adult bank voles kept in different regimes of light, which could be connected with the number of Leydig cells' LH receptors.     

N-methyl-D-aspartic acid stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N-methyl-D-aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10 microM) and AP5 (100 microM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 microM NMDA in Mg2+-replete compared with 5 microM in low-Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.     

Aromatase activity and the effect of estradiol and testosterone on DNA synthesis in endometrial carcinoma cell lines

Human endometrial and breast carcinoma cell lines were examined for aromatase activity and the effects of sex steroids (estradiol and testosterone) on DNA synthesis. Aromatase activity was high (greater than 500 fmol/10(7) cells/24 h) in the cell lines MCF-7 and OMC-2, moderate (100-499 fmol/10(7) cells/24 h) in the cell lines HEC-59 and Ishikawa, and low (less than 100 fmol/10(7) cells/24 h) in the HHUA cell line. A substantial stimulation of DNA synthesis by estradiol (10(-9)M) was observed in cell lines HEC-59, OMC-2, and MCF-7, with an increase in [3H]thymidine uptake of over 250%. The Ishikawa cell line was stimulated moderately (115-249%). No estradiol-induced increase in DNA synthesis was observed in HHUA. Responsiveness of DNA synthesis to testosterone was observed in cell lines that showed the greatest response to estradiol, namely HEC-59, OMC-2, and MCF-7. Otherwise, estrogen-responsiveness did not always correlated with a significant aromatase activity. These data suggest that some but not all endometrial carcinomas may possess an aromatase-dependent growth stimulating system.     

Hypothalamus-pituitary-ovarian axis in cyclic rats lacking progesterone actions

Antagonizing diestrous progesterone actions in cyclic rats by s.c. injections of the antiprogesterone RU486 (2 mg twice a day from metestrus through proestrus) increased LH and decreased FSH basal serum concentrations. Ovariectomy at metestrus (0800 h) increased serum levels of both gonadotropins in controls and reversed the RU486-induced dissociation of basal gonadotropin secretion. RU486-dissociated gonadotropin secretion is also dependent upon LHRH, since treatment (s.c.) with 1 mg GnRH antagonist (ORG 30276) twice a day on metestrus and diestrus completely prevented both the RU486-induced increase in LH and the decrease in FSH serum concentrations. The LHRH content in the medial basal hypothalamus and median eminence increased on proestrous morning in RU486-treated rats. The LH pituitary response to an exogenous i.v. bolus of 25 ng LHRH (Peninsula 7201; Peninsula Laboratory, Inc., Merseyside, UK) at 1700 h on diestrus was enhanced in rats treated with RU486. No differences in pituitary FSH response were noted with respect to oil-injected rats. The pituitary content of both gonadotropins decreased in RU486-treated rats on proestrous morning. All these effects due to RU486 in cyclic rats were reversed by ovariectomy. Testosterone serum levels increased significantly from diestrus onward, and the estradiol concentration increased on proestrous morning in RU486-treated rats. Ovariectomy as well as LHRH antagonist treatment eliminated the effects of RU486 on ovarian steroid production. Moreover, antiestrogen tamoxifen treatment reversed RU486-dissociated gonadotropin secretion, while antiandrogen flutamide treatment had no effect. The results of this experiment have confirmed previous findings that RU486 treatment dissociates basal gonadotropin secretion in cyclic rats. In addition, the present results show that: (1) this effect of RU486 is not due to a direct effect of this compound or to the blockade of progesterone action at a central level; (2) the effect of RU486 on pituitary gonadotropin secretion depends on ovarian substances other than progesterone and LHRH, since it is reversed by ovariectomy and completely abolished by LHRH antagonist treatment; (3) the reduction in FSH serum levels in rats treated with RU486 seems to be exerted by inhibin and estradiol at the pituitary level by reducing FSH synthesis and secretion; and (4) the hypersecretion of LH in rats treated with RU486, as compared to that resulting from ovariectomy, seems to be the consequence of, first, a lack of progesterone inhibitory action on LH secretion, and, second, an inappropriate feedback system involving increased hypothalamic LHRH activity and pituitary sensitivity to LHRH of moderately high levels of estradiol in the presence of abnormally high levels of testosterone.     

Inferior vena cava filters in cancer patients: indications and outcome

Estrogen, like other steroids, may induce rapid nongenomic cellular effects. We studied the effect on intracellular cAMP of short-term exposure (5 min) of cultured rat pulmonary vascular smooth muscle cells (VSMC) to estradiol 17 beta. At confluence, VSMC were incubated in phosphate buffer saline for 1 hr before exposure to different hormones. The reaction was stopped with 0.1 N HCl and cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The 5-min incubation with estradiol 17 beta (0.3-30 microM) significantly increased basal intracellular cAMP in a concentration-dependent manner. The stimulatory effect of estradiol on cAMP was time-dependent, increasing with prolonged exposure to the hormone, and was not affected by the protein synthesis inhibitor, actinomycin D (5 micrograms/ml), at 5 and 30 min. Comparable concentrations of testosterone or estradiol 17 alpha had no significant effect on cAMP. The estrogen receptor partial agonist, tamoxifen also significantly increased basal cAMP in a concentration-dependent manner, but inhibited the effect of estradiol. Furthermore, forskolin elicited a concentration-dependent increase in cAMP (396.6 +/- 53% at 10 microM concentration), which was significantly potentiated in presence of estradiol. The effect of estradiol is unlikely to be mediated by G-protein activation, because the G protein inhibitor, pertussis toxin (100 ng/ml), did not significantly affect estradiol-induced increase in cAMP. Removal of Ca++ from the incubation medium inhibited the stimulatory effect of estradiol 17 beta suggesting that estradiol may increase pulmonary VSMC cAMP via a Ca(++)-dependent pathway. We suggest that the effect of estradiol 17 beta in these experiments is nongenomic in nature, and is possibly mediated by direct interaction of the hormone with specific membrane binding sites.