Yoshihisa Hasegawa

Isolation of porcine follicular fluid inhibin of 32K daltons.

Purification of ovarian inhibin from porcine follicular fluid was performed by using an bioassay based upon the suppression of spontaneous FSH release from cultured cells of rat anterior pituitary. The presence in the follicular fluid of four molecular forms of inhibin activity corresponding to Mr 100K, 80K, 55K and 32K was revealed by SDS-gel electrophoresis under non-reducing conditions. The smallest inhibin amongst them, named 32K inhibin, eliciting about 70% of the total activity in the follicular fluid, was separated by gel filtration in the presence of 8 M urea. By subsequent ion-exchange chromatography, followed by RP-HPLC, 32K inhibin was purified to homogeneity with a 8,000 fold purification factor in a yield of 12%. The purified 32K inhibin was found to comprise two polypeptide subunits (Mr 20K and 13K), linked by disulfide bridges and to specifically suppress the secretion of FSH, but not of LH from the pituitary cells.

CLONING AND SEQUENCING OF HUMAN LH/HCG RECEPTOR CDNA

We have isolated and sequenced a cDNA encoding the human luteinizing hormone--choriogonadotropin (LH/hCG) receptor. The deduced amino acid sequence (699 residues) containing seven putative transmembrane segments displays sequence similarity to G protein-coupled receptors. The receptor consists of 335 residue extracellular domain which contains six N-linked glycosylation sites. While the protein is 85 and 87% identical overall with the previously cloned rat and porcine LH/hCG receptor respectively, the most highly conserved regions are the putative transmembrane segments (91 and 94% similarity, respectively).

Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes.

The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.

Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone

Abstract A peptide having gonadotropin-releasing activity was isolated in a yield of 2.5 μg from an extract of 2,000 chicken hypothalami. The biopotency was monitored using rat anterior pituitary cell culture system. The peptide differs from mammalian Luteinizing Hormone-Releasing Hormone (LH-RH) in its behavior during chromatographic separation (ionexchange and high performance liquid chromatography) and in its reaction towards anti-LH-RH antiserum directed against the C-terminal region of the LH-RH molecule. The peptide (chicken LH-RH) stimulates secretion of both LH and FSH from rat anterior pituitary cells. The biological potency of this peptide was about 4 % of that of the authentic decapeptide estimated in the rat anterior pituitary system. The amino acid composition is (Ser, Pro, Glx 2 , Gly 2 , Leu, Tyr, His, Trp), which differs from mammalian LH-RH only in that one Arg residue is replaced by a Glx residue. Based on the behavior on CM cellulose chromatography and the reaction towards anti-LH-RH antiserum, one possible structural candidate for this peptide (chicken LH-RH) is [Gln 8 ]-LH-RH.

Differences in salmon GnRH and chicken GnRH-II contents in discrete brain areas of male and female rainbow trout according to age and stage of maturity

We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2-10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.

Localization of avian LHRH-immunoreactive neurons in the hypothalamus of the domestic fowl, Gallus domesticus, and the Japanese quail, Coturnix coturnix

SummaryThe localization of LHRH-containing perikarya and nerve fibers in the hypothalami of the domestic fowl and Japanese quail was investigated by means of the specific immunoperoxidase ABC method, using antisera against chicken LHRH-I ([Gln8]-LHRH), chicken GnRH-II ([His5-Trp7-Tyr8]-LHRH [2–10]) and mammalian LHRH ([Arg8]-LHRH). Chicken LHRH-I-immunoreactive perikarya were sparsely scattered in the nucleus preopticus periventricularis (POP), nucleus filiformis (FIL) and nucleus septalis medialis (SM), and in bilateral bands extending from these nuclei into the septal area in both species. A few reactive perikarya were also observed in the nucleus accumbens (Ac) and lobus parolfactorius (LPO). Numerous cLHRH-I-immunoreactive fibers were widely scattered in the preoptic, septal and tuberal areas, and were densely concentrated in the external layer of the median eminence and in organum vasculosum of the lamina terminalis (OVLT) in both species. Anti-mammalian LHRH serum cross-reacted weakly with perikarya and fibers immunoreactive to anti-cLHRH-I serum in normal chicken and quail. Anti-cGnRH-II[2–10] serum immunoreacted with magnocellular neurons distributed in the rostral end of the mesencephalon along the midline close to the nervus oculomotorius (N III). These perikarya were apparently different from cLHRH-I immunoreactive neurons. No immunoreactive cells and fibers against anti-cGnRH-II[2–10] were observed in the hypothalamus and median eminence of the chicken or quail. Anti-cGnRH-II[2–10] bound specifically with cGnRH-II. The morphological evidence suggests that cGnRH-II may not be secreted into the portal circulation to act as hypothalamic hormone.

Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cell.

Erythroid differentiation factor (EDF), inhibin beta A-homodimer, induced expression of follicle stimulating hormone receptors on rat granulosa cells prepared from diethylstilbestrol primed immature female rats. After 3 day incubation with EDF, the number of FSH receptors on the granulosa cells was increased to about 3.5 times of the control value in a dose dependent manner with an ED50 value of 61 ng/ml. On the other hand, EDF related peptides, i.e., bovine 32K Da inhibin A and TGF beta, had no effect on the FSH receptor induction. The present observation suggests that EDF may play a role in the initiation of the cytodifferentiation of ovarian granulosa cells.

Isolation of bovine follicular fluid inhibin of about 32 kDa

Inhibin of about 32 kDa from bovine follicular fluid (bFF) was purified by using chromatographies operated under protein-dissociating conditions, which we have established for our previous purification of porcine follicular fluid (pFF) inhibin. On a gel filtration, bFF inhibin activity was eluted at 3 distinct regions corresponding to apparent molecular weight of 96, 55 and 32 kDa, representing 17%, 27% and 24% of the total inhibin activity in the follicular fluid, respectively. The smallest inhibin, named 32 kDa bFF inhibin, that evidently suppressed the basal secretion of FSH from the pituitary cells, was purified to homogeneity with a 5330-fold purification factor in a recovery yield of ca. 11%. 32 kDa bFF inhibin thus purified consists of 2 polypeptide chains (A-chain: 20 kDa and B-chain: 13 kDa), linked by disulfide bridges. N-Terminal sequences were Ser-Thr-Pro-Pro- for the A-chain and Gly-Leu-Glu-Cys- for the B-chain. The identical N-terminal sequences were also found in 32 kDa pFF inhibin, except that Pro-3 of the bFF A-chain is substituted by Ala in the 20 kDa chain of pFF inhibin.

Demonstration of high molecular weight forms of inhibin in bovine follicular fluid (bFF) by using monoclonal antibodies to bFF 32K inhibin

Monoclonal antibodies specifically recognizing each 20K and 13K subunit of bovine follicular fluid (bFF) 32K inhibin have been prepared. By immunoblotting procedures using these antibodies, we have demonstrated in bFF at least six inhibin forms, the apparent molecular weights of which are estimated to be 120K, 108K, 88K, 65K, 55K and 32K daltons, respectively. Amongst them 65K, 55K and 32K inhibin forms comprise two polypeptide subunits linked by disulfide bridge(s). In these inhibin forms a polypeptide of 13K daltons, a shorter subunit of bFF 32K inhibin, is attached by disulfide linkage(s) to polypeptides of 57K, 44K and 20K daltons, which are immunologically related to a larger subunit of the 32K inhibin, to yield 65K, 55K and 32K inhibins, respectively. On the other hand the higher molecular weight forms, 120K, 108K and 88K inhibins, are composed of three polypeptide subunits. In these forms a polypeptide of 62K daltons, immunologically related to the shorter subunit of bFF 32K inhibin, is attached by disulfide bridge(s) as the third component to the respective smaller inhibin forms which are composed of two subunits. These findings suggest that the complex formation of the subunit precursors may be extremely important and that restricted proteolytic cleavages following the complex formation may yield such divergent forms of inhibin in bFF.

Inhibin alpha-subunit monomer is present in bovine follicular fluid

The 26 kDa form of the inhibin alpha-subunit monomer was isolated from bovine follicular fluid. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis revealed that the 26 kDa form of the alpha-subunit monomer is composed of 6 kDa and 20 kDa fragments linked together by a disulfide bond(s). The NH2-terminal sequence and amino acid analyses of each fragment showed that those fragments are derived from residues 18 to 60 and 227 to 360, respectively, of the inhibin alpha-subunit precursor (residues 1 to 360) as deduced from the cDNA sequence. Thus, it is concluded that removal of the putative signal peptide (residues 1 to 17) and the middle portion (residues 61 to 226) from the alpha-subunit precursor generates the 26 kDa form of the alpha-subunit. In the in vitro rat pituitary assay, the 26 kDa alpha-subunit exhibited neither inhibin-like nor FSH-release stimulating activity.