We have recently developed sensitive and specific radioimmunoassays (RIAs) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II) and have measured the contents of both GnRHs in the rainbow trout brain. Our results showed that contents of the two GnRHs are variable among different brain regions. Therefore, in order to confirm the differential distribution of the two GnRHs by a different technique, we examined the distribution of immunoreactive sGnRH and cGnRH-II in the brain of masu salmon by using immunocytochemical techniques. sGnRH immunoreactive (ir) cell bodies were scattered in the transitional areas between the olfactory nerve and the olfactory bulb, the ventral olfactory bulb, between the olfactory bulb and the telencephalon, the ventral telencephalon, and the preoptic area. These sGnRH-ir cell bodies were dispersed in a strip-like region running rostrocaudally in the most ventral part of the ventral telencephalon. sGnRH-ir fibers were distributed in the various brain regions from the olfactory bulb to the spinal cord. They were especially abundant in the olfactory bulb, ventral telencephalon, preoptic area, hypothalamus, deep layers of the optic tectum, and thalamus. sGnRH-ir fibers also innervated the pituitary directly. cGnRH-II-ir cell bodies were found in the nucleus of the medial longitudinal fasciculus (nMLF). The distribution of cGnRH-II-ir fibers was similar to that of sGnRH-ir fibers, except that cGnRH-II-ir fibers were absent in the pituitary. The number of cGnRH-II-ir fibers was much fewer than that of sGnRH-ir fibers. The results of the present immunocytochemical study are in basic agreement with those of our previous RIA study. Thus, we suggest that in masu salmon, sGnRH not only regulates gonadotropin (GTH) release from the pituitary but also functions as a neuromodulator in the brain, whereas cGnRH-II functions only as a neuromodulator.

Follistatin is an activin-binding protein that prevents activin from binding to its receptors and neutralizes its activity. Follistatin also binds bone morphogenetic proteins (BMPs). In this study, we report the identification of a novel follistatin-like protein from mouse. The mouse cDNA encodes a 256-residue precursor and most likely a mouse homologue of human FLRG, which was found at the breakpoint of the chromosomal rearrangement in a B-cell line. Whereas follistatin has three follistatin domains, which are presumed to be growth factor binding motifs, FLRG possesses only two follistatin domains. Northern blotting revealed that mRNAs for FLRG were abundantly expressed in heart, lung, kidney, and testis in mouse. The recombinant mouse FLRG proteins were found to have binding activity for both activin and bone morphogenetic protein-2. Like follistatin, FLRG has higher affinity for activin than for BMP-2. The FLRG protein inhibited activin-induced and BMP-2-induced transcriptional responses in a dose-dependent manner. Glutathione S-transferase fusion proteins encoding various regions of FLRG were produced and studied. Ligand blotting using 125 I-activin revealed that the COOHterminal region containing the second follistatin domain was able to bind activin. Our finding implies that cellular signaling by activin and BMPs is tightly regulated by multiple members of the follistatin family.

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 [3HI thymidine mRNA for iBA 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 #A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation ofDNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes. (J.

Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin-related proteins, and myostatin antibodies. Although follistatin shows potent myostatin-inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (K(d)) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (K(d)) of a follistatin-derived myostatin inhibitor, designated FS I-I, to activin and myostatin are 64.3 microM and 46.8 nM, respectively. Transgenic mice expressing FS I-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I-I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I-I mice. These results indicate that myostatin blockade by FS I-I has a therapeutic potential for muscular dystrophy.

Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: Evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species (reproduction/neuropeptide) KAORU MIYAMOTO*, YOSHIHISA HASEGAWA*, MITSuo NOMURA*, MASAO Since the structural determination of porcine hypothalamic luteinizing hormone (LH)-releasing hormone (LHRH) in 1971 (1), the concept has widely been acepted that gonadotropin secretion is controlled by a sole gonadotropin-releasing hormone (GnRH), such as mammalian LHRH. On the other hand, we have demonstrated that gonadotropin secretion is not controlled by LHRH under certain physiological and experimental conditions (2). However, there has been no structural evidence that two or more than two GnRHs coexist and function in any vertebrate.The present paper represents the structural demonstration of the existence of two different GnRHs at least in avian species.Recently, we (3, 4) and King and Millar (5-7) have independently isolated chicken LHRH and determined its structure as [Gln8]LHRH. We have purified chicken LHRH by monitoring its biopotency towards rat anterior pituitary cells. During the course of the purification, another gonadotropin-releasing activity was found in the chicken hypothalamic extract, which was well separated from that due to chicken LHRH on ion-exchange chromatography.The present paper describes the isolation and structural determination of the second GnRH in chicken hypothalamic extract (designated chicken GnRH-II).Chicken GnRH-II has been purified by successive gel filtration, ion-exchange HPLC and reverse-phase HPLC. A pure peptide has been isolated in a yield of 7 pg (6 nmol), starting from 10,000 chicken hypothalamic fragments.The amino acid composition of the peptide indicated a decapeptide structure consisting of SerGlxPro1Gly2Tyrl-His2Trp2. By amino acid analyses and terminal analyses of enzymatic fragments derived from the purified decapeptide, the complete structure of chicken GnRH-II has been determined to be: pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH2.The present paper also describes the gonadotropin-releasing activity of chicken GnRH-II compared with that of mammalian LHRH. MATERIALS AND METHODSAssay of Gonadotropin-Releasing Activity. Gonadotropinreleasing activity was estimated on the rat anterior pituitary cell culture system as reported (3).Anterior pituitaries were obtained from female Holzman rats (180-to 220-g body weight), agitated in trypsin solution (0.25%), and finally dispersed with 1% Viokase (GIBCO) solution. Cells were collected and washed three times with Dulbecco's modified Eagle's medium (DME medium) containing gentamycin (100 ,ug/ml), fungizone (25 ttg/ml), 5% horse serum, 5% human cord serum, 2.5% bovine calf serum, 0.1% glutamine, and 1% nonessential amino acids (GIBCO). Cells were resuspended in an appropriate volume of the medium described above, and 0.2-ml portions (containing 5 x 104 cells) were put into 96 multi-well tissue culture plates (Nunc,. Th...