Anne Corrigan

Chemical and biological characterization of the inhibin family of protein hormones.

Publisher Summary This chapter discusses the chemical and biological characterization of the inhibin family of protein hormones, which is a family of peptides isolated from the follicular fluid or rete testis fluid on the basis of their ability to inhibit the secretion of the follicle-stimulating hormone (FSH) by cultured rat anterior pituitary cells. It also reviews the possible roles of inhibin and fibre-reinforced plastic (FRP)/activin in placenta, brain, and bone marrow. Inhibin-related dimers are broadly distributed anatomically and have powerful activities in several biological systems where inhibin and FRP/activin often exhibit opposite effects. While the physiologic roles of inhibin to regulate FSH secretion in the female rat and immature male rat are strongly supported, the significance of these hormones within the gonad, brain, placenta, and bone marrow have yet to be placed in in vivo context. Although the panoply of functions of inhibin and FRP/activin are certainly incompletely understood at this time, this family has already demonstrated a powerful mechanism for the generation of signal diversity whereby differential subunit association can result in the generation of dimers with opposing biological actions in multiple tissues.

Detection and purification of inhibin using antisera generated against synthetic peptide fragments

Publisher Summary Inhibin is a hormone whose best established physiological role is selective suppression of the release of follicle-stimulating hormone (FSH) from the pituitary. Subsequent to the identification of inhibin, two laboratories isolated a protein from porcine follicular fluid that selectively released FSH from pituitary cell cultures. Characterization of this protein––named FSH releasing protein (FRP)/activin––showed that it was a homo- or heterodimer of inhibin β subunits. This chapter also describes the production of antibodies to the α, βA, and βB subunits of inhibin; development of a radioimmunoassay (RIA) specific for inhibin, using an antibody directed to the α subunit; use of these antibodies for Western blot analysis; and a method for concentrating inhibin and FRP from biological fluids. It also discusses a method that was developed for the rapid isolation of inhibin from ram rete testis fluid (RTF) using immunoaffinity chromatography with an antibody directed against the α subunit.

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

SummaryThe organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.

Brain distribution of radioimmunoassayable gonadotropin-releasing hormone in female goldfish: Seasonal variation and periovulatory changes

A radioimmunoassay (RIA) for [Trp7, Leu8]gonadotropin-releasing hormone (sGnRH) was developed to determine the gonadotropin-releasing hormone (GnRH) content in discrete brain areas of female goldfish at different stages of ovarian development. Temporal changes in serum gonadotropin (GtH) and GnRH concentrations in discrete brain areas were measured during spontaneous ovulation. There were no clear parallel changes in brain GnRH with seasonal ovarian development in goldfish. However, under a 10 degrees temperature acclimation regimen, the GnRH content in the hypothalamus and pituitary decreased as the ovary progressed from the regressed to the mature condition; on the other hand. GnRH content in the spinal cord increased in sexually mature fish compared with that in regressed fish. Significant decreases in GnRH concentration were observed in certain brain areas (olfactory bulbs, telencephalon, hypothalamus, and pituitary) of fish undergoing spontaneous ovulation compared with those of nonovulatory fish. The simultaneous changes of GnRH concentration in these brain areas suggested that the GnRH neuronal system may function as an integrated unit for the activation of GtH secretion during ovulation in goldfish.

Activin-A Modulates Gonadotropin-Releasing Hormone Secretion from a Gonadotropin-Releasing Hormone-Secreting Neuronal Cell Line

The recent development of GnRH-secreting neuronal cell lines (GT1-1, GT1-3 and GT1-7 clones) has provided a model system for the study of the neural regulation of GnRH expression and secretion. We report here that activin-A stimulates GnRH secretion by GT1-7 cells in a dose-dependent manner, with an EC50 of approximately 2.5 ng/ml. The maximal response (50% stimulation) was achieved after 2 days of incubation with 20 ng/ml activin-A. Activin-A treatment increased total GnRH (secreted + cellular) in GT1-7 cells, possibly reflecting a stimulation of GnRH biosynthetic rates. The secretory effect of activin-A was also accompanied by a change in the cellular morphology to a more neuronal phenotype. The addition of TGF-beta (10 ng/ml), which is structurally related to activins, did not significantly increase secretion of GnRH by GT1-7 cells illustrating the specificity of the activin effect on this cell line. Although inhibin (20 ng/ml) alone did not directly affect the spontaneous secretion of GnRH, it was able to partially block the stimulatory effect of activin. The present study with the GT1-7 clonal cell line suggests that activin, and perhaps inhibin, might act at hypothalamic sites to regulate reproduction through the control of GnRH production and/or secretion.

Regulation and Actions of Smad7 in the Modulation of Activin, Inhibin, and Transforming Growth Factor-β Signaling in Anterior Pituitary Cells1

Activins and transforming growth factor-β (TGFβ) are crucial autocrine, paracrine, and endocrine modulators of anterior pituitary function. Activins regulate most pituitary cells and lactotropes are targets of TGFβ. Smad2 and Smad3 are two cellular mediators of activin/TGFβ signaling, whereas Smad7 is as an inducible, negative modulator of the pathway. This study was undertaken to evaluate Smad7 regulation in the pituitary. Activin A rapidly and transiently increased Smad7 messenger RNA (mRNA) levels of rat anterior pituitary (RAP), clonal gonadotrope (αT3–1 and LβT2), and corticotrope (AtT20) cells with an EC50 of 0.1–0.2 nm. In RAP cells, activin A or TGFβ1 had equivalent effects that were additive. Follistatin, known to bind and inactivate activins, prevented Smad7 induction by activin. Inhibin A partially antagonized activin A, perhaps reflecting gonadotrope-selective actions. This antagonism was also evident with αT3–1 and LβT2 gonadotropes. Forskolin had no measurable effect in RAP cells, but increa...

Actions of activins, inhibins and follistatins: implications in anterior pituitary function.

1. The anterior pituitary is well documented to be under the control of central and peripheral factors that dynamically interact to affect cell‐specific modulation of pituitary functions. However, it is becoming increasingly evident that these extrinsic factors work in concert with a variety of local products that exert autocrine/paracrine control on pituitary cells.

Interleukin-1β Regulates Pituitary Follistatin and Inhibin/Activin βB mRNA Levels and Attenuates FSH Secretion in Response to Activin-A

Activins and follistatins regulate all levels of the reproductive axis, including the pituitary where they stimulate and inhibit FSH production, respectively. Gonadotropes are known to express inhibin/activin βB and activin-B (βBβB) functions as an autocrine modulator of FSH production. By contrast, the mRNA for the activin-binding protein, follistatin, is present in most pituitary cells and folliculo-stellate cells may be the major source of the protein secreted by the anterior pituitary. Interleukin-1β (IL-1β) is one of several cytokines known to also influence the reproductive axis. IL-1β inhibits the hypothalamo-pituitary-gonadol (HPG) axis by suppressing GnRH and gonadal steroid production. Because several pituitary cell types, including follistatin-producing folliculo-stellate cells, are targets of IL-1β, cytokine effects on gonadotrope function were evaluated using cultured rat anterior pituitary cells. Activin-A (0.01 to 1 nM; 24 h) increased basal FSH secretion ∼2-fold. IL-1β (0.005 to 0.5 mM) by...

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues.

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

Competitive antagonists of the corticotropin releasing factor (CRF) scanned with a i-(i+3) Glu Lys Bridge

CRF [1] is involved in a wide spectrum of central nervous system (CNS)-mediated effects, suggesting that this peptide plays an important role within the brain, especially in response to stressful stimuli [2]. Systematic SAR investigations have resulted in the development of CRF antagonists such as [3], members of the (standard) family [4] and conformationally restricted analogs [5] that are effective in the CNS. Those results, predictive methods and physicochemical measurements have suggested that CRF and its family members (urotensins and sauvagine) assume an conformation when interacting with the CRF receptors. To further test this hypothesis, we have scanned the whole rat/human sequence with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown to be compatible with maintenance or enhancement of structure in at least some unpredictable cases.