The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.
N Zmora, D Gonzalez-Martinez, JA Munoz-Cueto, T Madigou, E Mananos-Sanchez, SZ Doste, Y Zohar, O Kah and A Elizur
S C van Buul-Offers, K de Haan, M G Reijnen-Gresnigt, D Meinsma, M Jansen, S L Oei, E J Bonte, J S Sussenbach and J L Van den Brande
In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5′). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one 115' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5′ without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs 115 and 116 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5′.
Inbreeding of 2 lines containing construct 115' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting.
In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.
Journal of Endocrinology (1995) 144, 491–502
Lordosis behaviour was induced in immature 20-day-old male rats by sequential treatment with oestradiol benzoate (OB) and progesterone, but prepubertal male rats were behaviourally less sensitive to the OB and progesterone treatment than were female rats. Thus, the sex difference in the lordosis response was present early during development. Castration at various times after birth showed that the capacity of immature rats to show lordosis is normally inhibited by an action of testicular secretions exerted during the first 10 days of life. Treatment of day 0 castrated rats with OB, either as a single injection given on the day of birth or as daily injections given on the first 10 days after birth, was much more effective in inhibiting the display of lordosis behaviour at 30 and 37 days of age than was treatment with testosterone benzoate (TB). Treatment with dihydrotestosterone benzoate neonatally had no inhibitory effect. Treatment of intact male rats or day 0 castrated OB-or TB-treated rats with the anti-oestrogen ethamoxytriphetol (MER-25) during the first 10 days of life antagonized the inhibitory effect of the testes and of the OB or TB treatment on the development of the lordosis response. It is suggested that during normal development oestradiol formed in the brain from testosterone in the circulation acts during the first 10 days of life to inhibit the capacity of male rats to show lordosis when adult.
RYOKO KAKIHANA, STEPHEN BLUM and SEYMOUR KESSLER
The development of the pituitary-adrenocortical stress response was studied in CBA/J × DBA/2J hybrid mice. On the basis of the plasma corticosterone response 15 min after a subcutaneous injection of histamine dihydrochloride (50 mg/kg), the first three neonatal weeks could be divided into stress-nonresponsive (3–211 days) and stress-responsive 16–21 days) periods. During the former period, corticosterone levels in the brains of the non-stressed control mice were 63% higher than those of comparable mice during the latter period. Histamine stress significantly increased corticosterone concentrations in the brain during both these periods, but the increase was much greater (88%) during the stress-responsive period than during the stress-nonresponsive period (29%).
B. T. DONOVAN
The effect of hypophysectomy or of division of the pituitary stalk on the growth and function of the corpora lutea in the ovaries of the pseudopregnant ferret was studied, and compared with the changes seen in the ovaries and uterus of normal animals. Hypophysectomy caused a fall in the weight of the ovaries and uterus and regression of the corpora lutea. Isolation of the pituitary gland from the brain was compatible with full development of the corpora lutea and did not interfere with the growth of the uterus during the first 4 weeks of pseudopregnancy. Later on, the ovarian and uterine weights fell below those of control animals. Blank operations, and stalk section with subsequent regeneration of the portal vessels, did not disturb luteal function. It is concluded that the pituitary gland of the pseudopregnant ferret secretes a luteotrophic hormone and that an additional factor, possibly oestrogen, may be required for optimal luteal activity.
A. J. ZOLOVICK
Agents known to alter synthesis, storage, release, reuptake or catabolism of the monoamines (Lippmann, 1968; Rubinstein & Sawyer, 1970), and excess amounts of 5-hydroxytryptamine (5-HT), can both interfere with ovulation and ovarian development (Brown, 1967; Vaughan, Benson & Norris, 1970). Conversely, manipulation of sex steroid levels alters hypothalamic catecholamine content (Lichtensteiger, Korpela, Langemann & Keller, 1969), and cyclic variations in hypothalamic catecholamines (Lichtensteiger, 1969) and monoamine oxidase activity have been observed during the oestrous cycle (Zolovick, Pearse, Boehlke & Eleftheriou, 1966).
Whether one monoaminergic system alone regulates gonadotrophin secretion or whether secretion is dependent upon a more complex integrative mechanism, as suggested by Lippmann (1968), remains to be determined. In most investigations, one or a combination of several pharmacological agents have been employed to alter brain levels of the various monoamines. However, these agents do not permit the selective manipulation of only one monoaminergic system without affecting others. Recently, intracisternal
R J A Helliwell and L M Williams
The pineal hormone, melatonin, is important in the timing of seasonal reproduction in the sheep. Melatonin of maternal origin readily crosses the placenta; its function in the fetal sheep is, however, unclear. To gain an insight into the role of melatonin in ovine development we have identified specific melatonin receptors throughout gestation using 2-[125I]iodomelatonin and quantitative in vitro autoradiography. Specific binding was found at the earliest time studied at 30 days of gestation, over the developing thyroid (term=145 days). At 31 days of gestation specific labelling was found over the thyroid and pituitary glands, the spinal nerves, nasal cavity and developing bronchi. This binding was diminished by over 50% in the presence of 10−4 m GTPγS (an analogue of guanosine triphosphate) indicating that the 2-[125I]iodomelatonin binding at this early stage of gestation represents a receptor coupled to a regulatory G-protein. By 40 days of gestation specific binding was found over the nasal epithelium, cochlear epithelium, regions of the brain, especially the hind brain and the vestibulocochlear and glossopharyngeal nerves, and both the pars distalis and pars tuberalis of the pituitary. As gestation proceeded, labelling over the pars distalis appeared to become more scattered in nature while that on the pars tuberalis remained consistent. Saturation studies of both the neuronal and pituitary binding sites at 121 days of gestation and in the newborn lamb revealed a single class of high-affinity binding sites with K d values in the picomolar range. Also at 121 days of gestation, binding over the fetal pars tuberalis was diminished in a dose-dependent manner by GTPγS, again confirming that specific binding is indicative of a receptor coupled to a regulatory G-protein. These data demonstrate a potential for sensitivity to melatonin from early in gestation, as well as the developmentally specific expression of the melatonin receptor in certain tissues, and suggest a wider role for melatonin in ovine fetal development than previously considered.
Journal of Endocrinology (1994) 142, 475–484
In larger mammals, thyroid hormone-binding plasma proteins are albumin, transthyretin (TTR) and thyroxine (T4)-binding globulin. They differ characteristically in affinities and release rates for T4 and triiodothyronine (T3). Together, they form a 'buffering' system counteracting thyroid hormone permeation from aqueous to lipid phases. Evolution led to important differences in the expression pattern of these three proteins in tissues. In adult liver, TTR is only made in eutherians and herbivorous marsupials. During development, it is also made in tadpole and fish liver. More intense TTR synthesis than in liver is found in the choroid plexus of reptilians, birds and mammals, but none in the choroid plexus of amphibians and fish, i.e. species without a neocortex. All brain-made TTR is secreted into the cerebrospinal fluid, where it becomes the major thyroid hormone-binding protein. During ontogeny, the maximum TTR synthesis in the choroid plexus precedes that of the growth rate of the brain and occurs during the period of maximum neuroblast replication. TTR is only one component in a network of factors determining thyroid hormone distribution. This explains why, under laboratory conditions, TTR-knockout mice show no major abnormalities. The ratio of TTR affinity for T4 over affinity for T3 is higher in eutherians than in reptiles and birds. This favors T4 transport from blood to brain providing more substrate for conversion of the biologically less active T4 into the biologically more active T3 by the tissue-specific brain deiodinases. The change in affinity of TTR during evolution involves a shortening and an increase in the hydrophilicity of the N-terminal regions of the TTR subunits. The molecular mechanism for this change is a stepwise shift of the splice site at the intron 1/exon 2 border of the TTR gene. The shift probably results from a sequence of single base mutations. Thus, TTR evolution provides an example for a molecular mechanism of positive Darwinian evolution. The amino acid sequences of fish and amphibian TTRs are very similar to those in mammals, suggesting that substantial TTR evolution occurred before the vertebrate stage. Open reading frames for TTR-like sequences already exist in Caenorhabditis elegans, yeast and Escherichia coli genomes.
CM Reijnders, JG Koster and SC van Buul-Offers
The insulin-like growth factors, IGF-I and IGF-II, and their binding proteins play an important role in the growth and development of the central nervous system. In the brain, colocalization of IGFs and IGFBPs often occurs, suggesting that IGFBPs can modulate IGF action. In one strain of our human (h)IGF-II transgenic mice, which carry an hIGF-II transgene driven by the H-2Kb promoter, we found overexpression of hIGF-II in the brain, as measured by Northern blot analysis. To clarify the localization and influence of the hIGF-II transgene on different components of the GH-IGF axis in the brain, we studied the expression pattern of the hIGF-II transgene, endogenous IGF-I and IGF-II, and IGFBP-2, -3 and -5 in the brain of prepubertal 4-week-old mice, using nonradioactive in situ hybridization. We found that the hIGF-II transgene is exclusively expressed in neurons of the piriform cortex, the cerebral cortex, the medulla oblongata and the granular layer of the cerebellum. In general, this pattern is comparable to the expression pattern of endogenous IGF-I, with a few exceptions: there is no expression of IGF-I in the granular layer of the cerebellum, whereas the Purkinje cells of the cerebellum and thalamus both express IGF-I but no hIGF-II transgene. This hIGF-II transgene expression pattern contrasts markedly with endogenous IGF-II expression, which is mainly located in nonneuronal cells such as the meninges and choroid plexus, and in some nuclei of the medulla oblongata. The hIGF-II transgene affects neither endogenous IGF-I and IGF-II expression, nor the expression of IGFBP-3, which is located in the choroid plexus. Although the hIGF-II transgene is expressed in neuronal structures similar to IGF-I and IGFBP-5, it is not able to regulate IGFBP-5 expression, as has previously been reported for IGF-I. In the medulla oblongata, the IGFBP-2 expression level showed 10-fold upregulation by the transgene, suggesting a modulating role for IGFBP-2 at the hIGF-II transgene action in this region.
LIDIA RUBINSTEIN and K. AHRÉN
The secretion of growth hormone from anterior pituitary transplants under the kidney capsule of gonadectomized and hypophysectomized male rats was investigated with special regard to the importance of the mass of functioning pituitary tissue. Body growth and mammary gland development after testosterone stimulation were studied.
In rats with the pituitary gland autotransplanted to the kidney capsule body growth was markedly reduced. After administration of testosterone a few groups of alveoli only were seen in the mammary glands.
Hypophysectomized rats with four pituitary transplants (an autotransplant and three homotransplants) under the kidney capsule showed slightly better body growth than rats with an autotransplanted hypophysis. When compared with rats with intact pituitary glands body growth was markedly reduced. Mammary gland development after testosterone stimulation was as poor in rats with four pituitary transplants as in rats with an autotransplanted hypophysis.
These results suggest strongly that the normal secretion of growth hormone is regulated by the hypothalamus and that the deficiency of growth hormone in rats with the pituitary gland transplanted remote from the brain is due mainly to a loss of 'specific' stimuli from the hypothalamus and not to a 'non-specific' reduction in the amount of functioning pituitary tissue.