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The level of gonadotrophin measured by radioimmunoassay in fully mature male goldfish was about 15 ng carp gonadotrophic hormone (c-HG)/ml plasma and 30 μg c-HG/g pituitary (wet weight). After treatment for 35 days with methallibure (added to the water at the rate of 2 p.p.m./day), the level of gonadotrophin in the blood was very low and remained at the pretreatment level in the pituitary. This suggested that releasing factor might control either synthesis or discharge of gonadotrophin from the pituitary. These results have also shown that type B spermatogonia and meiosis were gonadotrophin dependent but that the development of spermatocytes into spermatids did not require high concentrations of gonadotrophin. Spermiation was suppressed when the concentration of c-HG dropped below 10 ng/ml plasma.

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Laboratoire de Physiologie des Poissons and *Laboratoire de Physiologie de la Lactation, I.N.R.A., 78350 Jouy en Josas, France

(Received 11 October 1976)

Fish prolactin bioassays lack specificity (Sage & Bern, 1972), have poor sensitivity, are difficult to use (Ensor & Ball, 1968; Clarke, 1973) and contradictory conclusions have been drawn with them. Nicoll & Bern (1968) obtained no lactogenic effect of teleost pituitary gland in the pigeon-crop and the rabbit mammary gland tests, but Chadwick (1966) demonstrated the occurrence of a mammotrophic effect. Other workers have used heterologous radioimmunoassay (RIA) systems (McKeown & Van Overbeeke, 1972); these RIA systems do not seem to cross-react specifically with fish prolactin (Nicoll, 1975). We present a new approach using rabbit mammary gland prolactin receptors.

The technique used was a modification of the radioreceptor assay (RRA) for lactogenic hormones (Shiu, Kelly & Friesen, 1973). At the beginning of lactation the rabbit was injected with

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J Chyb, T Mikolajczyk and B Breton

In order to determine the factors of ovarian origin which can modulate the postovulatory secretion of the FSH-like gonadotropin (GtH I) and the LH-like gonadotropin (GtH II), freshly ovulated female rainbow trout were divided into two groups. In the first group the fish were stripped in order to eliminate the eggs and ovarian fluid from the body cavity, while in the second group the eggs were kept in the body cavity. Subsequently, fish from both groups were implanted with testosterone (10 mg/kg), 17beta-estradiol (10 mg/kg) or 17,20beta-ddihydroxy-4-regnen-3-one (17,20betaP) (1 mg/kg) or injected every 2 days with desteroidized ovarian fluid (1.5 ml/kg). The secretion of GtH I dramatically increased in stripped fish, reaching its maximum levels 2 weeks after ovulation. The preservation of eggs in the body cavity led to the suppression of this increase. The profiles of GtH II secretion were opposite to those encountered for GtH I because the increase of GtH II was observed only in unstripped fish. The administration of steroids showed that testosterone is able to inhibit GtH I release and stimulate that of GtH II in stripped fish, having no effect on the release of these gonadotropins in non-stripped animals. 17beta-Estradiol failed to modify GtH I secretion, however it decreased the release of GtH II in fish containing retained eggs in the body cavity. 17,20betaP had a delayed stimulating influence on GtH I release in unstripped fish. Finally, multiple injections of desteroidized ovarian fluid into stripped fish led to a significant decrease of GtH I release and to an increase of GtH II secretion. This study demonstrates that factors, which are present in ovarian fluid, modulate the post-ovulatory secretion of both gonadotropins--their net action is negative on GtH I and positive on GtH II. Among the steroids, testosterone is of major importance, being able to inhibit GtH I release and to stimulate that of GtH II. We also show that non-steroidal factors present in the ovarian fluid can influence the release of both gonadotropins, which indirectly supports the previous findings about the existence of inhibin/activin-like factors in fish.

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The lactogenic properties of extracts of the pituitary glands of salmon and trout were evaluated by using the organ culture technique with rabbit mammary explants. Crude extracts and fractions obtained after chromatography on Ultrogel and selected for their capacity to compete with ovine prolactin in a rabbit mammary gland radioreceptor assay were added to the culture medium. The criteria of lactogenesis were lactose synthetase activity, casein synthesis, measurements of the concentration of casein messenger RNA and the histology of mammary glands. All these tests led to the conclusion that salmon and trout pituitary glands contain a prolactin-like principle capable of initiating milk synthesis in the rabbit mammary cell.

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A Levoye, B Mouillac, G Rivière, D Vieau, M Salzet and C Breton

In annelids, it has been established that arginine-vasopressin (AVP)/oxytocin (OT) superfamily peptides are involved in the maintenance of water and electrolyte homeostasis as well as reproduction. At present, there is little information on their receptors. In this study, we report the characterization of a 1.7 kb cDNA for an AVP-related receptor from the leech Theromyzon tessulatum. The open reading frame encodes a 435-aminoacid transmembrane protein that displays seven segments of hydrophobic amino acids, typical of G-protein-coupled receptors. The overall predicted protein exhibits about 30% amino-acid identities to other invertebrate, as well as vertebrate, AVP/OT receptor family members, and displays conserved characteristic features belonging to the AVP/OT receptor superfamily. RT-PCR expression experiments showed that mRNA is expressed in the genital tract, the ovary and the brain. The receptor expression is stage specific, showing a weak expression after the two first blood meals, increasing dramatically after the last blood meal during the period of sexual maturation and disappearing after egg laying. Thus, the leech AVP-related receptor may mediate reproductive functions. When expressed in COS-7 cells, the receptor binds ligands with the following rank order of potency: AVP= Arg-vasotocin >Arg-conopressin >mesotocin = OT = Lys-conopressin=isotocin>annetocin. This shows an AVP-like pharmacological profile. The transfected receptor mediates AVP-induced accumulation of inositol phosphates, indicating that the leech AVP-related receptor is functional. This study describes the characterization of a novel AVP/OT superfamily receptor in annelids, which are considered the most distant group of coelomate metazoans possessing a functional AVP/OT-related endocrine system.