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Neurophysin I in Addition to Oxytocin from the Ovaryin

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Neurophysin I in Addition to Oxytocin from the Ovaryin Evidence for the secretion of immunoreactive neurophysin I in addition to oxytocin from the ovary in cattle D. Schams, E. Schallenberger and J. J. Legros Institut für Physiologie, Süddeutsche Versuchs- und Forschungsanstalt für Milchwirtschaft, Technische Universität München, 8050 Freising- Weihenstephan, Federal Republic of Germany and * Unité de Psychoneuroendocrinologie, Institut de Médecine et Laboratoire de Radioimmunologie, Université de Liège, Sart-Tilman, 4000 Liège, Belgium Summary. In Exp. I, blood samples were collected simultaneously from the posterior vena cava and jugular vein or aorta from 7 heifers every 5\p=n-\20min for 2\p=n-\5h. Concomi- tant pulsatile secretion of oxytocin and immunoreactive neurophysin I was detected in the vena cava, but not in the jugular vein or aorta. Concentrations of oxytocin and immunoreactive neurophysin increased earlier and were higher in the vena cava than in the jugular vein or aorta after the injection of a luteolytic dose of prostaglandin F-2\g=a\ analogue during the mid-luteal phase of the oestrous cycle, demonstrating its ovarian but not pituitary origin. In Exp. II, blood samples were collected from the jugular vein every 12 h during 1 week after oestrus. Follicular growth had been stimulated during the preceding oestrous cycle with PMSG (10 heifers and cows) or with FSH (5 animals); 6 heifers served as controls. There was a high correlation between the number of follicles or CL and the increase in oxytocin and immunoreactive neurophysin I. Although PMSG had a greater luteotrophic effect than did FSH on progesterone secre- tion, a similar stimulation of oxytocin and immunoreactive neurophysin I was not observed. It is concluded that immunoreactive neurophysin I and oxytocin are secreted from the ovary in concentrations dependent upon the number of corpora lutea (and of follicles) present. During the mid-luteal period the secretion occurs in a concomitant pulsatile fashion. Introduction A cycle-dependent release pattern of oxytocin can be measured in the peripheral blood plasma in ewes (Schams, Lahlou-Kassi & Glatzel, 1981 ; Sheldrick & Flint, 1981 ; Webb, Mitchell, Falconer & Robinson, 1981) and in cows (Schams, 1983). Only basal concentrations of oxytocin were measurable after ovariectomy (Schams et ai, 1982). It has been demonstrated that the corpora lutea of sheep and cows (Wathes & Swann, 1982; Wathes, Swann, Birkett, Porter & Pickering, 1983) contain high concentrations of oxytocin which is secreted into the ovarian vein (Flint & Sheldrick, 1982), exhibiting pulsatile fluctuations parallel with progesterone (Walters, Schams & Schallen- berger, 1984; Schallenberger, Schams, Bullermann & Walters, 1984). In the hypothalamo- neurohypophysial system oxytocin and oxytocin-correlated neurophysin I are synthesized together as part of a precursor molecule, which is cleaved before exocytosis of the neurosecretory granules (Richter, 1983). Ovarian neurophysin has been demonstrated in extracts of bovine corpora lutea (Wathes et al, 1983). High peripheral plasma concentrations of immunoreactive neurophysin I/II were measured in ewes before luteolysis (Fairclough et al., 1980). Downloaded from Bioscientifica.com at 09/29/2021 04:30:39AM via free access The aim of the present study was to provide further evidence for ovarian neurophysin and oxytocin secretion which might indicate biosynthesis within a common precursor molecule in the hypothalamo-neurohypophysial system and in the ovary. Materials and Methods Animals and experiments Twenty-three regularly cyclic heifers (18-24 months of age, body weight 450-550 kg) and 5 non- lactating cows (2-4 years old, body weight 550-650 kg) of the local Braunvieh or Fleckvieh breeds were used. All animals were tethered indoors during the experiment. They were fed hay, corn silage and energy supplement and had access to water ad libitum. Blood sampling. All blood samples were placed into heparinized tubes and centrifuged at 4°C ; the plasma was stored at 20°C until hormone analysis. Experiment I: short-term— secretion of oxytocin and immunoreactive neurophysin I. Vinyl medical grade cannulae were inserted during the mid-luteal phase into the posterior vena cava and into the abdominal aorta or jugular vein in 7 heifers 1 day before the start of the experiment. The detailed surgical procedure is given by Walters et al. (1984). Blood samples (10 ml) were collected simulta¬ neously from each vessel every 5-20 min. The release pattern of oxytocin and immunoreative neurophysin I was studied during series of frequent samplings of 2-5 h in 4 animals and after induction ofluteolysis with 500 pg cloprostenol (a PGF-2a analogue ; Estrumate : ICI, England) in 3 heifers. Experiment II: oxytocin and immunoreactive neurophysin I during and after superovulation. An i.m. injection of 1500-3100 i.u. PMSG (Anteron, 1000 i.u./mg: Schering, West Berlin) was given during the mid-luteal phase in 8 heifers and 2 cows. Five heifers were stimulated twice daily for 5 days with an i.m. injection of 5 mg of a crude porcine pituitary FSH preparation (Armour, U.S.A.). Six heifers served as controls. At 2 days after start of the treatments, luteolysis was induced by i.m. injection of 500 pg cloprostenol. Details of similar approaches to induce superovulation in cattle are given elsewhere (Schams et al., 1979b). Symptoms of oestrus were observed about 2 days after the prostaglandin in all experimental animals. Blood was collected every 12 h from jugular catheters from the day of oestrus (= Day 0) until Day 6 of the following cycle. The number of corpora lutea was then counted after exteriorizing the ovaries at laparoscopy. Hormone determinations The concentrations of oxytocin, neurophysin I and progesterone were determined radio- immunologically as described by Schams et al. (1979a), Schams (1983), Legros (1976) and Hoffmann, Kyrein & Ender (1973), respectively. Oxytocin. The antiserum used (final dilution 1/80 000) was raised in a rabbit against synthetic oxytocin coupled to bovine thyroglobulin and showed no cross-reaction with related peptides such as lysine- or arginine-vasopressin or pituitary hormones (e.g. prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone or adrenocorticotrophic hormone). Oxytocin was extracted from blood plasma with SEP-PAK C18 cartridges (recovery was 71-3 ± 3-7%), which also allows concentration of the samples. Total incubation volume of the assay was 0-4 ml. Separation of free and bound hormone was performed by the charcoal method. The reference preparation (synthetic oxytocin, 200 i.u./ml, kindly donated by Sandoz, Basle, Switzerland) had the same potency as the biologically and immunologically well defined 4th International Standard from the National Institute for Biological Standards and Control, London. The lower limit of assay sensitivity was 0-25 pg (0125 pU)/tube. The inter-assay CV of pooled control plasma with low and high oxytocin concentrations varied between 10-8 and 17-3%. The intra-assay CV was 7-3 ± 3T%. Downloaded from Bioscientifica.com at 09/29/2021 04:30:39AM via free access Immunoreactive neurophysin I. The antiserum against bovine oxytocin-neurophysin was raised in a rabbit and used at a final dilution of 1/8000. The cross-reaction with bovine neurophysin II (vasopressin-neurophysin) was <0-5% and with other bovine anterior pituitary hormones (prolactin, GH, LH, FSH and thyreotrophic hormone) or oxytocin and vasopressin was <0T%. Bovine neurophysin I was a gift from Dr P. Cohen, Institut Pierre et Marie Curie, Paris, France, and was used as reference preparation and for labelling. Neurophysin was labelled by the lactoper- oxidase method and purified on a Sephadex G-25 column. The assay was performed using 200 µ unextracted plasma. Separation of free and bound hormone was performed by the double-antibody method. Sensitivity of the assay was 0-5 ng/ml. The intra- and inter-assay CVs were 9% and 15-5%, respectively. Progesterone. The antiserum was raised in a rabbit against lloc-hydroxyprogesterone- hemisuccinate-bovine serum albumin. Endogenous oestrogens, androgens and corticosteroids exhibited no cross-reactivity with the antiserum. A weak cross-reaction with desoxycorticosterone (7-5%) was observed. The specificity was further ensured by an extraction step with light petroleum. The reagent blank was < 10 pg/ml. Average recovery with labelled progesterone added to bovine plasma was 78%. Sensitivity was 20 pg/ml. The mean intra- and inter-assay CVs were 8-5% and 14-8%, respectively. Statistics Differences between groups were tested with Student's / test and Wilcoxon range test. Significances are given only when proved by both methods. Results Experiment I Immunoreactive neurophysin and oxytocin exhibited a pulsatile secretion pattern (Text-fig. 1) in the plasma of the vena cava : the concentrations of neurophysin were higher (P < 0-05) than those in the aorta or jugular vein (Text-fig. lb). After the injection of cloprostenol immunoreactive neurophysin I and oxytocin concentrations increased more (P < 0-005) and appeared first in the vena cava then in the jugular vein (Text-fig. 2). Initial concentrations remained lower in the peri¬ pheral circulation. Experiment II The results of this experiment are summarized in Table 1. The animals receiving PMSG stimu¬ lation were classified according to the number of corpora lutea (Group III = 1-6 and Group IV = > 6) which was not dependent on the PMSG dose used. In control animals (Group I) bearing one corpus luteum, progesterone, oxytocin and immunoreactive neurophysin I concentrations increased throughout Days 4-6 of the cycle. After FSH stimulation (Group II) oxytocin, immunoreactive neurophysin and progesterone concentrations were significantly higher, even during oestrus, than in Group I animals. The oxytocin values were the highest recorded in the study. Progesterone concentrations of Group III animals were significantly different from those of Group I after Day 0 but not from those of Group II, although the number of corpora lutea was much lower than in Group II (14 compared with 3-6), thus indicating an additional luteotrophic potency of PMSG. This stimulating effect was not seen for oxytocin and immunoreactive neurophysin I concentrations.
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