Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species

Peptides, Vol. 9, pp. 927-932. ©Pergamon Press plc, 1988. Printed in the U.S.A.

0196-9781/88 $3.00 + .00

Identification of Neurohypophysial Peptides in the Ovaries of Several Mammalian and Nonmammalian Species WAYNE

B. W A T K I N S A N D V E R N O N J. C H O Y

Postgraduate School o f Obstetrics and Gynaecology, National Women's Hospital Claude Road, Auckland, N e w Zealand R e c e i v e d 7 J a n u a r y 1988 WATKINS, W. B. AND V. J. CHOY. Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species. PEPTIDES 9(5) 927-932, 1988.--Ovarian tissue from a variety of mammalian and nonmammalian species were extracted in acid. All extracts contained both oxytocin- and vasopressin-like immunoreactivities as determined by radioimmunoassay. Analysis by high performance liquid chromatography revealed the presence of oxytocin in all ovarian extracts examined. This was in contrast to the corresponding posterior pituitary gland which other workers have shown do not necessarily contain the oxytocin peptide. It is suggested that oxytocin may play an important role in ovarian function in species of differing phylogeny. Ovary

Neurohypophysis

Oxytocin

Vasopressin

PITUITARY oxytocin has long been recognized for its activity on smooth muscle contractility in the control of lactation and labour. More recently it has been shown that at the end of the luteal phase in the cycling ewe oxytocin and the oxytocin-associated neurophysin (24) are released into the periphery in pulsatile episodes coinciding with prostaglandin F2a (PGF2a) (9). This release of oxytocin is from the ovary (40). Moreover, administration of Estrumate, a stimulus for PGF2a release, causes the coordinate release of oxytocin and neurophysin into the vein draining an ovary with a corpus luteum (CL) (10,40). Confirmation that authentic oxytocin originates from the CL has been by sequence analysis (39) and mass spectrometry examination (11) of the peptide fraction from sheep CL extracts. In addition, biosynthesis pulse-label experiments indicate that 35[S]-cysteine is incorporated into oxytocin after incubation with ovine and bovine luteal cells (34). Oxytocin has been detected by radioimmunoassay and high performance liquid chromatography (HPLC) in the ovaries of cows (31,37), women (38), sows (27), monkeys (19), goats (6) and rabbits (22). Another neurohypophysial peptide, vasopressin (VP), has been detected in cattle (31,37), human (38) and pig (27) ovaries. There is also evidence for the presence of these two peptides in the oviduct and follicular fluid of women (26,30). In light of the above evidence that neurohypophysial peptides are significant products of the mammalian ovary, and are probably important in reproductive processes, it was considered appropriate to extend these earlier findings and to further examine the nature of neurohypophysial peptides in the ovarian tissue of a number of mammalian and nonmammalian species.

HPLC

Mammalian

Nonmammalian

METHOD

Tissue Collection and Peptide Extraction Ovarian or the equivalent tissue from representatives of the following species were collected and stored at -20°C until extraction: New Zealand green-lipped mussel (Perna canaliculus); New Zealand orange roughy (Hoplostethis atlanticus: trachyichthyidae); toad (Bufo buff)); 14-month-old laying chicken (288 Shaver Whites) (Gallus domesticus); and domestic pig (Sus scrofa). In the case of the pigs the CL were collected immediately after slaughter. Toad ovaries were collected and processed whole. The female orange roughy, in a state of advanced vitellogenesis, were dredged off the Kaikoura Coast of New Zealand in May 1986. An extract of green-lipped mussels gonads were obtained from McFarlane Laboratories (Auckland, New Zealand) and represented a sample from a pool of 500 mussels. All tissues were thawed and homogenised in 2 M acetic acid (1 g tissue/20 ml acid) and then extracted at room temperature for 4 hr. The supernatant was collected after centrifugation (20,000×g, 15 min) and was lyophilised. Prior to HPLC analysis, the lyophilised extracts were reconstituted in 0.1% trifluoroacetic acid (TFA) at a concentration of 5 mg/0.25 ml, centrifuged for l0 min at 3000xg, and filtered (0.45/xm filter, Millipore, Bedford, MA).

HPLC Fractionation HPLC fractionation of the tissue extracts was carried out on a Waters Liquid chromatograph (Waters Associate Inc., Milford, MA) comprising a Model 720 systems controller, a Series 441 absorbance detector, and Models M45 and 6000A

927

928

W A T K I N S A N D CHOY

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Fraction No.

FIG. 1. HPLC elution profile of pig CL extracts when chromatographed on a Radial-Pak column with acetonitrile in 0.1% TFA (solvent B) at a flowrate of 1 ml/min. After injection of the sample, the column was eluted for 10 min with 0.1% TFA (solvent A), then with two linear gradients of solvent B; initially 0-25% at 2.5% min, then 25-35% at 0.33% min. Dotted line, % solvent B; solid line, UV absorbance at 280 nm; closed circles, OXT-IR; open circles, AVP-IR; solid bars, position of AVT, LVP, AVP and OXT as determined by separate runs.

solvent delivery systems. This system was fitted with a Radial-Pak liquid chromatography cartridge (Waters, 8NVC18 5 p,) and eluted with 0.1% T F A as solvent A, and 60% acetonitrile in 0.1% T F A as solvent B. All solvents were degassed and filtered (0.45 /~m, Millipore) before use. All fractionations were at ambient temperatures. After application of the sample, the system was eluted for 10 min with 100% solvent A at a flow rate of 1 mi/min, followed by a gradient of 0-25% solvent B over the next 10 min, and then a gradient of 25-35% solvent B over 30 min. The eluant was monitored by UV detection at 280 nm. Fractions were collected at 0.33 min intervals and freeze-dried for later radioimmunoassay. The elution positions of the following standard peptides were determined on separate HPLC runs: oxytocin (OXY), arginine VP (AVP), lysine VP (LVP), mesotocin (MES), arginine vasotocin (AVT) (Bachem Inc., Torrance, CA); The standards were run at least 5 times at various times prior to and after chromatography of the ovarian extracts. In addition, MES was included as an internal standard added to each extract. The positions of the neuropeptides in the extracts were compared to that of MES in all runs.

Radioimmunoassays The AVP radioimmunoassay procedure has been described in detail elsewhere (5,28). AVP was radioiodinated with 125[I]-iodide using the chloramine-T method. Synthetic AVP was dissolved in 0.01 M acetic acid at a concentration of 5/zg/5/zl. The following reagents were added sequentially: 25/zl 0.5 M phosphate buffer, pH 7.5; 0.5 mCi 125[I]-iodide in 5 /zl (New England Nuclear, Boston, MA); 20 /.d chloramine-T (17 mg/5 ml 0.5 M phosphate buffer, pH 7.5), with 30 sec reaction time; 200/zl 10% (w/v) bovine serum albumin (BSA) (Sigma, St. Louis, MO) in 0.5 M phosphate buffer, pH 7.5. Labelled AVP was separated from excess 125[I]-iodide and iodinated BSA by gel filtration on a 0.8× 15 cm column of Sephadex G25 (Pharmacia, Uppsala, Sweden) eluted with tris/BSA buffer p H 7.4 at a flow rate of 10 ml/hr. The fraction containing the greatest AVP-immunoreactivity (AVP-IR) was collected and stored lyophilised at -20°C. The AVP antiserum (4) had a titre of 1:250,000. The assay range was 0.6--250 pg/tube. Intraassay variation at 20.7

pg/tube was 0.7 pg/tube (SEM, n=8); interassay variation at 21.3 pg/tube was 1.7 pg/tube (SEM, n=12). Crossreactivities of related neurohypophysial peptides were: AVT, 110%; isotocin (ISO), 0.116%; LVP, 3.9%; MES, 0.018%; OXY, 0.001%; 148%. The radioimmunoassay for oxytocin has been described in detail elsewhere (28). This assay has a sensitivity of 1.25 pg/tube, intraassay variation of 3.8%, and interassay variation of 7.4%. Cross-reactivities of related neurohypophysial peptides were: AVT, 0.001%; AVP, 0.005%; LVP, 0.006%; OXY antiserum (RIII5) was kindly supplied by Dr. I. C. A. F. Robinson (National Institute for Medical Research, London, England). Samples from each HPLC run were assayed together to minimise assay variability.

RESULTS AND DISCUSSION The HPLC elution profiles, as determined by UV absorption at 280 nm and OXY- and AVP-radioimmunoassay, for the pig CL extracts, and the toad, mussel, orange roughy, and chicken ovarian extracts are shown in Figs. 1-3. In all species studied, the HPLC elution profile as determined by the OXY radioimmunoassay had a peak of OXYimmunoreactivity (OXY-IR) that corresponded to the position of standard OXY. In each species except the fish, a peak of AVP-IR corresponded to that of standard AVP. Instead of AVP, orange roughy extracts had a peak of AVP-IR that ran in the same position as AVT standard (Fig. 2c). In contrast to the other species, the profiles (Fig. 2a,b) for the chicken had two peaks of AVP-IR that ran in the same positions as standard AVT and standard AVP, respectively. The concentration of OXY- and AVP-material was similar in some species (Table I). However, there was more OXY than AVP in the mussel and orange roughy. Comparison of ovarian and pituitary peptides for each species shows that these glands do not produce the same peptides (Table 2). The pig neurohypophysis contains OXY and LVP (12), whereas the pig CL contains an AVP-like peptide; this has been reported previously (27). The European wild boar and warthog pituitary contains both AVP and LVP (12,13). Whether CL extracts from these species contain AVP and/or LVP remains to be determined.

OVARIAN N E U R O H Y P O P H Y S I A L PEPTIDES

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FIG. 2. (a) HPLC elution profile of chicken ovary extract. For details see Fig. 1. (b) HPLC elution profile of chicken postovulatory follicle extract. For details see Fig. 1. (c) HPLC elution profile of orange roughy ovary extract. For details see Fig. 1. We have previously reported that the chicken neurohypophysis contains AVT as well as significant amounts of AVP in addition to MES (5). The data in the present report indicate for the first time that chicken endocrine tissue is capable of elaborating OXY. Both OXY and AVT induce premature oviposition when the peptides are given intravenously (29). Furthermore, when homogenates of postovulatory follicles are injected into chickens, premature oviposition occurs (36). The presence of OXY in the ovary (Fig. 2a) and postovulatory follicles (Fig. 2b) is evidence for the active role of this peptide in this process. Oviposition in the chicken is not inhibited after neural lobe hypophysectomy (25,32). One possible conclusion from these data is that OXY of ovarian origin is important for the process of egg-laying. The neural lobe of the pituitary of bony and cartilaginous

fish contains either isotocin, glumitocin, valitocin, apargtocin and/or AVT (1). While the neurohypophysial hormones have hemodynamic and electrolytic effects in fish under experimental conditions, their physiological role is less defined (20). Extracts offish neural lobes and OXY induce spawning in several species (20, 35, 41) and it has been suggested that the male mating behaviour causes release of neurohypophysial hormones in the female (7,8). The presence of OXY- and AVT-like peptides in the fish ovary is consistent with a role for peptides of ovarian origin in spawning. Serotonin has been localised by fluorescence and immunohistochemical methods in the mussel and scallop but little attention has been directed towards investigating the presence of neuropeptides in bivalves (21,33). Electron dense granules, which may be involved in insulin storage, have been identified at the ultrastructural level in the mussel

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FIG. 3. (a) HPLC elution profile of mussel gonad extract. For details see Fig. 1. (b) HPLC elution profile of toad ovary extract. For details see Fig. 1. TABLE 1 OXYTOCINANDARGININEVASOPRESSININ OVARIANTISSUEOF VARIOUS SPECIES Tissue Pig corpus luteum Chicken ovary Chicken postovulatory follicle Orange roughy ovary Mussel gonad Toad ovary

OXT pg/mg/wet weight

AVP pg/mg/wetweight

2.0 - 3.9 (n=4) 0.97-20.9 (n=4) 4.5 -11.4 (n=3)

0.25-0.8 (n=4) 0.05--2.52(n=4) 0.3 -3 (n=2)

0.3 -109 (n=12) 83.6(n=1) 3.7 (n=l)

0 -2.73 (n=12) 3.1 (n=l) 2.1 (n=l)

(14,15). Other peptides described in bivalves include methionine enkephalin and endorphin immunoreactivity in the digestive tract of the mussel (Mytilus edulis L.) (2), and AVT-like material in the central nervous system of the marine mollusc (Aplysia californica) (23). The role of OXY, or indeed any of these neuropeptides, in the normal physiology of the mussel is not known, although given the possible reproductive function in other species, OXY, like serotonin, may be important in controlling spawning (16). The role of OXY in the toad ovary is not known. In amphibians the neurohypophysial hormones have an effect on water and electrolyte balance (3). Contractility of the oviduct has also been observed with neurohypophysial peptides (17,18). The observation of significant concentrations of both

TABLE 2 POSTERIOR PITUITARYAND OVARIANPEPTIDE HORMONES Species Pig Chicken Orange Roughy Toad

Pituitary*

Ovary

LVP,OXT AVT,AVP,MES AVT,ISO AVT,MES

AVP,OXT AVT,AVP,OXT AVT,OXT AVP,OXT

Key: AVP, arginine vasopressin; AVT, arginine vasopressin; LVP, lysine vasopressin; OXT, oxytocin; MES, mesotocin; ISO, isotocin. *See (1, 5, 12).

OXY- and AVP-like material in the reproductive tissues of a wide variety of species, vertebrate and invertebrate, mammalian and nonmammalian and avian is indicative of a universal role for these peptide hormones in reproductive physiology. The wide variation in the hormone content in ovarian tissue from the same species may be representative of the reproductive state of the animal at the time of collection. Further work to ascertain their precise role, particularly their interaction with steroid hormones will be required to address some of the questions raised by the results of the present investigation. Furthermore, the conclusions are based solely of the specificity of the two radioimmunoassays employed. In this present study we are unable to comment upon the presence or absence of some other neurohypophysial peptides, e.g., ISO and MES.

OVARIAN NEUROHYPOPHYSIAL

PEPTIDES

931 ACKNOWLEDGEMENTS

This work was financed by the Medical Research Council of New Zealand. We thank Dr. I. C. A. F. Robinson for anti-oxytocin serum.

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19. Khan-Dawood, F. S.; Marut, E. L.; Dawood, M. Y. Oxytocin in the corpus luteum of the Cynomologus monkey (Macaca fuscicularis). Endocrinology 115:570-574; 1984. 20. Maetz, J.; Lahlou, B. Actions ofneurohypophysial hormones in fishes. In: Greep, R. O.; Astwood, E. B., eds. Handbook of physiology, vol. 1. section 7. Washington, DC: American Physiological Society; 1974:521-544. 21. Matsutani, T.; Nomura, T. Serotonin-like immunoreactivity in the central nervous system and gonad of the scallop, Patinopecten yessoensis. Cell Tissue Res. 244:515-517; 1986. 22. Miller, J. B.; Khan-Dawood, F. S.; Dawood, M. Y. Identification of oxytocin in rabbit ovarian tissue. Abstracts 65th Annual Meeting Endocrine Society, No. 246; 1983. 23. Moore, G. J.; Thornhill, J. A.; Gill, V.; Lederis, K.; Lukowiak, K. An arginine vasotocin-like neuropeptide is present in the nervous system of the marine mollusc, Aplysia califi)rnica. Brain Res. 206:213-218; 1981. 24. Moore, L. G.; Watkins, W. B. Development of radioimmunoassays of ovine neurophysins. Correlation of neurophysin release with oxytocin- and vasopressin-related stimuli. Endocrinology 112:113-120; 1983. 25. Opel, H. Oviposition in chickens after removal of the posterior lobe of the pituitary by an improved method. Endocrinology 76:673-677; 1965. 26. Peek, J. C.; Choy, V. J.; Watkins, W. B.; Graham, F. M. Levels of oxytocin-like activity and progesterone in follicular fluid from in vitro fertilization cycles. J. in vitro fert. embryo transfer 4:103-106; 1987. 27. Pitzel, L.; Welp, K.; Holtz, W.; Konig, A. Neurohypophyseal hormones in the corpus luteum of the pig. Neuroendocrinol. Lett. 6:1-5; 1984. 28. Robinson, I. C. A. F. The development and evaluation of a sensitive and specific radioimmunoassay for oxytocin in unextracted plasma. J. Immunoassay 1:323-347; 1980. 29. Rzasa, J.; Ewy, Z. Effect of vasotocin and oxytocin on oviposition in the hen. J. Reprod. Fertil. 21:54%550; 1970. 30. Schaeffer, J. M.; Liu, J.; Hsueh, A. J. W.; Yen, S. S. C. Presence of oxytocin and vasopressin in human ovary, oviduct and follicular fluid. J. Clin. Endocrinol. Metab. 59:970-973; 1984. 31. Sequeira, R. P.; Chaiken, I. M. Molecular characterization of oxytocin, vasopressin and neurophysins in the ovary. Fed. Proc. 43:913; 1984. 32. Shirley, H. V.; Nalbandov, A. V. Effects of neurohypophysectomy in domestic chickens. Endocrinology 58:477-483; 1956. 33. Stefano, G. B.; Aiello, E. Histofluorescent localization of serotonin and dopamine in the nervous system and gill of Mytilus edulis L. (Bivalvia). Biol. Bull. 148:156; 1975. 34. Swann, R. W.; O'Shaughnessy, R. J.; Birket, S. D.; Wathes, D. C.; Porter, D. G.; Picketing, B. T. Biosynthesis of oxytocin in the corpus luteum. FEBS Lett. 174:262-266; 1984. 35. Swift, D. R.; Pickford, G. E. Seasonal variations in the hormone content of the pituitary gland of the perch (Perca fluviatilis L.). Gen. Comp. Endocrinol. 5:354-365; 1965. 36. Tanaka, K.; Nakada, T. Participation of the ovarian follicle in control of time of oviposition in the domestic fowl. Poultry Sci. 53:2120-2125; 1974. 37. Wathes, D. C.; Swann, R. W.; Picketing, B. T. Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy. J. Reprod. Fertil. 71:551-557; 1984.

932 38. Wathes, D. C.; Swann, R. W.; Pickering, B. T.; Porter, D. G.; Hull, M. G. R.; Driffe, J. O. Neurohypophysial hormones in the human ovary. Lancet II:410-412; 1982. 39. Watkins, W. B.; Choy, V. J.; Chaiken, J. M.; Spiess, J. Isolation and sequence analysis of oxytocin from the sheep corpus luteum. Neuropeptides 7:87-95; 1986.

W A T K I N S A N D CHOY 40. Watkins, W. B.; Moore, L. G.; Flint, A. P. F.; Sheldrick, E. L. Secretion of neurophysins by the ovary in sheep. Peptides 5:61-64; 1984. 41. Wilhelmi, A. E.; Pickford, G. E.; Sawyer, W. H. Initiation of spawning reflex response in Fundulus by the administration of fish and mammalian neurohypophysial preparations and synthetic oxytocin. Endocrinology 57:243-252; 1955.