Fish Physiologyand Biochemistryvol. 6 no. 4 pp 213-219 (1989) Kugler Publications, Amsterdam/Berkeley
A u t o n o m i c innervation of the ovary of the Atlantic cod, Gadus morhua
Kazumasa Uematsu*, Susanne Holmgren and Stefan Nilsson
Comparative Neuroscience Unit, Department of Zoophysiology, University of GOteborg, Box 250 59, S-400 31 GOteborg, Sweden Keywords: autonomic nerves, ovary, teleost fish, cholinergic, adrenergic
Abstract
The autonomic innervation of the ovary of the Atlantic cod was investigated using histochemical and physiological/pharmacological methods. The paired ovary receives autonomic innervation via branches of the posterior splanchnic nerve (vesicular nerve). Histochemical studies demonstrated vasoactive intestinal polypeptide (VIP)-immunoreactive, 5-hydroxytryptamine-immunoreactive and adrenergic nerve fibers, but a number of antisera raised against other peptides failed to reveal any specific reaction in the tissue preparations. It is concluded that the cod ovary receives a double antagonistic autonomic innervation of excitatory cholinergic fibers and non-adrenergic inhibitory fibers. The nature of the inhibitory neurotransmitter is not known.
Introduction
The autonomic innervation of the ovary in teleosts possessing a hollow, muscular ovary has been studied in a number of species (Uransocopus scaber, Lophius piscatorius: Young 1931, 1936; Gadus morhua: Nilsson 1970; Sarotherodon nUoticus, Limanda yokohamae: Uematsu 1985, 1986). The paired ovary is innervated by branches of the posterior splanchnic nerve ('vesicular nerve'), which leaves the sympathetic chains in the posterior trunk region, and runs along the ureter to the urinary bladder and gonads (Young 1931; Nilsson 1970; Uematsu 1985, 1986). Ganglion cells are present in the vesicular nerve, and in the Atlantic
cod these form a distinct ganglion at the level of the exit of the nerve from the wall of the kidney (Nilsson 1970). Fluorescence histochemistry for catecholamines shows small fluorescent (presumably adrenergic) nerve, cell bodies in the sympathetic chain ganglia at the level of the 20th spinal nerve. Larger, non-fluorescent (non-adrenergic) nerve cell bodies are abundant in the vesicular nerve ganglion (Nilsson 1976). Preliminary pharmacological studies, including nerve stimulation experiments, suggest the presence of a cholinergic excitatory control of the ovarian wall by autonomic nerve fibers running the vesicular nerve (Nilsson 1970; Uematsu 1985). Both aand ~-adrenoceptors have been demonstrated in the
Correspondence to: Dr S. Nilsson, Comparative Neuroscience Unit, Department of Zoophysiology, University of Goteborg, Box 250 59, S-400 31 G6teborg, Sweden. *Presentaddress:Faculty of Applied BiologicalScience, Hiroshima University,Saijo-cho, Higachi-Hiroshima, Hiroshima 724, Japan.
214 ovarian musculature, but there is no evidence for an autonomic innervation beside the cholinergic excitatory control that has been suggested (Nilsson 1970; Uematsu 1985). This study was started to elucidate the nature of the autonomic innervation of the teleostean ovary, using both histochemical and physiological/pharmacological techniques.
Materials and methods
Atlantic cod, Gadus morl~ua, (300-1100 g body mass) were catlght off the Swedish west coast and kept in large tanks with recirculating sea water at IO~
Immunohistochemis[ry Whole mount preparations were made as described by Costa et al. (1980). The ovary was dissected out 9and placed in cold cod Ringer's solution (Holmgren and Nilsson 1974) until used. The ovarian wall was cut open, pinned flat on a piece of dental wax and fixed for 18-20h floating in a solution of 207o formaldehyde and 15% picric acid in 0.1 M phosphate buffer (pH = 7.3). The tissue was repeatedly rinsed in 8007oethanol, dehydrated, treated with xylene for 30 min and rehydrated. After the adherent eggs had been removed as far as possible, 4 x 6 mm pieces of the muscular wall was incubated with the primary antibodies in a moist chamber for 18 h at room temperature. The preparations were repeatedly rinsed in phosphate-buffered saline (PBS, pH = 7.3) for 30 min. and subsequently incubated for lh with a secondary antibody conjugated with FITC (Swine anti-rabbit IgG, DAKO immunoglobulins). After repeated rinsing to remove excess secondary antibody, the preparations were mounted on glass slides in carbonate-buffered glycerol (l:l, pH = 8.5), and viewed in a Leitz Dialux microscope equipped for epi-fluorescence. Photographs were taken on Kodak Tri-X film using a Leitz Vario-Orthomat automatic camera. Sections (10 tzm) for microscopical investigation were produced from pieces of the ovarian wall and
the vesicular nerve fixed in parabenzoquinone in 0. IM sodium cacodylate buffer (pH = 7.2) at 4~ for 2 - 3 h (Bishop et al. 1978), rinsed in the buffer containing 20~ sucrose for 24-48 h at 4~ and snap-frozen in liquid nitrogen for cryostat sectioning. Incubations were made with primary andsecondary antisera according to the method of Coons (1956), which essentially follows the procedure described for whole mount preparations. Control experiments were carried out by replacing the specific antiserum with antiserum preincubated for 48 h at 4~ with the appropriate hapten. The staining with the antiserum was in all cases completely abolished by this preincubation procedure. Identification of immunoreactive material was made with VIP (vasoactive intestinal polypeptide) antiserum L85 (Dockray) diluted 1:250 or 1:400, 5-HT (5-hydroxytryptamine, serotonin) antiserum 71934 (Immuno Nuclear Corp) diluted 1:50, and DBH (dopamine-/3-hydroxylase) antiserum RaDBH (Rush) diluted l:100. No immunoreactivity could be detected with antisera raised against bombesin (L89, Dockray), met-enkephalin (INC 451, Immuno Nuclear Corp and L146, Dockray), gastrin/cholecystokinin (CCK) (L112, Dockray), neurotensin (NtAb, Dockray), physalaemin (PS XII, Lazarus), somatostatin (1001, Yamada), substance P (G10, original, and CRB-SP, Cambridge Research Biochemical Ltd), neuropeptide Y (AB22, Peninsula), FMRF-amide (L155, Dockray) or LPLRF (L197, Dockray). Of these antisera, L89, INC 451, L146, L l l 2 , NtAB, G10, CRB-SP, AB22 and L155 have previously been shown to produce specific reactions in fish tissues studied at this laboratory.
Falck-Hillarp histochemistry Falck-Hillarp histochemistry for monoamines was performed essentially as described by Falck and Owman (1965). Pieces of the ovarian wall and the ureter were snap-frozen in liquid propane cooled in liquid nitrogen and freeze-dried for 3 days. After treatment with formaldehyde vapor at 80~ for lh and paraffin embedding, sections (7 #m) were cut
215 and viewed in the Leitz microscope as mentioned, with the barrier filter at 460 nm.
porcine vasoactive intestinal polypeptide (VIP) (a kind gift from Prof. V Mutt, Stockholm). Concentrations are expressed in tool.1 -I (M).
Isolated strip preparations Statistical evaluation The ovarian wall was cut open and the eggs were removed carefully. Strips, 1-2 mm wide and 10-15 mm long, were cut along the longitudinal axis, and mounted in organ baths in 5 ml cod Ringer's solution (Holmgren and Nilsson 1974) at 10~ bubbled with O2/CO 2 (97/3~ The strips were attached to GRASS FT03 isometric transducers for recording of isometric tension changes on a GRASS Polygraph mod 7. Cumulative concentration-response curves were produced as described previously (e.g., Holmgren and Nilsson 1974), and the affinity (PD2) and relative intrinsic activity (c~) determined for agonists. The antagonistic properties of atropine were estimated by using a Schild plot (Schild 1947) to determine PAx-values. To estimate effects of deterioration of the preparations between consecutive concentration-response curves, a second and third control curve was produced without addition of the antagonist in parallel with the antagonist treated preparations. Changes in a-value have been corrected for the change in maximal response thus determined. Transmural electrical stimulation of intrinsic nerves was made transmurally via a pair of stainless steel electrodes delivering rectangular pulses of 1 ms duration, 20-30V at 10 Hz for 8 sec every 2 min from a GRASS $6 stimulator.
Drugs The following drugs were used in this study: acetylcholine chloride (Sigma), L-adrenaline bitartrate (Sigma), atropine sulphate (Sigma), L-isoprenaline bitartrate (Sigma), 5-hydroxytryptamine creatinine sulfate (Sigma), methysergide (l-methyi-lysergic acid butanolamine bimaleate), L-noradrenaline bitartrate (Sigma), phentolamine methane suiphonate (a kind gift from CIBA-Geigy), proprano1ol hydrochloride (Sigma), tetrodotoxin (Sigma),
Differences in PD2-values between agonists were tested by Student's t-test. Differences of p < 0.05 were regarded as statistically significant.
Results
General disposition of the cod ovary The cod ovary consists of two lobes which fuse posteriorly and taper off in a short oviduct opening at the tip of the urino-genital papilla. One or two branches from the vesicular nerve enter the vertical septum between the two ovarian lobes. These branches run from the single posterior splanchnic (vesicular) nerve, which arises from the sympathetic chain ganglia at the level of the 20th or 21st pair of spinal nerves. Ganglion cell masses are found along the nerve, especially at the point .of exit from the kidney (cf. Nilsson 1976).
lmmunohistochemistry lmmunoreactive (IR) nerve fibers in the ovarian wall were detected with antisera raised against VIP; 5-HT and DBH. A dense plexus of VIP-IR fibers was present in the smooth muscle of the ovarian wall, while 5-HT- and DBH-IR fibers occurred as single strands only (Fig. I A - D ) . Antisera raised against bombesin, met-enkephalin, gastrin/CCK, neurotensin, physalaemin, somatostatin, substance P, neuropeptide Y, FMRFamide or LPLRF failed to reveal IR material in the ovarian preparations.
Falck-Hillarp histochemistry Single fine nerve fibers emitting the blue-green
216
Fig. I. lmmunohistochemical (A-D) and Falck-Hillarp fluorescence histochemical (E) demonstration of nerve fibers in the wall of the cod ovary. A. VIP-IR varicose nerve fibers (whole mount). B. Bundle of VIP-IR nerve fibers (section). C. Single varicose 5-HT-IR fibers (whole mount). D. DBH-IR nerve fibers (section). E. plexus of nerve fibers showing the blue-green fluorescence characteristic of catecholamines (section). Calibration bars in all figures = 10 r~m. 10 - s - 10-4M) p r o d u c e d c o n c e n t r a t i o n - d e p e n d e n t c o n t r a c t i o n s o f the p r e p a r a t i o n s (Fig. 2), with a p D 2 o f 5.40 + 0.17 (n = 7) a n d 5.90 _+ 0.28 (n = 6) respectively. T h e r e was no s t a t i s t i c a l l y significant d i f f e r e n c e in the PD2-values. T h e c o n t r a c t i l e effect o f c a r b a c h o l was a n t a g o n i z e d by a t r o p i n e ( I 0 - v - 10-6M, n = 6), which
Fig. 2. Recordings of drug-induced isometric tension changes in isolated longitudinal preparations of the wall of the cod ovary. Tension calibrations in milli-Newton (mN); log molar concentrations shown (lO-S, 3 x 10 -s, 10 -7 . . . or 10-8, 10 7 10 6 . . . M).
fluorescence c h a r a c t e r i s t i c o f a d r e n e r g i c nerve endings were o b s e r v e d in the o v a r i a n wall (Fig. 1E), but no fluorescent fibers c o u l d be seen in the vesicular nerve.
Isolated strip preparations A c e t y l c h o l i n e ( 1 0 - 7 - 3 x 10-4M)
and
carbachol
p r o d u c e d a p a r a l l e l shift o f the c o n c e n t r a t i o n r e s p o n s e curve (Fig. 3). E s t i m a t i o n o f PAx-values shows p A 2 = 8.8 a n d PAl0 = 7.5. C o n t r a r y to what is e x p e c t e d for a c o m p e t i t i v e a n t a g o n i s t , a t r o pine p r o d u c e d a d e c r e a s e in the a p p a r e n t intrinsic activity ( a = 0.64 + 0. I0, n = 6 a n d a = 0.51 + 0.12, n = 6 ( c o r r e c t e d a g a i n s t c o n t r o l curves) at a t r o p i n e c o n c e n t r a t i o n o f 10-TM a n d 10-6M respectively). 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) likewise h a d a s t i m u l a t o r y effect on the p r e p a r a t i o n s , p r o d u c i n g a c o n c e n t r a t i o n - d e p e n d e n t r e s p o n s e with p D 2 = 7.27 _+ 0.27 (n = 7) (Fig. 2). T h e effect was a n t a gonized by m e t h y s e r g i d e (10-6M, n = 7, Fig. 4). T h e a d r e n e r g i c a g o n i s t s i s o p r e n a l i n e (10 - 1 ~
217 100 -
/o /.
o--o
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y
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Adrenaline Isoprenaline
5o
Z!
Contr Atr 1 Atr 2
--
-8
27
26
--'4
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"
i 100 j
~--s
\ -io
\
\.
L9
\o
L8
-7
-6
LOG MOLAR CONC
-5
-4
LOG MOLAR CONC
Fig. 3. Concentration-response curves for carbachol acting on
Fig. 5. Concentration-response curves for the inhibitory action
isolated strip preparations from the cod ovary, either alone ( 0 ) or in the presence of atropine ( 9 10-7 (Atr I); 9 10 6M (Air 2)).
of isoprenaline ( 9 ) and adrenaline ( o ) on isolated strip preparations from the cod ovary.
'L LLU \LL 8,
100,
/2V" 111
I I
O. A T R 10"r M
10"6M
8
///
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-8
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--6
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9
0. -9
71 LL LL&L L _
25
-'4
1st control 2nd control unblocked + meth
-'3
22
LOG MOLAR CONC
Fig. 4. Concentration-response curves for 5-hydroxytryptamine (5HT) acting on isolated strip preparations from the cod ovary, either alone ( o , control run; 9 second run) or in the presence of methysergide ( , , , control run; , , methysergide (10 6M) run),
10-TM) and adrenaline (10 - s - 10-SM) had an inhibitory effect on the strips, causing a concentration-dependent decrease in tension of the preparations (Fig. 2). Isoprenaline (pD 2 = 9.07 +_ 0.32, n = 7) had a significantly higher affinity for the adrenoceptor than adrenaline (pD 2 = 6.81 _+ 0.18, n = 5) (Fig. 5). Noradrenaline had variable weak effects on the preparations, causing either relaxation, contraction or both. Transmural electrical stimulation (20 Hz, 1 ms, 10V) caused contraction of the preparations (Fig. 6). Atropine (10-6M) reduced or abolished the response, measured as a reduction in the amplitude varying between 0 and 100% (mean 35 +_ 13%,
ATR I0 "6 M
PHENT IO"6 M
mN
TTX
IO"6 M
~
O-
ATR-PHENTIO "6 M
TTXIO "6 M
PROPIO'6M
Fig. 6. Recordings of isometric tension changes produced by transmural electrical stimulation of isolated longitudina] preparations of the wall of the cod ovary. Tension calibrations in milli-Newton (raN). Molar concentralions of the drugs shown. ATR, atropine; P H E N T , phentolamine; TTX, tetrodotoxin; P R O P , propranolol.
n = 9), and atropine (10-SM) caused a blockade between 18 and 100~ (mean 81 _+ 10070, n = 10). The remaining response could always be blocked by the (x-adrenoceptor antagonist phentolamine (10 - 6 - 10-5M). Thus, a combination of cholinoceptor and (x-adrenoceptor blockade produced a total inhibition of the contraction, revealing an inhibitory response to the transmural stimulation. This inhibitory response (as well as the excitatory response to transmural stimulation), was abolished by tetrodotoxin (10-6M, n = 5 in both cases) (Fig. 6). The amplitude of the contraction caused by
218
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o - - o Iso O - - O Adr
~
o
g 50
\ 100
-io
29
28
\ 27
s
\ 25
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Fig. 7. Concentration-response curves for the inhibitory action of isoprenaline ( o ), adrenaline ( 4; ), and noradrenalinr ( 9 ) on isolaled slrip preparalJons from the cod ovary, [ransmurally stimulated with ]0'Hz, I ms and 20V pulses for 8 s every 2 min.
transmural electrical stimulation was reduced in a concentration-dependent w a y by the adrenergic agonists isoprenaline'(pD 2 = 8.22 _+ 0.21, n = 6), adrenaline (pD 2 = 6.60 _+ 0.12, n = 6 ) a n d noradrenaline (pO 2 4.79 +_ 0.05, n = 5) (Fig. 7). The maximal reduction of the amplitude caused by the three drugs was very similar (isoprenaline 65 _+ ll~ n = 6, adrenaline 67 _+ 9%, n = 6, noradrenaline62 _+ 14%, n = 5).
Discussion The general disposition of the cod ovary is very much like that in other species of the 'higher' teleosts, such as Perciformes and Pleuronectiformes. As in the Japanese dab, Limanda yokohamae, the cod ovary is innervated by a branch of the posterior splanchnic nerve ('vesicular nerve'), which runs along the ureter to innervate both the urinary bladder and the ovary. In the cod, the nervous pathways leave the central nervous system at the level of about the 20th pair of spinal nerves, and both adrenergic and non-adrenergic nerve cells have been described in the sympathetic chain ganglia and the vesicular nerve ganglion (Nilsson 1976). As in the case of the teleost urinary bladder, the ovarian wall is known to contract in response to acetylcholine or carbachol, and this effect is antagonized by atropine, thereby demonstrating the
presence of muscarinic cholinoceptors (Nilsson 1970). In the present study, however, the maximum contraction force produced by carbachol was reduced by atropine, and the PA2/PAi0 difference was greater than can normally be expected during competitive antagonism. The results are compatible with the view that the cholinergic agonists affect also other systems, i.e., releasing other transmitter substances from intramural nerves. Electrical stimulation of the intrinsic nerves by transmural electrodes produced contractions of the ovarian wall, similar to what has been previously reported in cod and other teleosts (Nilsson 1970; Uematsu 1985, 1986). The effect is blocked by tetrodotoxin, showing that it is indeed nervous, but contrary to these other studies, the response cannot be totally abolished by atropine. The remaining response, which can be of variable magnitude, is always abolished by the tx-adrenoceptor antagonist phentolamine, suggesting the involvement of an adrenergic excitatory component in the ovarian innervation in the cod. The function of such an adrenergic innervation is not clear: the effects of exogenous noradrenaline are weak and variable, while adrenaline is inhibitory. Furthermore, although adrenergic neurons could be demonstrated both by immunohistochemical localization of DBH and by Falck-Hillarp fluorescence histochemistry, the adrenergic innervation is very sparse and its importance in the control, excitatory or inhibitory, of the ovarian wall is doubtful. In the study by Nilsson (1970) of the autonomic innervation of the cod urinary bladder, an adrenergic inhibitory control was postulated, based on the effect of the fl-adrenoceptor antagonist propranolol on the inhibitory responses to vesicular nerve stimulation in the presence of atropine. Later studies put this conclusion in doubt (Lundin and Holmgren 1986), and suggested a non-adrenergic, non-cholinergic inhibitory component in the urinary bladder innervation. An inhibitory effect o f transmural stimulation of the cod ovary could not be abolished by propranolol, suggesting also in this case a non-adrenergic inhibitory innervation. The nature of this inhibitory transmitter substance is unknown. VIP, which is the only peptide identified with certainty in nerve
219 fibers ovarian wall, has little or no effect either on isolated strip preparations or the transmurally stimulated strips. It is concluded that the cod ovary receives a double antagonistic autonomic innervation of excitatory cholinergic fibers and non-adrenergic inhibitory fibers. The nature of the inhibitory neurotransmitter is unclear. Histochemical studies demonstrated VIP-immunoreactive, 5-HT-immunoreactive and adrenergic nerve fibers, but a number of antisera raised against other peptides failed to reveal any specific reaction in the tissue preparations.
Acknowledgements This study was supported by the Swedish Natural Science Research Council. We wish to thank Mss. Lena Utter and Inger Holmqvist for skilled technical assistance.
References cited Bishop, A.E., Polak, J.M., Bloom, S.R. and Pearse, A.G.E. 1978. A new universal technique for the immunocytochemical localization of peptidergic innervation. J. Endocrinol. 77: 25 - 26P. Coons, A.C. 1956. Histochemistry `'`"ith labelled antibody. Int. Re',. Cytol. 5: 1-23. Costa, M., Buffa, R., Furness, J.B. and Solcia, E, 1980. Immunohistochemical localization of polypeptides in peripheral autonomic nerves using ',,,,hole mount preparations. Histochemistry 65: 157-169. Falck, B. and O w m a n , Ch. 1965. A detailed morphological description of the fluorescence method for the cellular demonstration of biogenic amines. Acta Univ. Lundensis 7: 1-23.
Furchgott, R.F. 1967. The pharmacological differentiation of adrenergic receptors. Ann. N.Y. Acad. Sci. 139: 553-570. Grove, D.J., O'Neill, J.B. and Spiller, P.B. 1974. The action of 5-hydroxytryptamine on longitudinal gastric smooth muscle of the plaice, Pleuronectesplatessa. Comp. Gen. Pharmacol. 5: 229-238. Holmgren, S. 1983. The effects of putative non-adrenergic, noncholinergic a u t o n o m i c transmitters on isolated strips from the stomach of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 74C: 229-238. Holmgren, S. and Nilsson, S. 1974. Drug effects on isolated artery strips from two teleosts, Gadus morhua and Salmo gairdneri. Acta Physiol. Scand. 9 0 : 4 3 1 - 4 3 7 . Holmgren, S., Grove, D.J. and Nilsson, S. 1985. Substance P acts by releasing 5-hydroxytryptamine from enteric neurons in the stomach of the rainbow trout, Sahno gairdneri. Neuroscience 14: 683-693. Jensen, J. and Holmgren, S. 1985. Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua. C o m p . Biochem. Physiol. 82C: 8 1 - 8 9 . Lundin, K. and Holmgren, S. 1986. Non-adrenergic, innervation of the urinary bladder of the Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. 84C: 315-323. Nilsson, S. 1970. Excitatory and inhibitory innervation of the urinary bladder and gonads of a teleost, Gadus morhua. Acta Physiol. Scand. 83: 446-453. Nilsson, S. 1976. Fluorescent histochemistry and cholinesterase staining of sympathetic ganglia in a teleost, Gadus morhtta. Acta ZooI., Stockh. 57: 6 9 - 7 7 . Nilsson, S. and Holmgren, S. 1983. Splanchnic nervous control of the stomach of the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. 76C: 271-276. Uematsu, K. 1985. Effects of drugs on the responses of the ovary to field and nerve stimulation in a tilapia, Sarotherodon niloticus. Bull. Jap. Soc. Sci. Fish. 51: 4 7 - 5 3 . Uematsu, K. 1986. The autonomic innervation of the o`"ary of the dab, Limandayokohamae. Jap. J. lchthyol. 33:293 303. Young, J.Z. 1931. On the autonomic nervous system of the teleostean fish, Uranbscopus scaber. Quart. J. Microsc. Sci. 74: 491-535. Young, J .Z. 1936. The innervation and reaoions to drugs of the viscera of teleostean fish. Proc. Roy. Soc. London, Ser. B 120: 303-318.
A u t o n o m i c innervation of the ovary of the Atlantic cod, Gadus morhua
Kazumasa Uematsu*, Susanne Holmgren and Stefan Nilsson
Comparative Neuroscience Unit, Department of Zoophysiology, University of GOteborg, Box 250 59, S-400 31 GOteborg, Sweden Keywords: autonomic nerves, ovary, teleost fish, cholinergic, adrenergic
Abstract
The autonomic innervation of the ovary of the Atlantic cod was investigated using histochemical and physiological/pharmacological methods. The paired ovary receives autonomic innervation via branches of the posterior splanchnic nerve (vesicular nerve). Histochemical studies demonstrated vasoactive intestinal polypeptide (VIP)-immunoreactive, 5-hydroxytryptamine-immunoreactive and adrenergic nerve fibers, but a number of antisera raised against other peptides failed to reveal any specific reaction in the tissue preparations. It is concluded that the cod ovary receives a double antagonistic autonomic innervation of excitatory cholinergic fibers and non-adrenergic inhibitory fibers. The nature of the inhibitory neurotransmitter is not known.
Introduction
The autonomic innervation of the ovary in teleosts possessing a hollow, muscular ovary has been studied in a number of species (Uransocopus scaber, Lophius piscatorius: Young 1931, 1936; Gadus morhua: Nilsson 1970; Sarotherodon nUoticus, Limanda yokohamae: Uematsu 1985, 1986). The paired ovary is innervated by branches of the posterior splanchnic nerve ('vesicular nerve'), which leaves the sympathetic chains in the posterior trunk region, and runs along the ureter to the urinary bladder and gonads (Young 1931; Nilsson 1970; Uematsu 1985, 1986). Ganglion cells are present in the vesicular nerve, and in the Atlantic
cod these form a distinct ganglion at the level of the exit of the nerve from the wall of the kidney (Nilsson 1970). Fluorescence histochemistry for catecholamines shows small fluorescent (presumably adrenergic) nerve, cell bodies in the sympathetic chain ganglia at the level of the 20th spinal nerve. Larger, non-fluorescent (non-adrenergic) nerve cell bodies are abundant in the vesicular nerve ganglion (Nilsson 1976). Preliminary pharmacological studies, including nerve stimulation experiments, suggest the presence of a cholinergic excitatory control of the ovarian wall by autonomic nerve fibers running the vesicular nerve (Nilsson 1970; Uematsu 1985). Both aand ~-adrenoceptors have been demonstrated in the
Correspondence to: Dr S. Nilsson, Comparative Neuroscience Unit, Department of Zoophysiology, University of Goteborg, Box 250 59, S-400 31 G6teborg, Sweden. *Presentaddress:Faculty of Applied BiologicalScience, Hiroshima University,Saijo-cho, Higachi-Hiroshima, Hiroshima 724, Japan.
214 ovarian musculature, but there is no evidence for an autonomic innervation beside the cholinergic excitatory control that has been suggested (Nilsson 1970; Uematsu 1985). This study was started to elucidate the nature of the autonomic innervation of the teleostean ovary, using both histochemical and physiological/pharmacological techniques.
Materials and methods
Atlantic cod, Gadus morl~ua, (300-1100 g body mass) were catlght off the Swedish west coast and kept in large tanks with recirculating sea water at IO~
Immunohistochemis[ry Whole mount preparations were made as described by Costa et al. (1980). The ovary was dissected out 9and placed in cold cod Ringer's solution (Holmgren and Nilsson 1974) until used. The ovarian wall was cut open, pinned flat on a piece of dental wax and fixed for 18-20h floating in a solution of 207o formaldehyde and 15% picric acid in 0.1 M phosphate buffer (pH = 7.3). The tissue was repeatedly rinsed in 8007oethanol, dehydrated, treated with xylene for 30 min and rehydrated. After the adherent eggs had been removed as far as possible, 4 x 6 mm pieces of the muscular wall was incubated with the primary antibodies in a moist chamber for 18 h at room temperature. The preparations were repeatedly rinsed in phosphate-buffered saline (PBS, pH = 7.3) for 30 min. and subsequently incubated for lh with a secondary antibody conjugated with FITC (Swine anti-rabbit IgG, DAKO immunoglobulins). After repeated rinsing to remove excess secondary antibody, the preparations were mounted on glass slides in carbonate-buffered glycerol (l:l, pH = 8.5), and viewed in a Leitz Dialux microscope equipped for epi-fluorescence. Photographs were taken on Kodak Tri-X film using a Leitz Vario-Orthomat automatic camera. Sections (10 tzm) for microscopical investigation were produced from pieces of the ovarian wall and
the vesicular nerve fixed in parabenzoquinone in 0. IM sodium cacodylate buffer (pH = 7.2) at 4~ for 2 - 3 h (Bishop et al. 1978), rinsed in the buffer containing 20~ sucrose for 24-48 h at 4~ and snap-frozen in liquid nitrogen for cryostat sectioning. Incubations were made with primary andsecondary antisera according to the method of Coons (1956), which essentially follows the procedure described for whole mount preparations. Control experiments were carried out by replacing the specific antiserum with antiserum preincubated for 48 h at 4~ with the appropriate hapten. The staining with the antiserum was in all cases completely abolished by this preincubation procedure. Identification of immunoreactive material was made with VIP (vasoactive intestinal polypeptide) antiserum L85 (Dockray) diluted 1:250 or 1:400, 5-HT (5-hydroxytryptamine, serotonin) antiserum 71934 (Immuno Nuclear Corp) diluted 1:50, and DBH (dopamine-/3-hydroxylase) antiserum RaDBH (Rush) diluted l:100. No immunoreactivity could be detected with antisera raised against bombesin (L89, Dockray), met-enkephalin (INC 451, Immuno Nuclear Corp and L146, Dockray), gastrin/cholecystokinin (CCK) (L112, Dockray), neurotensin (NtAb, Dockray), physalaemin (PS XII, Lazarus), somatostatin (1001, Yamada), substance P (G10, original, and CRB-SP, Cambridge Research Biochemical Ltd), neuropeptide Y (AB22, Peninsula), FMRF-amide (L155, Dockray) or LPLRF (L197, Dockray). Of these antisera, L89, INC 451, L146, L l l 2 , NtAB, G10, CRB-SP, AB22 and L155 have previously been shown to produce specific reactions in fish tissues studied at this laboratory.
Falck-Hillarp histochemistry Falck-Hillarp histochemistry for monoamines was performed essentially as described by Falck and Owman (1965). Pieces of the ovarian wall and the ureter were snap-frozen in liquid propane cooled in liquid nitrogen and freeze-dried for 3 days. After treatment with formaldehyde vapor at 80~ for lh and paraffin embedding, sections (7 #m) were cut
215 and viewed in the Leitz microscope as mentioned, with the barrier filter at 460 nm.
porcine vasoactive intestinal polypeptide (VIP) (a kind gift from Prof. V Mutt, Stockholm). Concentrations are expressed in tool.1 -I (M).
Isolated strip preparations Statistical evaluation The ovarian wall was cut open and the eggs were removed carefully. Strips, 1-2 mm wide and 10-15 mm long, were cut along the longitudinal axis, and mounted in organ baths in 5 ml cod Ringer's solution (Holmgren and Nilsson 1974) at 10~ bubbled with O2/CO 2 (97/3~ The strips were attached to GRASS FT03 isometric transducers for recording of isometric tension changes on a GRASS Polygraph mod 7. Cumulative concentration-response curves were produced as described previously (e.g., Holmgren and Nilsson 1974), and the affinity (PD2) and relative intrinsic activity (c~) determined for agonists. The antagonistic properties of atropine were estimated by using a Schild plot (Schild 1947) to determine PAx-values. To estimate effects of deterioration of the preparations between consecutive concentration-response curves, a second and third control curve was produced without addition of the antagonist in parallel with the antagonist treated preparations. Changes in a-value have been corrected for the change in maximal response thus determined. Transmural electrical stimulation of intrinsic nerves was made transmurally via a pair of stainless steel electrodes delivering rectangular pulses of 1 ms duration, 20-30V at 10 Hz for 8 sec every 2 min from a GRASS $6 stimulator.
Drugs The following drugs were used in this study: acetylcholine chloride (Sigma), L-adrenaline bitartrate (Sigma), atropine sulphate (Sigma), L-isoprenaline bitartrate (Sigma), 5-hydroxytryptamine creatinine sulfate (Sigma), methysergide (l-methyi-lysergic acid butanolamine bimaleate), L-noradrenaline bitartrate (Sigma), phentolamine methane suiphonate (a kind gift from CIBA-Geigy), proprano1ol hydrochloride (Sigma), tetrodotoxin (Sigma),
Differences in PD2-values between agonists were tested by Student's t-test. Differences of p < 0.05 were regarded as statistically significant.
Results
General disposition of the cod ovary The cod ovary consists of two lobes which fuse posteriorly and taper off in a short oviduct opening at the tip of the urino-genital papilla. One or two branches from the vesicular nerve enter the vertical septum between the two ovarian lobes. These branches run from the single posterior splanchnic (vesicular) nerve, which arises from the sympathetic chain ganglia at the level of the 20th or 21st pair of spinal nerves. Ganglion cell masses are found along the nerve, especially at the point .of exit from the kidney (cf. Nilsson 1976).
lmmunohistochemistry lmmunoreactive (IR) nerve fibers in the ovarian wall were detected with antisera raised against VIP; 5-HT and DBH. A dense plexus of VIP-IR fibers was present in the smooth muscle of the ovarian wall, while 5-HT- and DBH-IR fibers occurred as single strands only (Fig. I A - D ) . Antisera raised against bombesin, met-enkephalin, gastrin/CCK, neurotensin, physalaemin, somatostatin, substance P, neuropeptide Y, FMRFamide or LPLRF failed to reveal IR material in the ovarian preparations.
Falck-Hillarp histochemistry Single fine nerve fibers emitting the blue-green
216
Fig. I. lmmunohistochemical (A-D) and Falck-Hillarp fluorescence histochemical (E) demonstration of nerve fibers in the wall of the cod ovary. A. VIP-IR varicose nerve fibers (whole mount). B. Bundle of VIP-IR nerve fibers (section). C. Single varicose 5-HT-IR fibers (whole mount). D. DBH-IR nerve fibers (section). E. plexus of nerve fibers showing the blue-green fluorescence characteristic of catecholamines (section). Calibration bars in all figures = 10 r~m. 10 - s - 10-4M) p r o d u c e d c o n c e n t r a t i o n - d e p e n d e n t c o n t r a c t i o n s o f the p r e p a r a t i o n s (Fig. 2), with a p D 2 o f 5.40 + 0.17 (n = 7) a n d 5.90 _+ 0.28 (n = 6) respectively. T h e r e was no s t a t i s t i c a l l y significant d i f f e r e n c e in the PD2-values. T h e c o n t r a c t i l e effect o f c a r b a c h o l was a n t a g o n i z e d by a t r o p i n e ( I 0 - v - 10-6M, n = 6), which
Fig. 2. Recordings of drug-induced isometric tension changes in isolated longitudinal preparations of the wall of the cod ovary. Tension calibrations in milli-Newton (mN); log molar concentrations shown (lO-S, 3 x 10 -s, 10 -7 . . . or 10-8, 10 7 10 6 . . . M).
fluorescence c h a r a c t e r i s t i c o f a d r e n e r g i c nerve endings were o b s e r v e d in the o v a r i a n wall (Fig. 1E), but no fluorescent fibers c o u l d be seen in the vesicular nerve.
Isolated strip preparations A c e t y l c h o l i n e ( 1 0 - 7 - 3 x 10-4M)
and
carbachol
p r o d u c e d a p a r a l l e l shift o f the c o n c e n t r a t i o n r e s p o n s e curve (Fig. 3). E s t i m a t i o n o f PAx-values shows p A 2 = 8.8 a n d PAl0 = 7.5. C o n t r a r y to what is e x p e c t e d for a c o m p e t i t i v e a n t a g o n i s t , a t r o pine p r o d u c e d a d e c r e a s e in the a p p a r e n t intrinsic activity ( a = 0.64 + 0. I0, n = 6 a n d a = 0.51 + 0.12, n = 6 ( c o r r e c t e d a g a i n s t c o n t r o l curves) at a t r o p i n e c o n c e n t r a t i o n o f 10-TM a n d 10-6M respectively). 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) likewise h a d a s t i m u l a t o r y effect on the p r e p a r a t i o n s , p r o d u c i n g a c o n c e n t r a t i o n - d e p e n d e n t r e s p o n s e with p D 2 = 7.27 _+ 0.27 (n = 7) (Fig. 2). T h e effect was a n t a gonized by m e t h y s e r g i d e (10-6M, n = 7, Fig. 4). T h e a d r e n e r g i c a g o n i s t s i s o p r e n a l i n e (10 - 1 ~
217 100 -
/o /.
o--o
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Adrenaline Isoprenaline
5o
Z!
Contr Atr 1 Atr 2
--
-8
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26
--'4
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"
i 100 j
~--s
\ -io
\
\.
L9
\o
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-7
-6
LOG MOLAR CONC
-5
-4
LOG MOLAR CONC
Fig. 3. Concentration-response curves for carbachol acting on
Fig. 5. Concentration-response curves for the inhibitory action
isolated strip preparations from the cod ovary, either alone ( 0 ) or in the presence of atropine ( 9 10-7 (Atr I); 9 10 6M (Air 2)).
of isoprenaline ( 9 ) and adrenaline ( o ) on isolated strip preparations from the cod ovary.
'L LLU \LL 8,
100,
/2V" 111
I I
O. A T R 10"r M
10"6M
8
///
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-8
;7
--6
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9
0. -9
71 LL LL&L L _
25
-'4
1st control 2nd control unblocked + meth
-'3
22
LOG MOLAR CONC
Fig. 4. Concentration-response curves for 5-hydroxytryptamine (5HT) acting on isolated strip preparations from the cod ovary, either alone ( o , control run; 9 second run) or in the presence of methysergide ( , , , control run; , , methysergide (10 6M) run),
10-TM) and adrenaline (10 - s - 10-SM) had an inhibitory effect on the strips, causing a concentration-dependent decrease in tension of the preparations (Fig. 2). Isoprenaline (pD 2 = 9.07 +_ 0.32, n = 7) had a significantly higher affinity for the adrenoceptor than adrenaline (pD 2 = 6.81 _+ 0.18, n = 5) (Fig. 5). Noradrenaline had variable weak effects on the preparations, causing either relaxation, contraction or both. Transmural electrical stimulation (20 Hz, 1 ms, 10V) caused contraction of the preparations (Fig. 6). Atropine (10-6M) reduced or abolished the response, measured as a reduction in the amplitude varying between 0 and 100% (mean 35 +_ 13%,
ATR I0 "6 M
PHENT IO"6 M
mN
TTX
IO"6 M
~
O-
ATR-PHENTIO "6 M
TTXIO "6 M
PROPIO'6M
Fig. 6. Recordings of isometric tension changes produced by transmural electrical stimulation of isolated longitudina] preparations of the wall of the cod ovary. Tension calibrations in milli-Newton (raN). Molar concentralions of the drugs shown. ATR, atropine; P H E N T , phentolamine; TTX, tetrodotoxin; P R O P , propranolol.
n = 9), and atropine (10-SM) caused a blockade between 18 and 100~ (mean 81 _+ 10070, n = 10). The remaining response could always be blocked by the (x-adrenoceptor antagonist phentolamine (10 - 6 - 10-5M). Thus, a combination of cholinoceptor and (x-adrenoceptor blockade produced a total inhibition of the contraction, revealing an inhibitory response to the transmural stimulation. This inhibitory response (as well as the excitatory response to transmural stimulation), was abolished by tetrodotoxin (10-6M, n = 5 in both cases) (Fig. 6). The amplitude of the contraction caused by
218
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o - - o Iso O - - O Adr
~
o
g 50
\ 100
-io
29
28
\ 27
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\ 25
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Fig. 7. Concentration-response curves for the inhibitory action of isoprenaline ( o ), adrenaline ( 4; ), and noradrenalinr ( 9 ) on isolaled slrip preparalJons from the cod ovary, [ransmurally stimulated with ]0'Hz, I ms and 20V pulses for 8 s every 2 min.
transmural electrical stimulation was reduced in a concentration-dependent w a y by the adrenergic agonists isoprenaline'(pD 2 = 8.22 _+ 0.21, n = 6), adrenaline (pD 2 = 6.60 _+ 0.12, n = 6 ) a n d noradrenaline (pO 2 4.79 +_ 0.05, n = 5) (Fig. 7). The maximal reduction of the amplitude caused by the three drugs was very similar (isoprenaline 65 _+ ll~ n = 6, adrenaline 67 _+ 9%, n = 6, noradrenaline62 _+ 14%, n = 5).
Discussion The general disposition of the cod ovary is very much like that in other species of the 'higher' teleosts, such as Perciformes and Pleuronectiformes. As in the Japanese dab, Limanda yokohamae, the cod ovary is innervated by a branch of the posterior splanchnic nerve ('vesicular nerve'), which runs along the ureter to innervate both the urinary bladder and the ovary. In the cod, the nervous pathways leave the central nervous system at the level of about the 20th pair of spinal nerves, and both adrenergic and non-adrenergic nerve cells have been described in the sympathetic chain ganglia and the vesicular nerve ganglion (Nilsson 1976). As in the case of the teleost urinary bladder, the ovarian wall is known to contract in response to acetylcholine or carbachol, and this effect is antagonized by atropine, thereby demonstrating the
presence of muscarinic cholinoceptors (Nilsson 1970). In the present study, however, the maximum contraction force produced by carbachol was reduced by atropine, and the PA2/PAi0 difference was greater than can normally be expected during competitive antagonism. The results are compatible with the view that the cholinergic agonists affect also other systems, i.e., releasing other transmitter substances from intramural nerves. Electrical stimulation of the intrinsic nerves by transmural electrodes produced contractions of the ovarian wall, similar to what has been previously reported in cod and other teleosts (Nilsson 1970; Uematsu 1985, 1986). The effect is blocked by tetrodotoxin, showing that it is indeed nervous, but contrary to these other studies, the response cannot be totally abolished by atropine. The remaining response, which can be of variable magnitude, is always abolished by the tx-adrenoceptor antagonist phentolamine, suggesting the involvement of an adrenergic excitatory component in the ovarian innervation in the cod. The function of such an adrenergic innervation is not clear: the effects of exogenous noradrenaline are weak and variable, while adrenaline is inhibitory. Furthermore, although adrenergic neurons could be demonstrated both by immunohistochemical localization of DBH and by Falck-Hillarp fluorescence histochemistry, the adrenergic innervation is very sparse and its importance in the control, excitatory or inhibitory, of the ovarian wall is doubtful. In the study by Nilsson (1970) of the autonomic innervation of the cod urinary bladder, an adrenergic inhibitory control was postulated, based on the effect of the fl-adrenoceptor antagonist propranolol on the inhibitory responses to vesicular nerve stimulation in the presence of atropine. Later studies put this conclusion in doubt (Lundin and Holmgren 1986), and suggested a non-adrenergic, non-cholinergic inhibitory component in the urinary bladder innervation. An inhibitory effect o f transmural stimulation of the cod ovary could not be abolished by propranolol, suggesting also in this case a non-adrenergic inhibitory innervation. The nature of this inhibitory transmitter substance is unknown. VIP, which is the only peptide identified with certainty in nerve
219 fibers ovarian wall, has little or no effect either on isolated strip preparations or the transmurally stimulated strips. It is concluded that the cod ovary receives a double antagonistic autonomic innervation of excitatory cholinergic fibers and non-adrenergic inhibitory fibers. The nature of the inhibitory neurotransmitter is unclear. Histochemical studies demonstrated VIP-immunoreactive, 5-HT-immunoreactive and adrenergic nerve fibers, but a number of antisera raised against other peptides failed to reveal any specific reaction in the tissue preparations.
Acknowledgements This study was supported by the Swedish Natural Science Research Council. We wish to thank Mss. Lena Utter and Inger Holmqvist for skilled technical assistance.
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Furchgott, R.F. 1967. The pharmacological differentiation of adrenergic receptors. Ann. N.Y. Acad. Sci. 139: 553-570. Grove, D.J., O'Neill, J.B. and Spiller, P.B. 1974. The action of 5-hydroxytryptamine on longitudinal gastric smooth muscle of the plaice, Pleuronectesplatessa. Comp. Gen. Pharmacol. 5: 229-238. Holmgren, S. 1983. The effects of putative non-adrenergic, noncholinergic a u t o n o m i c transmitters on isolated strips from the stomach of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 74C: 229-238. Holmgren, S. and Nilsson, S. 1974. Drug effects on isolated artery strips from two teleosts, Gadus morhua and Salmo gairdneri. Acta Physiol. Scand. 9 0 : 4 3 1 - 4 3 7 . Holmgren, S., Grove, D.J. and Nilsson, S. 1985. Substance P acts by releasing 5-hydroxytryptamine from enteric neurons in the stomach of the rainbow trout, Sahno gairdneri. Neuroscience 14: 683-693. Jensen, J. and Holmgren, S. 1985. Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua. C o m p . Biochem. Physiol. 82C: 8 1 - 8 9 . Lundin, K. and Holmgren, S. 1986. Non-adrenergic, innervation of the urinary bladder of the Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. 84C: 315-323. Nilsson, S. 1970. Excitatory and inhibitory innervation of the urinary bladder and gonads of a teleost, Gadus morhua. Acta Physiol. Scand. 83: 446-453. Nilsson, S. 1976. Fluorescent histochemistry and cholinesterase staining of sympathetic ganglia in a teleost, Gadus morhtta. Acta ZooI., Stockh. 57: 6 9 - 7 7 . Nilsson, S. and Holmgren, S. 1983. Splanchnic nervous control of the stomach of the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. 76C: 271-276. Uematsu, K. 1985. Effects of drugs on the responses of the ovary to field and nerve stimulation in a tilapia, Sarotherodon niloticus. Bull. Jap. Soc. Sci. Fish. 51: 4 7 - 5 3 . Uematsu, K. 1986. The autonomic innervation of the o`"ary of the dab, Limandayokohamae. Jap. J. lchthyol. 33:293 303. Young, J.Z. 1931. On the autonomic nervous system of the teleostean fish, Uranbscopus scaber. Quart. J. Microsc. Sci. 74: 491-535. Young, J .Z. 1936. The innervation and reaoions to drugs of the viscera of teleostean fish. Proc. Roy. Soc. London, Ser. B 120: 303-318.
