Pituitary adenylate cyclase activating polypeptide innervation of the rat female reproductive tract and the associated paracervical ganglia: Effect of capsaicin

Neuroscience Vol. 73, No. 4, pp. 104~1060, 1996

~ Pergamon

IBRO Copyright © 1996. Published by ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0306-4522(96)00082-6 0306-4522/96 $15.00 + 0.00

PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE INNERVATION OF THE RAT FEMALE REPRODUCTIVE TRACT AND THE ASSOCIATED PARACERVICAL GANGLIA: EFFECT OF CAPSAICIN J. F A H R E N K R U G * and J. H A N N I B A L Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, DK-2400 Copenhagen NV, Denmark A~tract--Pituitary adenylate cyclase activating peptide (PACAP) is a novel vasoactive intestinal polypeptide-like peptide which is present in neuronal elements of a number of peripheral organs. PACAP occurs in two forms, PACAP-27 and the C-terminally extended PACAP-38, both derived from the same precursor which in addition gives rise to a structurally-related peptide, PACAP-related peptide. Using specific radioimmunoassays for PACAP-38, PACAP-27 and PACAP-related peptide we found that the three PACAP-precursor-derived peptides were present in tissue extracts from all regions of the rat female genital tract. PACAP-38 was the dominating peptide with the highest concentrations in the Fallopian tube and the ovary. Upon reverse phase high pressure liquid chromatography the immunoreactive material was found to co-elute with synthetic PACAP-38, PACAP-27 and PACAP-related peptide, respectively. By immunohistochemistry, PACAP was shown to be located in varicose nerve fibres associated with blood vessels, smooth muscle and epithelial cells. Within the local paracervical ganglion PACAP-immunoreactive fibres ramified often forming varicose, pericellular plexuses around non-PACAP-positive cell bodies. Also bundles of PACAP-immunoreactive fibres were transversing the ganglion. In the paracervical ganglion of normal rat only a few neuronal cell bodies showed immunostaining for PACAP, but after local colchichine-treatment a moderate number of positive perikarya appeared. The synthesis of PACAP in neurons of the paracervical ganglia was confirmed by in situ hybridization histochemistry with a digoxigenin-labelled cRNA probe. Double immunostaining for PACAP and vasoactive intestinal polypeptide disclosed a partial co-existence of the two peptides in nerve fibres of all tubular organs in the rat female genital tract and in cell bodies and nerve fibres in the paracervical ganglion. After neonatal capsaicin treatment the concentration of immunoreactive PACAP-38 as well as the number and intensity of PACAP-positive nerve fibres were reduced while vasoactive intestinal polypeptide immunoreactivity was unaffected. In conclusion, PACAP-immunoreactive nerve fibres have been demonstrated in all regions of the rat female genital tract associated with blood vessels, smooth musculature and epithelium. In some fibres, which seem to originate in the local paracervical ganglia, PACAP was co-localized with vasoactive intestinal polypeptide. PACAP released from these fibres could alone or in concert with vasoactive intestinal polypeptide play a role in neuroregulation of female reproductive organs acting directly on the musculature and vasculature. Other PACAP-containing fibres are sensory in nature, and some of these might influence ganglionic neurotransmission in the local paracervical ganglia. Copyright © 1996 IBRO. Published by Elsevier Science Ltd. Key words: co-existence, in situ hybridization, immunocytochemistry, neuropeptides, sensory nerves,

autonomic nerves.

Pituitary adenylate cyclase activating peptide (PACAP) is a neuropeptide first isolated from ovine hypothalamus based on its ability to stimulate adenylate cyclase in rat anterior pituitary cell cultures, z8 P A C A P is a member of the vasoactive intestinal polypeptide (VIP)-family of regulatory peptides and *To whom correspondence should be addressed. CGRP, calcitonin gene-related peptide; DTT, dithiothreitol; EDTA, ethylenediaminetetra acetate; HPLC, high pressure liquid chromatography; PACAP, pituitary adenylate cyclase activating polypeptide; PBS-BT, phosphate-buffered saline + 0.25% bovine serum albumin+0.1% Triton X-100; PRP, PACAP-related peptide; SSC, saline sodium citrate; VIP, vasoactive intestinal polypeptide.

Abbreviations:

occurs in two biologically active forms, which are both amidated at the C-terminus, PACAP-38, and the C-terminally truncated PACAP-27.17'~s Cloning of the ovine, human and rat P A C A P c D N A ' s has revealed that the encoded amino acid sequence of PACAP-38 is identical in all three species. 14'23 Both PACAP-38 and PACAP-27 are derived from a 175 amino acid precursor, which in addition contains a sequence, named PACAP-related peptide (PRP). This peptide displays some sequence similarity to P A C A P and other members of the VIP-family of peptides. 23'24 PACAP-immunoreactivity is present in neuronal elements in a number of peripheral organs such as the gut, 15'27'30'32 respiratory tract, 3'33 pancreas, 7 salivary

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gland, 31 adrenal gland s a n d the eye. 34 In a d d i t i o n P A C A P i m m u n o r e a c t i v i t y a n d P A C A P m R N A have been d e m o n s t r a t e d in nerve cell bodies o f the dorsal root ganglia at all levels, the trigeminal ganglion a n d vagal sensory n e u r o n s o f the rat. ~9 21 The P A C A P i m m u n o r e a c t i v e n e u r o n s in the spinal ganglia were f o u n d to be sensitive to capsaicin, suggesting a t r a n s m i t t e r role o f the peptide in p r i m a r y sensory neurons. 19 In a recent study we d e m o n s t r a t e d the presence of P A C A P - i m m u n o r e a c t i v e nerve fibres in the various organs of the h u m a n female genital tract. 29 In addition we observed t h a t P A C A P - 3 8 a n d P A C A P - 2 7 h a d a n inhibitory effect on vascular a n d n o n - v a s c u l a r s m o o t h muscle activity, with dose-response curves similar to VIP, suggesting a n action via a c o m m o n receptor. 29 A l t h o u g h these findings indicate t h a t P A C A P could be involved as a t r a n s m i t t e r in the local nervous control of b l o o d flow a n d motility in the female genital tract the exact physiological role for the peptide is still u n k n o w n . Also, the n a t u r e a n d origin of the P A C A P - c o n t a i n i n g nerve fibres in the female genital tract remain to be clarified. The present study was u n d e r t a k e n to investigate by r a d i o i m m u n o a s s a y a n d i m m u n o c y t o c h e m i s t r y the c o n c e n t r a t i o n a n d distribution of P A C A P i m m u n o r e a c t i v i t y in the female genital tract of normal rats in c o m p a r i s o n with rats treated neonatally with capsaicin, a n e u r o t o x i n k n o w n to cause permanent d e g e n e r a t i o n o f small-diameter sensory neurons. T h e presence o f P A C A P - i m m u n o r e a c t i v i t y a n d P A C A P m R N A in the associated paracervical ganglia was studied by i m m u n o c y t o c h e m i s t r y a n d in situ hybridization histochemistry. Finally we examined the possible co-existence of P A C A P with the structurally a n d functionally related peptide, VIP, which in the female reproductive tract is k n o w n primarily to have a local origin. 1'9 EXPERIMENTAL PROCEDURES

Animals and tissue specimens The genital tract of female Wistar rats (about 60 days old and weighing 180-200 g; Mollegaard Breeding, L1. Skensved, Denmark), housed in 12 h light: 12 h dark, were examined. All animals were killed between 8 and 11 a.m. eight animals were decapitated and the genital tract of each animal was dissected to include vagina, uterine cervix, uterine horns, Fallopian tubes and ovaries. The tissues were frozen on dry ice and stored at - 2 0 ° C until radioimmunoassay for PACAP-38 and PACAP-27, PRP and VIP. For immunocytochemistry six animals were anaesthetized with Mebumal (15 mg i.p.) and perfused via the ascending aorta with a room temperature solution of saline (0.9%) to which heparin (15,000 IU/1) was added (75-100ml) over three minutes. This perfusion was followed by 2% paraformaldehyde, 0.2% picric acid in 0.1M sodium phosphate buffer, pH 7.2 (400ml over 15min). After perfusion fixation, specimens from the above mentioned regions of the female genital tract were removed and postfixed in the same fixative for 24 h. The specimens were then rinsed repeatedly in tyrode solution, containing 10% sucrose, and frozen on dry ice for cryostat sectioning. For in situ hybridization specimens containing the paracervical ganglia, i.e. the parametrial connective tissue attached to the

uterovaginal muscle coat bilaterally, were obtained from four animals and immediately frozen on dry ice before cryostat sectioning. In order to enhance the immunostaining in the cell bodies of the paracervical ganglia four rats were treated with colchicine as follows: Under anaesthesia with Mebumal (15mgi.p.) a swab soaked in colchicine (10 mg/ml) was pushed through the vagina as far as the uterine cervix and left for 1 h to allow the colchicine to soak through the surrounding epithelium. The rats were anaesthetized and perfusion fixed 24 h later and specimens containing the paracervical ganglia dissected. To determine if PACAP-immunoreactivity is present in sensory fibres in the rat female reproductive system, animals were treated neonatally with the sensory neurotoxin, capsaicin (Sigma, St. Louis, USA). 10 female rats were injected intraperitoneally with capsaicin 50 mg/kg body weight at day 2 after birth as described by Nagy et all 2 Ten littermatched rats were injected with vehicle solution (Tween 80, saline, absolute alcohol, I : 8 : 1 by volume) at the same time as the experimental animals and were used as controls. After 8-12 weeks the rats were killed and the various regions from the genital tract were collected. The effectiveness of the capsaicin treatment was checked by disappearance of immunostaining with a commercial antiserum (code No. B47-1, Euro-diagnostica, Malmr, Sweden) for calcitonin gene-related peptide (CGRP) known to be stored in capsaicin-sensitive nerves. Tissue extraction, radioimmunoassays and chromatograph)' Before peptide analysis the frozen tissue specimens were weighed and extracted in boiling water/acetic acid. 1~ The extracted samples were reconstituted and analysed by radioimmunoassays specific for PACAP-38, PACAP-27, rat-PRP and VIP, respectively. 5'6'11The PACAP-38 antiserum (code No. 733C), which does not react with PACAP-27, was used in a final titre of 1:180,000. Iodinated PACAP 28 38, labelled with 1251by iodogen to a specific radioactivity of 31 Bq/fmol, was used as tracer. The detection limit of this assay was 5pM, and the working range 5-50pM. The PACAP-27 assay used antiserum (code No. 91084) in a final dilution of 1:420,000. PACAP 18-27, radiolabelled by the chloramine T method to a specific radioactivity of 25 Bq/fmol, was used as tracer. The PACAP-27 antiserum recognized the C-terminal amide and showed no crossreactivity with PACAP-38. The detection limit of the assay was 5 pM and the working range 5-75 pM. The PRP antiserum (code No. 81D3) used in a final titre of 1:75,000 was specific for rat PRP. The assay used rat [~25I]PRP, prepared by the chloramine T method as tracer, and had a detection limit of 5 pM and a working range from 5-70 pM. The VIP radioimmunoassay was performed according to the published method. 5'6 All antisera were raised in rabbits. Synthetic PACAP-38, PACAP-27, rat PRP and VIP (Peninsula Laboratories, St. Helens, UK) were used as standards. The radioimmunoassays were specific for the respective peptides and did not show any cross-reactivity with structurally-related peptides. The within- and betweenassay coefficients of variation were below 10%. All tissue extracts were assayed in duplicate in at least two different dilutions. For chromatography, dried extracts of Fallopian tube from 18 rats were pooled, redissolved in 0.1% v/v trifluoroacetic acid in distilled water and subjected to reverse phase high pressure liquid chromatography (HPLC), using gradient elution with 99% ethanol, containing 0.1% trifluoroacetic acid. H The column was calibrated with synthetic PACAP-38, PACAP-27 and rat PRP. The eluted fractions were collected, freeze-dried and stored at - 2 0 ° C until radioimmunoassays for PACAP-38, PACAP-27 and PRP. lmmunocytochemistry Single antigen imrnunocytochemistry. Sections of 12/~m thickness were processed for single antigen immunocyto-

PACAP innervation of the rat female reproductive tract chemistry using a mouse monoclonal PACAP-antibody (code No. MabJHH1), described and characterized previously.~°,~l Double antigen immunocytochemistry. For co-localisation between PACAP and VIP the sections were incubated in a mixture of the monoclonal PACAP-antibody and a rabbit anti-VIP-antiserum (code No. 291E) 4 for 24 h at 4°C. After incubation the sections were washed for 3 × 10min in phosphate-buffered saline + 0.25% bovine serum albumin + 0.1% Triton X-100 (PBS-BT) and incubated for 60 rain in a mixture of biotinylated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, USA), diluted 1: 50, and a fluoreseein isothiocyanate-conjugated swine anti-rabbit IgG (V205, DAKO), diluted 1:40. After washing for 3 x 10 rain in PBS-BT, the sections were finally incubated for 60 min with a streptavidin-Texas Red ® conjugated complex (Amersham, Copenhagen, Denmark), diluted 1 : 50. After washing for 3 × 10 min in PBS-BT, the sections were covered with coverglass in PBS/glycerol 1:1. The monoclonal PACAP antibody was directed against an epitope of amino acid 6-16 of PACAP, recognizing both PACAP-38 and PACAP-27J 1 Staining was abolished by omission of primary antibody or preabsorbtion with the respective antigens (20 #g/ml). In situ hybridization histochemistry In situ hybridization was performed using a slight modification of the previously described procedureJ TM Blocks containing the paracervical ganglion from four rats, two of which were treated with colchicine as described above, were cut in 12q~m-thick consecutive sections. Digoxigenin-llUTP-labelled antisense and sense RNA probes were prepared by in vitro transcription using T7 (sense) and SP6 (antisense) RNA polymerase. The template was a plasmid containing a cDNA encoding the whole PACAP sequence. 23 The plasmid was linearized with Hind III for antisense or Apa 1 for the sense probe. Transcription was performed at 37°C for 2h in 20/~1 containing transcription buffer (Boehringer Mannheim, Germany), 25 mM dithiothreitol (DTT), 20 U RNasin (Amersham, Copenhagen, Denmark), 1.5 mM NTP-mix (Boehringer Mannheim, Germany) and 3 #1 10 mM digoxigenin-I 1-UTP (Boehringer Mannheim, Germany). After removal of the DNA template by adding 1 #1 RNasin (30-40U), 2#1 t-RNA (10#g/#1) and 1 #1 DNase (Boehringer Mannheim, Germany) and incubating for further 15 min at 37°C, the probes were purified by water/phenol extraction followed by chloroform/isoamylalcohol extraction and finally NH4Ac/ ethanol precipitation. The labelled products were fragmented by incubation in hydrolysis buffer (30#1 0.2M Na2CO3, 20/~1 0.2 M NaHCO3) for 60 rain at 60°C and used in a dilution of 1:1000. After hybridization overnight at 53°C the sections were washed in 4 x saline sodium citrate (SSC), 4 mM DTT for a few minutes at room temperature followed by RNAse treatment for 30 min (RNAse A buffer; Sigma, St. Louis, USA). After a wash in 2 x SSC, 2 mM DTT at room temperature for 60 min, followed by washing in 0.01 x SSC, 2 mM DTT at 60°C in 60 rain and 1 × SSC, 2 mM DTT for I0 min at room temperature the sections were dehydrated through series of alcohol. After a rinse in PBS-BT 3 x 10min the sections were incubated at room temperature with an anti-digoxigenin alkaline phosphataseconjugated sheep antiserum (Boehringer Mannheim, Germany) diluted 1 : 200 for 60 min followed by washing in PBS-BT in 10 min. The sections were then rinsed in 0.1 M Tris-HC1/0.9% NaC1, pH 7.4 for 30min and in 0.1M Tris-HC1/0.1 M NaCI/0.05 M MgC12, pH 9.0 for 10min, and then incubated overnight at room temperature with Nitroblue Tetrazolium and 5-bromo-4-chloro-3-indotyl phosphatase. The colour reaction was terminated by incubation in 10 mM Tris-HC1/10 mM EDTA/0.9% NaCI, pH 7.5 for 2 × 30 rain. The section was dried and mounted in Pertex. For control purposes, hybridization was performed in parallel using antisense and sense probe on consecutive

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sections. Consecutive sections were also hybridized with an alkaline phosphatase-conjugated VIP-oligonucleotide probe as described previously, z4

Statistical analysis Tissue concentrations were expressed as means _+ S.E.M. The significance of the differences between means was calculated by Student's t-test and P-values < 0.05 were considered significant. RESULTS

Peptide tissue concentration PACAP-38, PACAP-27 and P R P were detected in tissue extracts from all the examined regions of the rat female genital tract (Fig. 1). PACAP-38 was in all regions the dominating preproPACAP-derived peptide. The highest concentrations of" PACAP-38 were found in the Fallopian tube and the ovary in which regions concentrations were several-fold higher than the corresponding VIP concentrations. In the remaining regions the PACAP-38 concentrations were low and in the vagina and uterine cervix approximately 15 times lower than VIP. When extracts from the Fallopian tube were fractionated on reverse phase H P L C immunoreactive components to synthetic PACAP-38, PACAP-27 and rat P R P were identified by the respective antisera (data no shown).

Immunocytochemistry Vagina. PACAP-immunoreactive nerve fibres were present throughout the vaginal wall (Figs 2D and 3C). The fibres were most abundant in the submucosa where a dense plexus running parallel to the squamous cell epithelium was seen. F r o m this plexus delicate varicose fibres were formed to terminate between the basal cell layers of the epithelium (Fig. 2D). PACAP-immunoreactive free nerve endings were also seen in the connective tissue. P A C A P containing nerve fibres were numerous in all layers associated with blood vessels, but were particularly abundant around veins in the lamina propria (Figs 2D and 3C). In the muscle layer few or single fibres parallelled fascicles of smooth muscle. The distribution pattern of VIP was similar to that of P A C A P , although VIP-immunoreactive nerve fibres clearly outnumbered PACAP. Double immunofluorescence labelling disclosed co-localization between P A C A P and VIP in many of the nerve fibres (Fig. 4A and C). Cervix. PACAP-immunoreactive fibres formed dense perivascular plexuses, especially around the arteries and larger arterioles in the cervical adventitia (Fig. 5B), but also around myometrial arterioles. In the wall of the large arteries, the P A C A P - i m m u n o reactive fibres ran in the outer media or along the border between the adventitia and media. A moderate number of PACAP-immunoreactive fibres were found in the smooth muscle fascicles in the myometrium, primarily orientated parallel to the long

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Fig. I. PACAP- and VIP-immunoreactivity in the rat female genital tract. Concentrations (pmol/g wet weight) of immunoreacfive PACAP-38 (black bars), PACAP-27 (hatched bars), PRP (open bars) and VIP (cross-hatched bars) in the female genital tract of normal rats. Each bar represents the mean + S.E.M. of extracts from eight rats. V: vagina; UC: uterine cervix; UH: uterine horn; FT: Fallopian tube; O: ovary.

Fig. 2. PACAP-immunoreactive fibres in the ovary, Fallopian tube, uterus and vagina. (A) Section of the ovary showing PACAP-positive fibres in the stroma and around arteries (arrows). F: follicle. (B) Transverse section of the Fallopian tube showing PACAP-positive fibres mainly in the muscularis and in association with blood vessels. Some fibres are also located beneath the tubal epithelium. (C) Longitudinal section of the rat uterine wall showing bundles of PACAP-positive fibres in the myometrium and fibres surrounding blood vessels (arrows). (D) Longitudinal section of the vaginal wall showing bundles of PACAP-positive fibres in the connective tissue and delicate varicose fibres underneath and between the cells of the squamous epithelium (arrows). Scale bars: A, C and D = 50 #m; B = 100 #m.

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Fig. 3. PACAP-immunoreactive fibres in the Fallopian tube and vagina after capsaicin. (A) Transverse section of the Fallopian tube showing varicose PACAP-positive fibres below the tubal epithelium and in the smooth muscle layers (arrows). (B) Section from a comparable region of the Fallopian tube from a capsaicin-treated rat demonstrating that PACAP-staining is reduced to a few faintly-stained fibres (arrows). (C) Longitudinal section of the vaginal wall showing network and bundles of PACAP-positive fibres in the connective tissue and muscle layer (thick arrows). Fibres are also seen around blood vessels and below the epithelium (thin arrows). E: epithelium. (D) Section from a comparable region of the vaginal wall from a capsaicin-treated rat demonstrating that PACAP-staining is reduced to a few faintly-stained fibres (arrows). E: epithelium. Scale bar: A, B, C and D = 50 pm.

axes of the smooth muscle fascicles. Occasionally single-beaded PACAP-immunoreactive fibres were encountered in the cervical epithelium. PACAPimmunoreactive fibres were less numerous than the VIP-immunoreactive fibres but the two peptides often occurred in the same fibres. Most of the vessels in the cervical adventitia were innervated by both peptides (Fig. 5B and D), but some contained VIP-immunoreactive fibres and lacked PACAP-innervation, while in others only PACAP-containing fibres occurred. Uterus. No apparent regional variation in density of PACAP-immunoreactive fibres from the cervical to the oviductal end of the uterine horn could be observed. Within the uterine wall PACAP-immunoreactive fibres had longitudinal and circular orientations apparently paralleling the orientation of myometrial smooth muscle. Besides being associated with the smooth muscle fascicles, numerous beaded perivascular PACAP-immunoreactive fibres often forming plexuses could be demonstrated in the myometrium (Fig. 2C). The density of the fibres de-

creased deeper in the wall of the uterus and no fibres seemed to course to the endometrium. PACAP- and VIP-containing fibres of the uterus had a similar distribution pattern, but the two peptides were not always co-localized in the same fibres. Occasionally VIP-containing and PACAP-containing fibres ran separately and some VIP-fibres appeared in the endometrium, which was devoid of PACAP-immunoreactive fibres. Fallopian tube. A large number of varicose PACAP-immunoreactive fibres were observed among bundles of smooth muscle in both the circular and longitudinal layers (Fig. 2B). Perivascular PACAPimmunoreactive fibres were also evident in the oviduct. Throughout the Fallopian tube delicate beaded PACAP-immunoreactive fibres were running below the tubal epithelium (Fig. 3A). VIP-containing fibres were distributed as the PACAP-containing fibres but were scarce, particularly below the epithelium. In only a limited number of fibres the two peptides seemed to be co-localized (Fig. 5A and C).

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Ovary. PACAP-immunoreactive fibres were seen in the ovarian stroma where many of the fibres formed perivascular plexuses (Fig. 2A). A few delicate fibres coursed near the follicles and the interstitial gland tissue. VIP-immunoreactive fibres were less abundant than the PACAP-containing fibres and primarily distributed in the peripheral stroma and in association with blood vessels. In none of the fibres the two peptides were found to co-exist. No PACAP immunoreactivity was observed in the follicular cells or the oocyte. Paracervical ganglia. Within the paracervical ganglia PACAP-immunoreactive fibres ramified among the principal neurons, and many of the fibres were intimately associated with cell bodies (Fig. 6A). Some of the PACAP-immunoreactive terminal-like structures appeared as clusters on the neuron somata or as tangles of fibres around the non-PACAPpositive cell bodies. Other fibres, often forming bundles, seemed to transverse the ganglia. A few neuronal cell bodies of normal rats showed immunostaining for PACAP (Fig. 6A), but after colchicinetreatment several positive perikarya appeared (Fig. 6C and D). A proportion of the PACAP-

immunoreactive ganglion cells were also VIP-positive (Fig. 4B and D). By in situ hybridization m R N A for PACAP was detectable in only a few cells of normal rats, whereas colchicine-treatment increased both the number and the intensity of PACAP mRNA-labelled cells (Fig. 7A). No PACAP mRNA-signal was observed using a sense probe (Fig. 7C). Using the VIP-probe a population of mRNA-positive cell bodies with a distribution which partially overlapped the PACAP mRNA-positive cells was demonstrated (Fig. 7B).

Effect of capsaicin In capsaicin-treated animals a reduction in PACAP-38 concentration was most prominent in the vagina and the Fallopian tube amounting to 43 and 42%, respectively (Fig. 8). A slight but significant decrease was also observed in the uterine horn, while PACAP concentration in the uterine cervix and the ovary was unaffected by capsaicin. No change in VIP-concentration was observed in capsaicin-treated rats when compared to littermate controls (data not shown). The peptide concentrations in tissue extracts from littermate controls were comparable to those

Fig. 4. PACAP- and VIP-immunoreactive cell bodies and fibres in the paracervical ganglia and the vagina. Double-immunostaining showing PACAP (A and B) and VIP (C and D) immunoreactive fibres in the vagina (A and C) and immunoreactive cell bodies and fibres in the paracervical ganglion (B and D). Examples of co-existence of PACAP and VIP in the same nerve fibres and cell bodies are indicated by thick arrows. Examples of fibres and cell bodies showing PACAP- but not VIP-immunoreactivity are indicated by open arrows. Examples of fibres showing VIP- but not PACAP-immunoreactivity are indicated by thin arrows. Scale bars: A and C = 50 #m; B and D = 25/tm.

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Fig. 5. PACAP- and VIP-immunoreactive fibres in the Fallopian tube and uterine cervix. Doubleimmunostaining showing PACAP (A and B) and VIP (C and D) immunoreactive fibres in the Fallopian tube (A and C) and around large arteries in the adventitia of the uterine cervix (B and D). Examples of co-existence of PACAP and VIP in the same nerve fibres are indicated by arrows. Scale bar: A, B, C and D = 50pm.

observed in normal rats. In the paracervical ganglia as well as in the Fallopian tube, uterine horn, uterine cervix, vagina and ovary the sensory neurotoxin resulted in a reduction in the number and intensity of the PACAP-immunoreactive fibres (Fig. 3B and D, Fig. 6B). The reduction was most prominent in the vagina and the Fallopian tube. The VIP-immunoreactive fibres and VIP-positive cell bodies in the rat female genital tract were unaffected by capsaicin.

DISCUSSION

In the present study we have shown that immunoreactive PACAP, mainly in the form of PACAP-38, is present throughout the rat female genital tract. PACAP-immunoreactivity displayed a characteristic distribution pattern, which differed from that of the structurally related peptide, VIP. By immunocytochemistry PACAP was found to be located in varicose nerve fibres with relation to blood vessels, smooth muscle and epithelial cells, suggesting that PACAP could participate in the nervous control of haemodynamic and non-vascular smooth muscle activity in the female reproductive organs. Thus

PACAP can be added to the list of peptides localized in nerves of the female reproductive tract (for review see Ref. 26). In vitro PACAP like VIP has been shown to inhibit the activity of vascular and non-vascular smooth muscle from the human female genital tract, 29 but the physiological relevance of these observations remains to be clarified. The effect of PACAP could be mediated via a receptor normally working for VIP, since several types of receptors that can interact with both PACAP and VIP have been described. ~2 One type of receptor binds VIP, PACAP-27 and PACAP38 with similar affinities. This receptor most likely corresponds to the cloned VIP type 1 (PACAP type 2 receptor), present in brain tissues, liver, lung and intestine. ~a A second VIP-receptor, named the VIP type 2 receptor, also displays similar affinity for VIP and PACAP but has only 50% amino acid sequence identity with the rat VIP type 1 receptor/6 This receptor is present in the rat anterior pituitary, olfactory bulb, thalamus, hypothalamus, particularly in the suprachiasmatic nucleus and hippocampus) 6 Finally a specific PACAP-receptor (PACAP type l receptor), which exists in several splice variants, has been cloned. 28 This receptor has a wide-spread distribution in the CNS but is also present in the anterior

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and intermediate lobes of the pituitary, in the adrenal medulla and in the testis. 28 Which type of receptor is expressed in the female reproductive tract is unknown at present, but our previous functional in vitro studies favour a common receptor PACAP/VIP-receptor. 29 The partial co-localization between PACAP and VIP in nerve fibres of the female genital tract could suggest a functional interplay between the two peptides in the local nervous control of blood flow and motility in the female genital tract. The abundance of PACAP-immunoreactive fibres and terminals in the vaginal epithelium and the presence of PACAP-immunoreactive nerve cell bodies in spinal ganglia at all levels 19 prompted us to examine the effect of capsaicin in order to obtain information on the possible origin and nature of PACAP-immunoreactive nerve fibres in the female genital organs and paracervical ganglia. Capsaicintreatment lowered the PACAP-concentration in all tubular reproductive organs and reduced the number and intensity of PACAP-immunoreactive nerve fibres

in all regions as well as in the paracervical ganglia. The capsaicin-sensitive fibres are small-diameter unmyelinated C-type primary afferent nerves most likely derived from the previously described PACAP-containing cell bodies in the dorsal root ganglia, while the origin of the capsaicin-resistant nerve fibres could be the PACAP-positive neurons in the paracervical ganglia, which were quite numerous after enhancing peptide concentration by colchichine treatment. Some of the PACAP-positive cell bodies in the paracervical ganglia also contained VIP. It is likely that fibres in the various structures of the female genital tract displaying co-existence of PACAP and VIP are projections from these neurons. That the cell bodies in paracervical ganglia are actually synthesizing PACAP and VIP was supported by the demonstration of their respective mRNA's by in situ hybridization histochemistry. The number of PACAPimmunoreactive nerve fibres has previously been shown to be reduced by capsaicin treatment in other tissues with a sensory innervation such as the eye, the

Fig. 6. PACAP-immunoreactive cell bodies and fibres in the paracervical ganglion after capsaicin or colchicine. (A) Paracervical ganglion of control rat showing PACAP-immunoreactive fibres and a few weakly-stained PACAP-positive cell bodies (arrows). Some principal neurons are enwrapped by PACAPimmunoreactive fibres and terminals while other neurons lack varicosities. Bundles of PACAP-immunoreactive fibres in the uterine cervical wall are indicated by open arrows. (B) Comparable sections of paracervical ganglion from a capsaicin-treated rat demonstrated that PACAP-staining is reduced to few faintly-stained fibres (arrows) and varicosities. (C and D) Paracervical ganglion of a colchicine-treated rat showing bundles of coarse PACAP-immunoreactive fibres transversing the ganglion as well as delicate fibres. Many principal neurons in the ganglion are enwrapped by clusters of PACAP-immunoreactive terminals. PACAP-positive cell bodies are indicated by arrows, some of which seem to innervate non-PACAP-positive cell bodies (open arrows). Scale bars: A, B and C = 50/~m; D = 25/~m.

PACAP innervation of the rat female reproductive tract

Fig. 7. PACAP- and VIP-mRNA in the paracervical ganglion. In situ hybridization histochemistry showing PACAP mRNA-containing cells in the paracervical ganglion of a colchicine-treated rat using Digoxigenin-UTP-labelled antisense cRNA probe (A), and on a consecutive section VIP mRNA in the same ganglion using an alkaline phosphatase conjugated oligonucleotide probe (B). Control on a consecutive section of the paracervical ganglion using a sense cRNA probe for PACAP. Scale bar: A, B and C = 100#m.

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airway epithelium, the facial skin, the tongue and the urinary bladder. ~9 In these tissues PACAP seemed to be stored in a subpopulation of CGRP-, and substance P-containing nerves supporting the view that some PACAP-fibres could be sensory in nature. ~9 Double staining with antibodies against PACAP and "sensory" neuropeptide and retrograde tracing studies combined with immunocytochemistry, which are now in progress, will shed further light on the nature and origin of the PACAP-immunoreactive nerves in the female genital tract. The occurrence of PACAP-immunoreactive fibres intimately associated with neurons in the paracervical ganglia suggests that the neuropeptide might also influence ganglionic neuronal transmission. Since many of these fibres were capsaicin-sensitive they could represent intraganglionic collaterals of primary sensory nerve fibres, offering the possibility of an operational axonal reflex involving activation of peripheral sensory endings in the reproductive organs followed by modulation of efferent neural activity in the paracervical ganglia. The capsaicin-resistant fibres in the paracervical ganglia could be autonomic preganglionic or fibres from the PACAP-positive cell bodies in the ganglion. In the ovary PACAP-immunoreactive fibres formed perivascular plexa and were associated with follicles. In accordance recent data have provided evidence that PACAP may participate in the regulation of reproductive functions in the rat ovary by augmenting steroidogenic activity in granulosa cells.35 The finding that the PACAP-concentration in ovarian extracts was unaffected by capsaicin, while

the number of PACAP-positive nerve fibre was reduced is puzzling. The discrepancy could be explained if endocrine cells contained immunoreactive PACAP which was undetectable by immunocytochemistry but measurable by the more sensitive radioimmunoassay. This could also explain the relatively high concentration of PACAP-immunoreactivity in ovarian extracts. The origin of the PACAP-immunoreactive fibres, which could reach the ovary by different routes, 26 remains to be clarified. By denervation experiments and tracing studies combined with immunocytochemistry evidence has been provided that VIP-immunoreactive nerve fibres in the tubular organs of the female genital tract originate from the local paracervical ganglia. ~,9 We found in the present study that the VIP-concentration and VIP-immunoreactive nerve fibres in the various regions of the female genital tract as well as cell bodies and fibres in the paracervical ganglia were unaffected by capsaicin-treatment, supporting the view that VIP-fibres in the female reproductive tract is of local origin and non-sensory in nature. In accordance an effector function of VIP in the local nervous control of blood flow and motility in the female genital tract is well documented in a number of studies (for review see Ref. 25).

CONCLUSION

In conclusion PACAP-immunoreactive nerve fibres have been demonstrated in all regions of the rat female genital tract associated with blood vessels, smooth musculature and epithelium. Some of the

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Fig. 8. PACAP-38-immunoreactivity in the female genital tract after capsaicin. Concentrations (pmol/g wet weight) of immunoreactive PACAP-38 in the female genital tract of capsaicin-treated rats (hatched) and littermate controls (black). Each bar represents the mean __+S.E.M. of extracts from 10 rats. V: vagina; UC: uterine cervix; UH: uterine horn; FT: Fallopian tube; O: ovary. Student's t-test was used to compare the PACAP-38 concentration in treated animals with controls in each region (*P < 0.05, **P < 0.01).

PACAP innervation of the rat female reproductive tract

fibres, which seemed to originate in the local paracervical ganglia, are partially co-localized with VIP. PACAP released from these fibres could alone or in concert with VIP play a role in neural regulation of female reproductive organs acting directly on the musculature and vasculature. Other PACAP-containing fibres are sensory in nature and some of these

1059

might influence ganglionic neural transmission in the local paracervical ganglia. Acknowledgements--The skilful technical assistance of Anita Hansen, Lea Larsen, Juliano Olsen and Anna Hlin Schram is gratefully acknowledged. The study was supported by the Danish Biotechnology Centre for Signal Peptide Research.

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(Accepted 5 February 1996)