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Occurrence of nerves containing vasoactive intestinal polypeptide immunoreactivity in the male genital tract
Pergamon Press
Life Sciences Vol . 21, pp . 503-508 Printed in the U .S .A .
OCCURRENCE OF NERVES CONTAINING VASOACTIVE INTESTINAL POLYPEPTIDE IMMUNOREACTIVITY IN THE MALE GENITAL TRACT L .-I . Larsson . Institute of Medical Biochemistry University of Aarhus, Aarhus Denmark J .Fahrenkrug and O.B . Schaffalitzky de Muckadell Department of Clinical Chemistry, Bispebjerq Hospital, Copenhagen, Denmark (Received in final form July 5, 1977)
Summa~ Immunocytochemistry and radioimmunochemistry demonstrates the occurrence of numerous nerves containing the vasoactive intestinal polypeptide (VIP) in the male genital tract. The nerves occur in association with arteries and smooth musculature of the organs . Available evidence suggests VIP to be a new neurotransmittor possibly involved in the regulation of blood flow and muscle contractility of the male genital organs . The vasoactive intestinal polypeptide (VIP) was originally isolated from the proximal small intestine by Said and Mutt (1,2) . For quite some time VIP was thought of solely as a gastrointestinal hormone candidate (3,4) but subsequent studies have indicated that VIP represents a major new neuronal peptide (5-10) . With electron microscopic immunocytochemistry VIP has been shown to occur in the granules of the terminals of nerves distinct from adrenergic and cholinergic nerves (Larsson : in preparation) . This localization is compatible with the view that VIP represents a new neurotransmittor . Its pronounced effects on blood vessel and smooth muscle contractility (2) prompted a study of its possible occurrence in the male genital tract . This study was supported by the Danish Medical Research Council . 503
50 4
Vasoactive Peptide in Male Genital Tract
Vol . 21, No . 4, 1977
Methods Male adult (2-3 kg) cats were anesthetized with chloralose or mebumal (Nembutal R ) and surgical biopsies from various portions of the genital tract were taken . Specimens from testicles, epididymis, ductus deferens, prostate, seminal vesicles and penis were rapidly frozen on dry ice (radioimmunochemistry) or in melting Freon-22 (immunocytochemistry) . Immunocytochemical specimens were freeze-dried, vapour-fixed with diethylpyrocarbonate and embedded in paraffin in vacuo . Deparaffinized 5u sections were hydrated and subjected to an indirect immunocytochemical procedure for the demonstration of VIP immunoreactivity (6) . The VIP antiserum (No . 5603) has been characterized in detail elsewhere (6,11) . As tested at both the immunocytochemical and radioimmunochemical level the antiserum does not react with either glucagon, gastric inhibitory polypeptide, secretin, gastrin, pancreatic polypeptide, somatostatin, substance P, insulin, motilin or cholecystokinin (6,11) . For immunocytochemistry it was applied at a dilution of 1 :5120 for 24 hours at 4oC . The site of antigen-antibody reaction was revealed by the PAP procedure of Sternberger (12) . Controls were those suggested by Sternberger (12) and included the application of antigen-inactivated antiserum (30 nmol VIP per ml antiserum diluted 1 :80) . The specimens were either examined in a conventional light microscope or in a Nomarski-type interference contrast microscope to enhance background details . For radioimmunoassay the tissue material was extracted as 125 1-labeled VIP was prepared by a previously described (6) . chloramine T method to a specific radioactivity of approximately 900 uCi per nmol peptide . Highly purified porcine VIP was used as standard . Free labeled VIP was separated from antibody-bound VIP by absorption to plasma-coated charcoal . The lowest VIP concentration to be distinguished from zero (958 confidence) was 3 .3 pmol/1 . The within and between assay reproducibility expressed as coefficient of variation was 0 .07 and 0 .15 at a level of 44 pmol/1 . All samples were assayed in triplicate in at least three dilutions . Results VIP immunoreactive nerves were detected in all organs examined (Table I) . All controls were negative . In the testicles, VIP nerves were almost exclusively confined to the capsule a1= though a few were seen to extend in between the most superficial seminiferous tubules . The VIP nerves became much more numerous in the epididymis where some nerves were found to be situated immediately beneath the basement membrane of the cylindrical epithelium and others in the connective tissue between the coiled ducts . In the latter location the nerves were rather numerous and tended to occur in tiny bundles . The ductus deferens contained an abundancy of VIP nerves that mainly were associated with the longitudinal and circular muscle layers . As in the epididymis quite a few nerves did also occur immediately beneath the surface epithelium (Fig . 1a), and arteries running alongside the ductus deferens were heavily endowed with VIP nerves, that were situated at the zone of transition between the vascular media and adventitia . The prostate and the seminal vesicles received a quantitatively important supply of VIP nerves
Vol . 21, No . 4, 1977
Vsaoactive Peptide is Male Geaital Tract
505
TABLE I Relative Frequency of Occurrence of Immunoreactive VIP Nerves in the Male Genital Tract . Location
Subepithelial
Testicle Epididymia Ductus deferens Prostate Seminal vesicle Penis ; corporc spongiosa Penis ; corpus cavernosum Glens penis
- a) + ++
Smooth muscle
Arteries
n .p . + ++
(+) + ++
++ ++
++ ++
n .p .
+
(+)
n .p .
-
-
n .p .
(+)
(+) (+)
Other Capsule : +
Connective tissue : +
a - denotes absence of nerves ; (+), occasional nerves ; +, fair amount of nerves (cf . Fig . 1a) ; and ++, abundant nerves ; n .p, indicates that the organs were devoid of recognizable smooth muscle .
Fiq . 1 . Section from feline vas deferens (a ; x 180) and seminal vesicle (b ; x 230) stained with VIP antiserum . Note the abundancy of black-stained VIP-nerves that occur in the subepithelial layer of the vas deferens and in the connective tissue between the glands of the seminal vesicle .
Vaeoactive Peptide in Male Genital Tract
506
Vol . 21, No . 4, 1977
which in both organs occurred associated with smooth muscle cells in the connective tissue between the glandular tubules (Fig . 1b) . At the periphery of both glands VIP nerves were found at the adventitial-medial border of arteries as well as In the in nerve trunks that also contained unreactive nerves . penis VIP nerves were quite abundant in the subcutaneous connective tissue where they sometimes were seen to innervate small In addition, some VIP nerves were seen around arterial branches . vessels as well as in the connective tissue trabecules of the corpora cavernosa . Apart from occasional nerves surrounding the urethra, the corpus spongiosum was devoid of VIP . In the glans penis occasional VIP nerves were sometimes seen associated with arteries, but they were never numerous in this portion of the organ . Radioimmunochemical analysis gave results in good agreement with those of the immunocytochemical study (Table II) .
TABLE II Distribution of VIP Immunoreactivity in the Feline Male Genital Tract Location
VIP
(pmol/g)
Testicle Testicular capsule Epididymis Ductus deferens Prostate Seminal vesicle Urethra Penis Glans penis
21 a 33 298 123 418 367 183 154 40
(2) b (1) (2) (1) (2) (1) (2) (2) (1)
a b
Concentrations are expressed as pmol per g wet weight . Numbers in brackets indicate the number of animals tested .
It is noteworthy that the concentrations measured in the epididymis, the ductus deferens, seminal vesicles, prostate and penis were of the same order of magnitude as those found in the Gel permeation muscular layer of the small intestine (6) . chromatography of extracts from the penis showed that the VIP immunoreactivity eluted in the position of highly purified porcine intestinal VIP (Fig . 2) .
Vol . 21, No . 4, 1977
Vaeoactiva Peptide in Male Genital Tract
507
effluent volume ( ml )
Fig . 2 . Elution diagram of VIP immunoreactivity from an extract of feline penis . Gel permeation chromatography on a Sephadex G50 superfine column as described elsewhere (6) . Filled circles denote tissue extract and open circles purified porcine VIP . Vo: void volume . Vt: total mobile phase . Discussion Our results demonstrate that nerves containing the vasoactive "intestinal" polypeptide are abundant in the male genital organs . In all organs examined the VIP nerves occur in three distinct locations : 1) In an innervation-like pattern around arteries, 2) Associated with smooth musculature of non-vascular origin and 3) In most divisions of the genital tract in the form of a distinct subepithelial plexus . Gel chromatographic evidence indicates that the observed immunoreactivity is due to the vasodilatory peptide originally isolated from the intestinal wall . Recent electron microscopical studies have shown that VIP occurs in the granules of the terminals of a unique type (p-type) of neuron . The scene is thus set for VIP to represent a new neurôtransmittor . Its known biological effects in combination with its localization to arteries and smooth muscle cells makes it tempting to speculate that VIP may function in the regulation of blood flow and muscle contractility of the male genital tract . References 1. 2. 3.
S .I . S .i . ed .) M.i .
SAID and V. MUTT, Science 169 1217-1218 (1970) . SAID, In : Gastrointestina~fHormones (J .C . Thompson, Austin, University o Texas Press, p. 591-597 (1975) . GROSSMAN, Gastroenteroloc~r 67 730-755 (1974) .
508
4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
Vaeoactive Peptide in Male Genital Tract
Vol . 21, No . 4, 1977
J .M . POLAR, A .G .E . PEARSE, J .-L . GARAUD and S .R . BLOOM, Gut 15 720-724 (1974) . .I S .SAID and R,N . ROSENBERG Science 192 907-908 (1976) . L .-I . LARSSON, J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, F . SUNDLER, R. HAKANSON, J.F . REHFELD, Proc . Nat . Acad . Sci. U .S .A . 7 3 3197-3200 (1976) . L .-I . LÂRSSON, L . EDVINSSON, J . FAHRENKRUG, R . HAKANSON, C . OWMAN, O. SCHAFFALITZKY DE MUCKADELL, F . SUNDLER, Brain Res . 113 400-404 (1976) . M .G . BRYANT, S .R . BLOOM, J,M, POLAK, R .E . ALBUQUERQUE, I . MODLIN, A .G .E . PEARSE, Lancet _I 991-993 (1976) . J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, A . FAHRENKRUG, Brain Res . 124 581-584 (1977) . L .-I . LARSSON, J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, Science (in press) . J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, J . Lab . Clin , _Med . (in press) . L .A . STERNBERGER, Immunoc tochemistr , Prentice-Hall Inc ., Englewood Cliffs, N,J, T4 .
Life Sciences Vol . 21, pp . 503-508 Printed in the U .S .A .
OCCURRENCE OF NERVES CONTAINING VASOACTIVE INTESTINAL POLYPEPTIDE IMMUNOREACTIVITY IN THE MALE GENITAL TRACT L .-I . Larsson . Institute of Medical Biochemistry University of Aarhus, Aarhus Denmark J .Fahrenkrug and O.B . Schaffalitzky de Muckadell Department of Clinical Chemistry, Bispebjerq Hospital, Copenhagen, Denmark (Received in final form July 5, 1977)
Summa~ Immunocytochemistry and radioimmunochemistry demonstrates the occurrence of numerous nerves containing the vasoactive intestinal polypeptide (VIP) in the male genital tract. The nerves occur in association with arteries and smooth musculature of the organs . Available evidence suggests VIP to be a new neurotransmittor possibly involved in the regulation of blood flow and muscle contractility of the male genital organs . The vasoactive intestinal polypeptide (VIP) was originally isolated from the proximal small intestine by Said and Mutt (1,2) . For quite some time VIP was thought of solely as a gastrointestinal hormone candidate (3,4) but subsequent studies have indicated that VIP represents a major new neuronal peptide (5-10) . With electron microscopic immunocytochemistry VIP has been shown to occur in the granules of the terminals of nerves distinct from adrenergic and cholinergic nerves (Larsson : in preparation) . This localization is compatible with the view that VIP represents a new neurotransmittor . Its pronounced effects on blood vessel and smooth muscle contractility (2) prompted a study of its possible occurrence in the male genital tract . This study was supported by the Danish Medical Research Council . 503
50 4
Vasoactive Peptide in Male Genital Tract
Vol . 21, No . 4, 1977
Methods Male adult (2-3 kg) cats were anesthetized with chloralose or mebumal (Nembutal R ) and surgical biopsies from various portions of the genital tract were taken . Specimens from testicles, epididymis, ductus deferens, prostate, seminal vesicles and penis were rapidly frozen on dry ice (radioimmunochemistry) or in melting Freon-22 (immunocytochemistry) . Immunocytochemical specimens were freeze-dried, vapour-fixed with diethylpyrocarbonate and embedded in paraffin in vacuo . Deparaffinized 5u sections were hydrated and subjected to an indirect immunocytochemical procedure for the demonstration of VIP immunoreactivity (6) . The VIP antiserum (No . 5603) has been characterized in detail elsewhere (6,11) . As tested at both the immunocytochemical and radioimmunochemical level the antiserum does not react with either glucagon, gastric inhibitory polypeptide, secretin, gastrin, pancreatic polypeptide, somatostatin, substance P, insulin, motilin or cholecystokinin (6,11) . For immunocytochemistry it was applied at a dilution of 1 :5120 for 24 hours at 4oC . The site of antigen-antibody reaction was revealed by the PAP procedure of Sternberger (12) . Controls were those suggested by Sternberger (12) and included the application of antigen-inactivated antiserum (30 nmol VIP per ml antiserum diluted 1 :80) . The specimens were either examined in a conventional light microscope or in a Nomarski-type interference contrast microscope to enhance background details . For radioimmunoassay the tissue material was extracted as 125 1-labeled VIP was prepared by a previously described (6) . chloramine T method to a specific radioactivity of approximately 900 uCi per nmol peptide . Highly purified porcine VIP was used as standard . Free labeled VIP was separated from antibody-bound VIP by absorption to plasma-coated charcoal . The lowest VIP concentration to be distinguished from zero (958 confidence) was 3 .3 pmol/1 . The within and between assay reproducibility expressed as coefficient of variation was 0 .07 and 0 .15 at a level of 44 pmol/1 . All samples were assayed in triplicate in at least three dilutions . Results VIP immunoreactive nerves were detected in all organs examined (Table I) . All controls were negative . In the testicles, VIP nerves were almost exclusively confined to the capsule a1= though a few were seen to extend in between the most superficial seminiferous tubules . The VIP nerves became much more numerous in the epididymis where some nerves were found to be situated immediately beneath the basement membrane of the cylindrical epithelium and others in the connective tissue between the coiled ducts . In the latter location the nerves were rather numerous and tended to occur in tiny bundles . The ductus deferens contained an abundancy of VIP nerves that mainly were associated with the longitudinal and circular muscle layers . As in the epididymis quite a few nerves did also occur immediately beneath the surface epithelium (Fig . 1a), and arteries running alongside the ductus deferens were heavily endowed with VIP nerves, that were situated at the zone of transition between the vascular media and adventitia . The prostate and the seminal vesicles received a quantitatively important supply of VIP nerves
Vol . 21, No . 4, 1977
Vsaoactive Peptide is Male Geaital Tract
505
TABLE I Relative Frequency of Occurrence of Immunoreactive VIP Nerves in the Male Genital Tract . Location
Subepithelial
Testicle Epididymia Ductus deferens Prostate Seminal vesicle Penis ; corporc spongiosa Penis ; corpus cavernosum Glens penis
- a) + ++
Smooth muscle
Arteries
n .p . + ++
(+) + ++
++ ++
++ ++
n .p .
+
(+)
n .p .
-
-
n .p .
(+)
(+) (+)
Other Capsule : +
Connective tissue : +
a - denotes absence of nerves ; (+), occasional nerves ; +, fair amount of nerves (cf . Fig . 1a) ; and ++, abundant nerves ; n .p, indicates that the organs were devoid of recognizable smooth muscle .
Fiq . 1 . Section from feline vas deferens (a ; x 180) and seminal vesicle (b ; x 230) stained with VIP antiserum . Note the abundancy of black-stained VIP-nerves that occur in the subepithelial layer of the vas deferens and in the connective tissue between the glands of the seminal vesicle .
Vaeoactive Peptide in Male Genital Tract
506
Vol . 21, No . 4, 1977
which in both organs occurred associated with smooth muscle cells in the connective tissue between the glandular tubules (Fig . 1b) . At the periphery of both glands VIP nerves were found at the adventitial-medial border of arteries as well as In the in nerve trunks that also contained unreactive nerves . penis VIP nerves were quite abundant in the subcutaneous connective tissue where they sometimes were seen to innervate small In addition, some VIP nerves were seen around arterial branches . vessels as well as in the connective tissue trabecules of the corpora cavernosa . Apart from occasional nerves surrounding the urethra, the corpus spongiosum was devoid of VIP . In the glans penis occasional VIP nerves were sometimes seen associated with arteries, but they were never numerous in this portion of the organ . Radioimmunochemical analysis gave results in good agreement with those of the immunocytochemical study (Table II) .
TABLE II Distribution of VIP Immunoreactivity in the Feline Male Genital Tract Location
VIP
(pmol/g)
Testicle Testicular capsule Epididymis Ductus deferens Prostate Seminal vesicle Urethra Penis Glans penis
21 a 33 298 123 418 367 183 154 40
(2) b (1) (2) (1) (2) (1) (2) (2) (1)
a b
Concentrations are expressed as pmol per g wet weight . Numbers in brackets indicate the number of animals tested .
It is noteworthy that the concentrations measured in the epididymis, the ductus deferens, seminal vesicles, prostate and penis were of the same order of magnitude as those found in the Gel permeation muscular layer of the small intestine (6) . chromatography of extracts from the penis showed that the VIP immunoreactivity eluted in the position of highly purified porcine intestinal VIP (Fig . 2) .
Vol . 21, No . 4, 1977
Vaeoactiva Peptide in Male Genital Tract
507
effluent volume ( ml )
Fig . 2 . Elution diagram of VIP immunoreactivity from an extract of feline penis . Gel permeation chromatography on a Sephadex G50 superfine column as described elsewhere (6) . Filled circles denote tissue extract and open circles purified porcine VIP . Vo: void volume . Vt: total mobile phase . Discussion Our results demonstrate that nerves containing the vasoactive "intestinal" polypeptide are abundant in the male genital organs . In all organs examined the VIP nerves occur in three distinct locations : 1) In an innervation-like pattern around arteries, 2) Associated with smooth musculature of non-vascular origin and 3) In most divisions of the genital tract in the form of a distinct subepithelial plexus . Gel chromatographic evidence indicates that the observed immunoreactivity is due to the vasodilatory peptide originally isolated from the intestinal wall . Recent electron microscopical studies have shown that VIP occurs in the granules of the terminals of a unique type (p-type) of neuron . The scene is thus set for VIP to represent a new neurôtransmittor . Its known biological effects in combination with its localization to arteries and smooth muscle cells makes it tempting to speculate that VIP may function in the regulation of blood flow and muscle contractility of the male genital tract . References 1. 2. 3.
S .I . S .i . ed .) M.i .
SAID and V. MUTT, Science 169 1217-1218 (1970) . SAID, In : Gastrointestina~fHormones (J .C . Thompson, Austin, University o Texas Press, p. 591-597 (1975) . GROSSMAN, Gastroenteroloc~r 67 730-755 (1974) .
508
4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
Vaeoactive Peptide in Male Genital Tract
Vol . 21, No . 4, 1977
J .M . POLAR, A .G .E . PEARSE, J .-L . GARAUD and S .R . BLOOM, Gut 15 720-724 (1974) . .I S .SAID and R,N . ROSENBERG Science 192 907-908 (1976) . L .-I . LARSSON, J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, F . SUNDLER, R. HAKANSON, J.F . REHFELD, Proc . Nat . Acad . Sci. U .S .A . 7 3 3197-3200 (1976) . L .-I . LÂRSSON, L . EDVINSSON, J . FAHRENKRUG, R . HAKANSON, C . OWMAN, O. SCHAFFALITZKY DE MUCKADELL, F . SUNDLER, Brain Res . 113 400-404 (1976) . M .G . BRYANT, S .R . BLOOM, J,M, POLAK, R .E . ALBUQUERQUE, I . MODLIN, A .G .E . PEARSE, Lancet _I 991-993 (1976) . J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, A . FAHRENKRUG, Brain Res . 124 581-584 (1977) . L .-I . LARSSON, J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, Science (in press) . J . FAHRENKRUG, O . SCHAFFALITZKY DE MUCKADELL, J . Lab . Clin , _Med . (in press) . L .A . STERNBERGER, Immunoc tochemistr , Prentice-Hall Inc ., Englewood Cliffs, N,J, T4 .