THE ANATOMICAL RECORD 233:18-24 (1992)

lmmunocytochemistry of Paraneurons in the Female Urethra of the Horse, Cattle, Sheep, and Pig ALFRED0 VITTORIA, TIZIANA COCCA, ELENA LA MURA, AND ALDO CECIO Department of Biological Structures, Functions and Technology, University of Naples Federico II, 80137 Napoli, Italy

ABSTRACT The aim of this study is to describe the presence of neuroendocrine (NE) cells (paraneurons), producing biogenic amines and/or peptidergic hormones, in the female urethra of cattle, sheep, pigs, and horses, by means of histochemical and double labeling immunofluorescent techniques. 5-Hydroxytryptamine-, chromogranin A-, cholecystokinin- and somatostatin-containing NE cells are present in the urethral epithelium of all the species studied, with the unique exception of the lack of somatostatin cells in the horse. Paraneurons containing 5-hydroxytryptamine colocalized with chromogranin A or cholecystokinin were also found in all subjects. Such active substances are hypothesized to play a role in the contraction of the urethral musculature, emission of urogenital fluids, and inhibition of endocrine and exocrine secretions. o 1992 Wiley-Liss, Inc. “Neuroendocrine (NE) cells” (Pearse, 1977) or “paraneurons” (Fujita, 1976, 1980) are amine- andlor peptide-producing cells scattered in the tunica mucosa of many organs of the body, such as lung, stomach, intestine, urinary bladder, and urethra. Their secretory granules contain three structural acidic proteins, chromogranins A, B, and C (Winkler, 1976; Lloyd and Wilson, 1983; O’Connor, 1983; Rindi et al., 1986), whose distribution shows intra- and interspecific variations, type A being more frequent. Chromogranins are considered “marker” proteins of paraneurons. The presence of NE cells in the human urogenital tract of both sexes has long been recognized through histochemical argyrophil and argentaffin techniques (Pretl, 1944; Feyrter, 1951). Later, they were studied through light and fluorescence histochemistry for monoamines in the male and female cat urethra (Owman et al., 1971) and in the urethroprostatic complex of the guinea pig (Hakanson e t al., 1974) and rabbit (Vittoria et al., 1983; Paino et al., 1985). Recent ultrastructural (Di Sant’Agnese and De Mesy Jensen, 1984a; Fetissof et al., 19831, immunohistochemical (Di Sant‘Agnese et al., 1985), and immunofluorescence (Cecio et al., 1989) studies demonstrated the existence of different types of NE cells in the urethroprostatic complex of man and other mammals. On the basis of these studies, 5-hydroxytryptamine (5-HT or serotonin) (Abrahamsson et al., 1986)-, somatostatin (Di Sant’Agnese and De Mesy Jensen, 1984b)-, calcitonin (Fetissof et al., 1986, 1987; Di Sant’Agnese, 1986), bombesin (Di Sant’Agnese, 1986), and thyroid-stimulating hormone (TSH) (Abrahamsson and Lilja, 1989)containing cells have been described in man, the first cytotype being more numerous than the others. As far as the other mammals are concerned, serotonin-containing cells were also present in the buffalo (Vittoria et al., 19861, dog (Hanyu et al., 19871, and sheep (Vittoria et al., 1985,1990) and, finally, somatostatin-containing cells were found ir, the sheep (Cecio et al., 1986; Vittoria et al., 1990). 0 1992 WILEY-LISS, INC.

There are, to our knowledge, only three immunohistochemical reports dealing with NE cells of the female urethra. They describe the presence of 5-HT-containing cytotypes in woman (Fetissof et al., 1983,1987)and dog (Hanyu et al., 1987). We have studied immunohistochemically the presence and distribution of NE cells containing serotonin and several peptides in the female urethra of cattle, sheep, pigs, and horses. Colocalizations in the same cytotype of the amine and chromogranin A or cholecystokinin (CCK) are also described. MATERIALS AND METHODS

The urethra of adult, female cattle, sheep, pigs, and horses was collected in a local slaughterhouse, soon after the death of the animals. Sampling was performed from the proximal and distal halves of the organ. The material was fixed in Bouin’s fluid or in 4% paraformaldehyde in phosphate buffer (pH 7.3,O.l M), routinely dehydrated, and embedded in Paraplast. Sections were cut a t 4-5 pm thickness and stained by means of histochemical, immunohistochemical, and immunofluorescence techniques. The histochemical study included the argentaffin method of Masson-Hamperl, as modified by Singh (19641, and the argyrophil method of Grimelius (1968) and Linder (1978). The latter has been recently applied to the study of NE cells (Cecio et al., 1985, 1988). Immunohistochemistry was performed by the peroxidase-antiperoxidase (PAP) procedure of Sternberger (19791, and the site of the immunological reaction was demonstrated by diaminobenzidine (DAB). The working dilutions of the antisera used were achieved starting from 1:lOO and, through gradual dilutions, up to the best definition of positivities and the best background conditions. Negative controls

Received June 17, 1991; accepted November 18, 1991.

PARANEURONS IN THE FEMALE MAMMALIAN IJRETHRA

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TABLE 1. Antisera used

Antigen Chromogranin A Serotonin

Source Incstar (Stillwater, MN) Incstar

Code No. 20086

Raised in Peculiarities of the antibodies used Rabbit Against native porcine chromogranin A

20079

Goat

Against serotonin conjugated to bovine serum albumine BSA Serotonin Incstar 20080 Rabbit Against serotonin conjugated to BSA; nonspecific staining of capillaries removed by BSA addition to antiserum CCK-8 Incstar 20078 Rabbit Against CCK (26-33)coupled to keyhole limpet haemocyanin (KLH) A 556 Somatostatin DakoiAccurate Rabbit Against synthetic cyclic (1-14) human somatostatin; (Westbury,NY) reacts with somatostatin of various mammalian species Bombesin Immunonuclear Co. 30H2T Rabbit Against synthetic bombesin conjugated to (Stillwater, MN) thyroglobulin; some collagen weakly stained without normal serum preincubation of tissues Calcitonin Immunonuclear Co. 25H2T Rabbit Against synthetic human calcitonin 05H2T Gastrin 1-17 Immunonuclear Co. Rabbit Against human gastrin (1-17) conjugated to KLH ACTH Incstar 24H2T Rabbit Against porcine pituitary ACTH (1-39) Substance P Incstar 20064 Rabbit Against synthetic substance P conjugated to KLH 6751002 Substance P U.C.B. Against synthetic substance P conjugated to Rabbit (Braine-L'Alleud,Belgium) thyroglobulin Against porcine VIP; no cross reactivity with motilin, VIP Incstar 20077 Rabbit pancreatic polypeptide, gastric inhibitory polypeptide, peptide histidine isoleucine, insulin, glucagon, somatostatin, gastrin, ACTH, serotonin, substance P, histamine and human or rat growth hormone releasing factor; light cross reactivity with secretin Insulin Incstar 20056 Guinea pig Against beef/pork insulin CGRP Milab B-GP-470-1 Guinea pig Against synthetic human CGRP; no cross reactivity (Malmo. Sweden) with calcitonin

were obtained by preabsorbing 10-100 p,g of each antigen with 1 ml of the diluted antiserum. Positivities were abolished in all cases. The colocalization of two substances in the same cell was studied by a double labeling immunofluorescence technique (Wessendorf and Elde, 1985). In the specific step, a first antibody, raised in goat and directed against 5-HT, was used in combination with a second, raised in rabbit, represented either by a n antichromogranin A antibody or a n anti-CCK one. Fluorescein isothyocyanate (FITC) and lyssamine rodamine isothyocyanate (LRITC) were the fluorochromes conjugated to the secondary antibodies. The sections were observed using a fluorescence microscope equipped with a reflecting illumination system and with two different filter sets. The first, for FITC, was composed of a 450490 nm bandpass excitation and a 525120 nm bandpass barrier filter; the second, for LRITC, was composed of a 546114 nm bandpass excitation and a 580 nm longpass barrier filter. The dilutions of the primary antibodies were lower than for light immunohistochemistry because of the scarce sensitivity of the immunofluorescence techniques. Control sections were obtained as described by the authors cited above to avoid simulations of coexistence due to 1) affinity of one of the primary antisera for the inappropriate antigen, 2 ) affinity of one of the secondary antisera for the inappropriate primary antiserum, 3) affinity of the two primary antisera

for each other, 4) affinity of the two secondary antisera for each other, 5 ) affinity of one of the primary antisera for one of the secondary, and 6) emission of both red and green lights by one of the two fluorochromes. The primary antisera used in this study and their dilutions and times of incubations are reported in Tables 1 and 2 . RESULTS

All the histochemical techniques employed gave positive results, the Linder argyrophil being more efficient in impregnating NE cells and the others in displaying fine cytoplasmic details of secretion. Argentaffin and argyrophil paraneurons were found along the mucosa of the urethra in the four species examined (Fig. 1). They were more numerous in the cow, less so in the horse, and showed intermediate patterns of diffusion in the sheep and pig. In all subjects, argyrophil paraneuions outnumbered the argentaffin paraneurons, and the distal half of the urethra contained many more NE cells than the proximal half. The general morphology of urethral NE cells has been satisfactorily studied with argyrophil techniques. They vary in shape, being round, triangular, or elongated. Round paraneurons are small, while elongated ones are voluminous and show one, two, or more cytoplasmic dendritic-like processes. The latter can be directed towards the basal membrane, the organ lumen, or the neighboring epithelium, and sometimes their

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A. VITTORIA ET AL.

TABLE 2. Technical methods

Antibody (anti-)

Chromogranin A Serotonin Serotonin CCK-8 Somatostatin Bombesin Calcitonin Gastrin 1-17

ACTH Substance P VIP Insulin CGRP

Dilution 1:1001/1:500 1:1001 1:1,500 1:3001/1:600 1:300 1:lOO 1:lOO 1:lOO 1:lOO 1:lOO 1:lOO 1:lOO 1:lOO

Incubation time Temperature (hr) (“C) 1.30 1.30 1.30 16 16 24 24 24 24 24 24 24 24

37 37 37 4 4 4 4 4 4 4 4 4 4

‘For immunofluorescence

endings show round or oval synaptic-like expansions clearly in contact with cell bodies or, more rarely, with cytoplasmic processes of other NE cells. Elongated, multipolar paraneurons are numerous in the horse. Generally, urethral NE cells are isolated in the exocrine epithelium, but sometimes they are found grouped in clusters in which few exocrine cells separate the neuroendocrine ones from each other. In all species examined, a large number of paraneuions reacted with the antichromogranin A (Fig. 2A) and antid-HT (Fig. 2B) antibodies. Both cellular types were widely diffused in the distal portion of the urethra, confirming, as expected, the results obtained through histochemistry. The former cellular type was more diffuse than the latter. Chromogranin A- and 5HT-containing paraneurons are generally voluminous and elongated in shape due to the presence of varyingly oriented cytoplasmic processes. The double-labeling immunofluorescent staining of urethral sections showed the presence of cells containing both the amine and the peptide in all species (Fig. 2C,D). In particular, they were numerous in the cow, rare in the sheep and horse, and intermediate in frequency in the pig. CCK-containing NE cells (Fig. 3A) were fairly numerous in all the animals studied with the unique exception of the pig, in which they were rare. Their morphology was very similar to that of chromogranin Acontaining paraneurons, but the former are much less frequent. By means of the double-labeling immunofluorescent technique, CCK was found colocalized with 5-HT (Fig. 3B,C) in cells fewer in number than those containing both the amine and chromogranin A. Somatostatin-containing NE cells (Fig. 4)were rare in the cow, sheep, and pig and were absent from the horse. They were generally round and small and sometimes showed a unique cytoplasmic process directed towards the basal membrane. The findings described above are summarized in Table 3. The antiadrenocorticotropic hormone (ACTH), bombesin, calcitonin, gastrin, substance P, vasoactive intestinal polypeptide (VIP), insulin, and calcitonin gene-related peptide (CGRP) antibodies gave negative results. DISCUSSION

The seminal colliculus of the male urethra of many mammals contains a large number of NE cells whose

peptides are retained to regulate the contraction of its walls. Such a segment probably plays a determining role in the emission of urogenital fluids (Di Sant’Agnese and De Mesy Jensen, 1985; Hanyu et al., 1987; Cecio and Vittoria, 1989; Vittoria et al., 1990). The distal half of the female urethra may be similar in function to the seminal colliculus in that it shows a high concentration of the same cells, as has been described for the female dog (Hanyu et al., 1987) and for the species here examined as well. NE cells of the female urethra show cytoplasmic processes reaching the organ lumen. Such cells are defined as “open” in type (Iwanaga et al., 1987) and have been also called “taste cells” (Fujita, 1976; Fujita et al., 1988) due to the hypothetical capability to receive luminal stimuli and to respond with the basal secretion of their products. On the other hand, “open” cells have been considered to be able to release active substances directly into the lumen through their cytoplasmic processes. This phenomenon has been hypothesized for gastric (Jordan and Yip, 1972; Uvnas-Wallensten, 19801, duodenal (Nilsson et al., 19871, and intestinal (Gronstad et al., 1985) paraneurons. Another modality of secretion in the neuroendocrine system is “synaptocrinia,” defined as the release of active substances in the area of contact between two cytoplasmic processes coming from different NE cells (Fujita et al., 1988). Such contacts have been noted, although rarely, in all the species studied here. In the urethra of male and female dogs, chromogranin A and 5-HT have been found to be colocalized in the vast majority of the NE cells (Hanyu et al., 1987). This finding is only partially in agreement with result from in the pelvic urethra of the male sheep, in which the percentage of colocalization of the two substances was not higher than 25% (Vittoria et al., 1990). These findings and those obtained in the present study led us to consider that the quantity of urethral NE cells containing the amine and chromogranin A colocalized might vary from one species to another. Interspecific differences in the types and distribution of chromogranins could also occur. The smooth musculature of the gut is one of the main “targets” of 5-HT, which strongly stimulates its contraction (Furness and Costa, 1982). Such a function is considered important in the onset of intestinal peristalsis (Gonella, 1981). By analogy, in the urogenital tract, serotonin could induce the contraction of the smooth musculature and the consequent expulsion of the urine andlor semen (Di Sant’Agnese and De Mesy Jensen, 1985; Hanyu et al., 1987). It has been demonstrated that the injection of the amine in the dog iliac artery causes contraction of the smooth musculature in both the urinary bladder and the urethra (Hanyu et al., 1987). Anather function of serotonin in the gastroenteropancreatic system is the stimulation of exocrine secretion (Furness and Costa, 1982). This function could also be extended to the urogenital tract and could be involved in the fertilizing capability of semen (Di Sant’Agnese et al., 1987). To our knowledge, the presence of CCK in urogenital paraneurons has never been described. CCK, as 5-HT, is known to induce the contraction of the smooth musculature of the intestinal wall (Rehfeld, 1980). In the urogenital tract, it could act synergistically

PARANEURONS IN T H E FEMALE MAMMALIAN URETHRA

21

Fig, 1, Epithelium of the female urethra. Ovine (A), bovine (B), swine (C),and equine (D) paraneurons positive via Masson-Hamper1 (A), Grimelius (B) and Linder ( C , D)techniques. x 1,300.

with the colocalized amine in inducing the emission of urogenital fluids. Somatostatin inhibits both endocrine and exocrine

gastrointestinal secretions (Reichlin, 1983a1, acting by means of a “paracrine” mechanism. The hormone is released from cytoplasmic processes in intercellular

22

A. VITTORIA ET AL.

Fig. 2. Bovine (A) and ovine (B) neuroendocrine cells containing, respectively, chromogranin A and serotonin. PAP, DAB. a, x 500. b, x 1,300. C, D: Single section of the bovine urethra showing five paraneurons containing both chromogranin A and serotonin. Double labeling immunofluorescent technique. C: Chromogranin A, FITC, D: Serotonin, LRITC. x 500.

Fig. 4. Swine paraneuron containing somatostatin. PAP, DAB. x 1.300.

TABLE 3. Frequency of occurrence of paraneurons in the female urethra' Masson-Hamper1 Grimelius Linder ChromograninA 5-HT Chrom. A-5-HT2 CCK CCK-5-HT2 Somatostatin

cow

SheeD

Pie

Horse

++ +++ ++++ ++++ ++++ +++ ++ ++ +

++ ++ +++ +++ +++ + ++ + +

++ ++ +++ +++ +++ ++ + +

+ ++ +++ +++ +++ + ++ +-

+

+ + + + , very numerous positive cells; + + + , numerous positive cells; + + ,few positive cells; + , rare positive cells; -, no positive cells. 'Double labeling stain.

spaces and acts on nearby cells (Reichlin, 198313). An analogous local inhibitory role has been hypothesized for the somatostatin produced by human prostatic paraneurons (Di Sant'Agnese and De Mesy Jensen, 1984b). The somatostatin-containing NE cells of the urethra of male sheep (Vittoria et al., 1990) and of the female animals studied here frequently showed the presence of a unique, brief cytoplasmic extension oriented towards the basal membrane. Thus a traditional endocrine mechanism of action is tenable for such cells. Fig, 3. A Equine paraneuron containing CCK and showing a long, narrow cytoplasmic process oriented towards the lumen. PAP, DAB. x 1,300. B, C : Single section of the equine urethra showing two neuroendocrine cells containing both CCK and serotonin. Double- labeling immunofluorescent technique. B: CCK, FITC; C: Serotonin, LRITC. x500.

LITERATURE CITED Abrahamsson, P.A., and H. Lilja 1989 Partial characterization of a Thyroid stimulating ~ ~ ~ ~peptide ~ in ~ neuroendocrine ~ l i cells of the human prostate gland, prostate,14:71-81,

k

~

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A. VITTORIA ET AL.

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Immunocytochemistry of paraneurons in the female urethra of the horse, cattle, sheep, and pig.

The aim of this study is to describe the presence of neuroendocrine (NE) cells (paraneurons), producing biogenic amines and/or peptidergic hormones, i...
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