Glycosylation of the male genital ducts and spermatozeugmata formation in the clearnose skate Raja eglanteria

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.     

Glycosylation of the Male Genital Ducts and Spermatozeugmata Formation in the Clearnose Skate Raja eglanteria

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.     

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.     

Evidence for immunoreactive relaxin in boar seminal vesicles using combined light and electron microscope immunocytochemistry.

Light-microscope immunocytochemistry using the peroxidase-antiperoxidase technique and a polyclonal rabbit antiserum raised against purified porcine relaxin showed that cytoplasmic immunostaining for relaxin could be visualized in the epithelial cells of the seminal vesicle. No relaxin immunoreactivity was seen in the testis, epididymis, ductus deferens, prostate or bulbo-urethral gland. A ten times higher concentration of porcine relaxin antiserum was necessary to achieve immunostaining in the seminal vesicle comparable to that in the corpora lutea of pregnant sows. Ultrastructural examination showed that the epithelial cells of the boar seminal vesicle resembled typical protein-secreting cells with prominent rough endoplasmic reticulum and well-developed Golgi apparatus. The most striking feature of these cells was the accumulation of granules with a limiting membrane, which ranged from 200 to 600 nm in diameter and contained flocculent material of moderate electron density. Electron-microscope immunocytochemistry using the protein A-gold technique and relaxin antiserum demonstrated that the granules were the only intracellular organelles that showed immunoreactivity for relaxin. These results indicate that a relaxin-like substance is present in boar seminal vesicles and that the subcellular site of its localization is the granules, suggesting that the seminal vesicle produces and stores a relaxin-like substance, but that it is present at much lower concentrations than in the corpora lutea of pregnant sows.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.     

Expression of cysteine sulfinate decarboxylase (CSD) in male reproductive organs of mice

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of taurine, but it is still controversial whether the male reproductive organs have the function to synthesize taurine through CSD pathway. The present study was thus undertaken to detect CSD expression in male mouse reproductive organs by RT-PCR, Western blot and immunohistochemistry. The results show that CSD is expressed both at the mRNA and protein levels in the testis, epididymis and ductus deferens. The relative levels of both CSD mRNA and protein increase from the testis to the epididymis and to the ductus deferens. Immunohistochemical results demonstrate that the main cell types containing CSD are Leydig cells of testis, epithelial cells and some stromal cells throughout the efferent ducts, epididymis and ductus deferens. These results suggest that male genital organs have the function to produce taurine through the CSD pathway, although quantifying the relation of CSD expression to taurine synthesis and the exact functions of taurine in male genital organs still need to be elucidated in future studies.     

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.     

Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract

The distribution of human carbonic anhydrase (HCA) isoenzymes I, II and VI in the human male reproductive tract was studied using specific antisera against affinity purified isoenzymes in conjunction with the peroxidase-antiperoxidase complex method. HCA VI-specific staining could not be demonstrated in any of the tissues studied, and HCA I was observed only in red blood cells. Immunostaining denoted HCA II in the epithelia of the seminal vesicle, ampulla of the ductus deferens and distal ductus deferens. Some cells in the epithelium of the corpus and cauda epididymidis also stained for HCA II. The staining for HCA II in the epithelium of the reproductive tract declined from the strongly positive seminal vesicle to the proximal part of the ductus deferens, which stained negatively. There were also HCA II-positive particles derived from the apical protrusions of the epithelium in the lumina of the seminal vesicle, ampulla of the ductus deferens and ductus deferens. The physiological role of HCA II is linked to the secretion of bicarbonate into the seminal plasma and thereby to the regulation of sperm motility and pH in the seminal plasma.     

Breeding and post-breeding structure of the vas deferens of the long-toed salamander Ambystoma macrodactylum

The vas deferens of Ambystoma macrodactylum is composed of a peritoneal epithelium, connective tissue layer with fibroblasts, circular smooth muscle, capillaries, cells containing lipid, and a luminal epithelium composed of a single layer of cuboidal cells covered by a net of interconnected ciliated squamous cells. The cuboidal cells have abundant rough endoplasmic reticulum, mitochondria, and PAS + secretory vesicles. Squamous cells of breeding males consistently have tufts of ～100 cilia located at one end of the long axis of each cell. These cilia may help distribute secretory products. The squamous cells, absent in post-breeding males, are apparently sloughed into the lumen. Lipid vesicles are present throughout the cytoplasm of the cuboidal and squamous epithelial cells and are also in some cells of the connective tissue layer. These vesicles increase dramatically in number during the first 4 weeks after breeding and may serve as an energy pool for the next breeding season. Enzyme-histochemical tests for testosterone synthesis were negative. In addition to the accumulation of lipid and the loss of squamous cells in the vas deferens, after breeding PAS + vesicle production is terminated. These alterations appear to represent energy conservation strategies employed by the sperm-depleted vas deferens.     

Development of secretory activity in the seminal vesicle of the male migratory grasshopper,Melanoplus sanguinipes (fabr.) (Orthoptera : Acrididae)

This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing patterns of its secretion during sexual maturation and after allatectomy in Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). In epithelia from seminal vesicles of newly fledged males, the rough endoplasmic reticulum is well developed, and Golgi complexes are elaborate, which indicates the gland is metabolically active. The cells also contain large glycogen deposits and the lumen microvilli are well differentiated. These ultrastructural features are more dominant in 24-hr-old adults where the cytoplasm is clearly differentiated into basal and apical regions. Basally, the cytoplasm is dominated by rough endoplasmic reticulum, large Golgi complexes, glycogen deposits and numerous mitochondria, while the apical cytoplasm is filled with large secretory and/or lysosomal vesicles. Between days 3 and 7, the ultrastructural features change little other than the rough endoplasmic reticulum cisternae, which become vesicular. Analysis by isoelectric focusing shows that the amount of secretory protein increases with age until day 3, at which time the gland contains its full complement of secretion. In seminal vesicles from allatectomized insects, ultrastructural features of cells and isoelectric focusing patterns of the secretion arc identical to those from normal males.     

Buck (Capra hircus) genes encode new members of the spermadhesin family

Spermadhesins are the major proteins of boar seminal plasma and form a group of polypeptides probably involved in reproduction. In previous work, a member of the spermadhesin family from buck seminal plasma, called BSFP, was characterized by mass spectrometry and N-terminal sequencing. The present study aimed to clone and characterize the BSFP gene and investigate its expression along the genital tract using real-time polymerase chain reaction (PCR). The cDNAs of the seminal vesicle, testis, epididymis, bulbourethral gland, and ductus deferens were prepared from a buck. Following 3'- and 5'-end amplifications using seminal vesicle cDNA, we cloned and sequenced four highly similar (97-98%) nucleotide sequences encoding spermadhesins, which were named Bodhesin-1(Bdh-1), Bdh-2, Bdh-3, and Bdh-4. All deduced amino acid sequences contained the CUB domain signature and were 49-52% similar to boar AWN. Among the four Bdh amino acid sequences, Bdh-2 was the most similar to the BSFP N-terminal fragment. By using real-time PCR, it was verified specific amplifications for all Bdh in the seminal vesicle, testis, epididymis, and bulbourethral gland, with the exception of Bdh-2 in epididymis. The amplicons had a melting temperature and size of approximately 78 degrees C and 130 bp, respectively. Bdh expression was higher in the seminal vesicle when compared to the other tissues. The present work confirms that goat is the fifth mammalian species, after pig, cattle, horse, and sheep, in which spermadhesin molecules are found. To the best of our knowledge, this is the first report on buck spermadhesin genes using molecular cloning and expression profile.     

Ultrastructure of the Olfactory Epithelium in the Australian Lungfish Neoceratodus forsteri

Four cell types are present in the olfactory epithelium of Neoceratodus forsteri, i.e., olfactory receptor cells, supporting cells, non-sensory ciliated cells, and basal cells. Only microvilli and no cilia were observed on the receptor cells. The neurotubules pass out into these microvilli. Conspicuous arrays of agranular endoplasmic reticulum are present in the nuclear region of the receptor cells. The supporting cells are provided with microvilli. These cells may be secretory. The non-sensory ciliated cells produce secretory granules containing acid mucopolysaccharides. A discontinuous zonula occludens appears to be present.     

Ultrastructure of the male reproductive system and spermatophore formation in Labidocera aestiva (Crust.: Copepoda)

Summary The male reproductive system of Labidocera aestiva produces a flask-shaped spermatophore connected to a chitin-like coupling apparatus. As immature spermatozoa leave the anterior region of the testis, they pass through the lumen of a long, sinuous duct composed of a ductus deferens and seminal vesicle. Ultrastructural examination of the ductus deferens reveals a highly glandular, columnar epithelium. The cells contain arrays of rough endoplasmic reticulum and abundant, well-developed Golgi complexes. This region produces and releases into the lumen, a flocculent substance and two granular secretions that constitute the seminal fluid. In its terminal part, the ductus deferens synthesizes another secretion that forms the spermatophore wall enclosing the spermatozoa and seminal fluid. Final synthesis of the spermatophore wall occurs within the thin-walled seminal vesicle, although this region functions primarily as a storage organ. Contiguous to the seminal vesicle is an elongate, highly glandular spermatophore sac. The chitin-like coupling apparatus, which functions to attach the spermatophore to the female, is formed in the anterior region of the sac by secretions from eight cell types. The posterior region of the sac stores the flask-shaped spermatophore and produces secretions that aid ejaculation of the entire spermatophore complex.Contribution No. 236, Harbor Branch Foundation, Inc.     

Immunochemical distribution and immunohistochemical localization of 20β-hydroxysteroid dehydrogenase in neonatal pig tissues

Immunochemical distribution of 20β-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20β-HSD antibody. 20β-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20β-HSD were detected in the testis, followed by the kidney and liver, by the [¹²⁵I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20β-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20β-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20β-HSD has a very important physiological role in testicular function during the neonatal stage.     

Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract

Summary The distribution of human carbonic anhydrase (HCA) isoenzymes I, II and VI in the human male reproductive tract was studied using specific antisera against affinity purified isoenzymes in conjunction with the peroxidase-antiperoxidase complex method. HCA VI-specific staining could not be demonstrated in any of the tissues studied, and HCA I was observed only in red blood cells. Immunostaining denoted HCA II in the epithelia of the seminal vescle, ampulla of the ductus deferens and distal ductus deferens. Some cells in the epithelium of the corpus and cauda epididymidis also stained for HCA II. The staining for HCA II in the epithelium of the reproductive tract declined from the strongly positive seminal vesicle to the proximal part of the ductus deferens, which stained negatively. There were also HCA II-positive particles derived from the apical protrusions of the epithelium in the lumina of the seminal vesicle, ampulla of the ductus deferens and ductus deferens. The physiological role of HCA II is linked to the secretion of bicarbonate into the seminal plasma and thereby to the regulation of sperm motility and pH in the seminal plasma.     

Observations on the ultrastructure of the copulatory organ of Archilopsis unipunctata (Fabricius, 1826) (Proseriata,Monocelididae)

The copulatory organ in adult specimens of Archilopsis unipunctata has been studied by transmission electron microscopy.This copulatory organ is of the conjuncta-duplex type with eversible cirrus. The seminal vesicle, lined with a nucleate epithelium, is surrounded by spirally arranged muscles. The fibres are enclosed in a sheath that is continuous with the septum of the bulbus and the basement lamina of the male canal epithelium. Distally to the seminal vesicle the bulbus is filled with the secretory cell-necks of the prostate glands. The male canal shows three different parts: seminal duct, ejaculatory duct and eversible cirrus. At the transition of seminal duct and ejaculatory duct two prostate ducts open into the lumen. The structure of the epithelium lining the different parts of the canal is described. The transition into the cirrus may be recognized by an abrupt change in the thickness, the electron density and the stratification in the basement lamina and by the disappearance of the epithelium absent indeed in the cirrus. The material found inside the cirrus-lumen is different according to the zone considered. The origin of this material and of the cirrus teeth is discussed.Abbreviations ab- apoptotic body - ba- bacteria - bb- basal bodies of cilia - bl- basement lamina - bw- body wall - c- cilia - cb- cell body - cgp- common genital porus - ci- cirrus - cip- cirrus plug - cl- lumen of cirrus - cm- circular muscles - cr- cytoplasmatic remnants - cs- cytoplasmatic sheets - ejd- ejaculatory duct - ep_ej- epithelium of ejaculatory duct - d- desmosomes - f- flagella of spermatozoa - fd- female duct - fp- female porus - gc- golgi complex - gl- glycogen particles - hd- hemidesmosomes - lm- longitudinal muscles - ly- lysosome-like body - m- muscles - m_b- muscles of the bulbus - m_c- muscles of the cirrus - m_c- muscles of the seminal vesicle - mi- mitochondria - ml- microvilli - ms- mesenchyme - n_sd- nuclei of the seminal duct - pd- prostate duct - pg- prostate glands - ri- ribosomes - s- septum - sb- secretory vesicle - sd- seminal duct - sp- spines - sv- seminal vesicle - v- vagina - vd- vas deferens     

Histogenesis of human extraparenchymal Leydig cells

From 64 consecutive autopsies of patients with neither testicular nor hormonal pathology, 26 showed extraparenchymal Leydig cells, located mainly in the epididymis and in the spermatic cord. The ultrastructural study of these specimens plus those obtained from 2 patients affected with functional testicular tumors leads to the following conclusions: (1) The origin of ectopic Leydig cells is not interstitial Leydig cells having infiltrated the testicular nerves and migrated along them towards ectopic locations. (2) The ectopic Leydig cells are considered to develop from undifferentiated precursor cells, located extraparenchymally, mainly inside and beside the testicular nerves. These precursor cells are similar to those observed in the testicular interstitium and have an ovoid shape and some cytoplasmic projections. The cytoplasm contains vesicles of smooth endoplasmic reticulum, lysosomes, lipid droplets and abundant microfilament bundles. The transformation from these cells into mature Leydig cells implies a progressive differentiation of the cytoplasmic components involved in steroid biosynthesis.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat

Summary The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vescicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. An immunohistochemical study.

The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vesicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.