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. 2009 Mar;214(3):330–338. doi: 10.1111/j.1469-7580.2008.01038.x

Peptidergic not monoaminergic fibers profusely innervate the young adult human testis

Yong-Guang Gong 1,*, Ming-Ming Feng 2,*, Xiang-Nong Hu 3, Yi-Qiu Wang 2, Min Gu 2, Wei Zhang 2, Ren-Shan Ge 4
PMCID: PMC2673784  PMID: 19245500

Abstract

Numerous studies have reported that intratesticular nerves exert important regulatory effects on the functions of the male gonad; however, as yet little is known about their distribution in the young adult human testis. The purpose of this study was to explore whether peptidergic and adrenergic nerves occur in the male gonad of this age, and, if present, to depict their distribution further. Thirty testes were collected from 15 reproductively healthy donors aged 21–32 years. Antibodies against protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), C-terminal flanking peptide of NPY (CPON) and vasoactive intestinal peptide (VIP) were employed for immunohistochemical detection of intratesticular peptidergic nerves, and those against dopamine-beta-hydroxylase (DBH) and 5-hydroxytryptamine (5-HT) for monoaminergic ones. The testicular parenchyma exhibited a rich innervation by PGP 9.5-positive fibers, mainly associated with Leydig cell nests, blood vessels, and seminiferous tubules. Numerous NPY- and CPON-immunoreactive (IR) nerves also appeared in the gonads, but the vast majority were confined to blood vessels. A small number of VIP-IR fibers were detected in some arterioles. By contrast, however, no fibers displaying DBH or 5-HT immunoreactivity were observed within the testis. Additionally, expression of PGP-9.5, NPY, CPON, VIP, DBH and 5-HT was found in Leydig cells, PGP 9.5 in spermatogonia, and NPY and CPON in peritubular myoid cells. Our results suggest that the young adult human testis is devoid of monoaminergic nerves but profusely innervated by peptidergic fibers, which may serve as major neuronal regulators for testicular functions at this age.

Keywords: C-terminal flanking peptide of neuropeptide Y, dopamine-beta-hydroxylase, 5-hydroxytryptamine, human, nerve, neuropeptide Y, protein gene product 9.5, testis, vasoactive intestinal peptide

Introduction

Testosterone synthesis and secretion, major functions of the male gonad, are classically considered to be under the control of the hypothalamic-pituitary-testicular axis and intratesticular paracrine and autocrine factors (Saunders, 2003; Holdcraft & Braun, 2004). However, testicular innervation, which derives from the superior spermatic nerves (SSNs) and the inferior spermatic nerves (ISNs) (Kuntz & Morris, 1946; Rauchenwald et al. 1995), has also been shown to have an important role in this process. In the rat, for instance, bilateral SSN section blunts the human chorionic gonadotropin stimulated testosterone production, reduces the number of testicular LH receptors (Campos et al. 1993), and inhibits the acute stress-induced rise of serum testosterone (Frankel & Ryan, 1981), whereas electrical stimulation of SSNs induces an increase in the testosterone levels of the spermatic veins in cats (Chiocchio et al. 1999). Recently, Chow et al. (2000) found that sectioning of the spermatic nerves results in significant regression of seminiferous epithelia, suggesting an indispensible role of the spermatic nerves for spermatogenesis. Investigation of their distribution in the male gonads thus becomes essential for deepening understanding their regulatory roles in male reproductive physiology.

Testicular neuronal supply reaches the gonad via SSNs and ISNs. SSNs derive from the mesenteric and renal plexuses and accompany the testicular artery to reach the testis. ISNs, originating from the pelvic and inferior mesenteric plexuses, run along the vas deferens and penetrate the epididymis to enter the gonad (Kuntz & Morris, 1946; Setchell et al. 1994). Most testicular nerves seem to be catecholaminergic (Bell & McLean, 1973; Campos et al. 1990a,b; Zhu et al. 1995; Wrobel & Moustafa, 2000), but peptidergic fibers have also been found in the testis of a vast majority of the species investigated so far (Tainio, 1994; Zhu et al. 1995; Wrobel & Brandl, 1998; Wrobel & Moustafa, 2000; Wrobel & Gürtler 2001; Saleh et al. 2002; Suburo et al. 2002). These two types of nerve fibers are, therefore, considered to be the most prominent subpopulations in the testis of most mammalians.

Interestingly and surprisingly, a significant age-related difference in testicular innervation has been reported in non-human mammalian species. In rhesus monkeys, for instance, the juvenile testes contain abundant adrenergic fibers, but the adult ones are sparsely innervated (Mayerhofer et al. 1996). Wrobel & Brandl (1998) discovered that 3–5-week-old piglets had the most intense and most constant intratesticular innervation, whereas the testes of adult boars were completely devoid of intrinsic nerves. The age-dependent variance of testicular innervation is postulated to reflect its distinct functions at different age stages. An important question then arises whether this also occurs to human beings. Using electron microscopy, Prince (1984,1992,1996) observed abundant adrenergic nerves in children's testes. Tainio (1994) reported the presence of intratesticular peptidergic and adrenergic fibers in aged men. However, testicular innervation of young adult men, the group with the highest reproductive activity, is still unknown. To resolve this question, the present study was carried out to explore whether peptidergic and adrenergic nerves occur in the young adult human testis and, if present, further to depict their distribution.

Materials and methods

Tissue preparation

Thirty testes were obtained from 15 reproductively healthy donors aged 21–32 years who underwent brain death. Immediately after removal and washing with PBS, samples of suitable size (1 × 0.5 × 0.5 cm) were taken from the whole testis. These tissue blocks were fixed in two steps as described by Wrobel & Moustafa (2000). The samples were first immersed for 30 min in fixative I, which contains 4% paraformaldehyde, 15% saturated picric acid and 0.1% glutaraldehyde in 0.1 m phosphate buffer. Fixative II, with the same composition as fixative I but without glutaraldehyde, was used for further fixation for several hours. The blocks were subsequently transferred into a graded series (10%, 20%, 30%) of saccharose-containing rinsing buffer and immersed in Tissue OCT compound for serial cryostat sections (14 µm thick). Before immunohistochemical analysis, all these testes underwent histological examination and showed normal spermatogenesis.

Immunohistochemical procedures

Cryosections were air-dried for 12 h at 24 °C and subsequently incubated in 0.3% hydrogen peroxide in methanol for 10 min at room temperature to remove endogenous peroxidase activity. All sections were treated for 30 min with rabbit normal serum (1 : 100 dilution) to reduce non-specific binding of the secondary antibody, and then incubated with primary antibodies against protein gene product 9.5 (PGP-9.5), neuropeptide Y (NPY), C-terminal flanking peptide of neuropeptide Y (CPON), vasoactive intestinal peptide (VIP), dopamine-beta-hydroxylase (DBH) and 5-hydroxytryptamine (5-HT) overnight (Table 1). All antibodies were diluted in PBS containing 0.01% bovine serum albumin, 0.03% Triton-X100, and 0.01% sodium azide (PBS-X). Finally, sections were incubated with diluted (1 : 50) extravidin-horseradish peroxidase (Sigma/Aldrich Quimica, Madrid, Spain). Between each step, the sections were completely washed with PBS-X. After diaminobenzidine reaction, nuclear counterstaining with Mayer's haematoxylin was applied. Sections were dehydrated, cleared, and mounted with Pet-mount. The specificity of the staining reaction was determined in control experiments. Negative control was obtained by substitution of the primary antibody with PBS or normal rabbit serum (1 : 100), omission of both primary and secondary antibodies, and blocking of the primary antibody by pre-incubation with the matching antigen in excess. Positive reactions against DBH and 5-HT were conducted in medullary chromaffin cells of rat adrenal glands (Verhofstad et al. 1985; Lloyd et al. 1986) and enterochromaffin (EC) cells of human antral gastric mucosae (Bordi et al. 2000; Tzaneva, 2003), respectively.

Table 1.

Characteristics of antibodies used in this study

Primary antibody Abbreviation Source Type Host Dilution
Protein gene product 9.5 PGP 9.5 Abcam Ltd. (UK) po Rabbit 1 : 1000
Neuropeptide Y NPY Abcam Ltd. (UK) po Rabbit 1 : 2000
Terminal flanking peptide of NPY CPON Abcam Ltd. (UK) po Rabbit 1 : 1000
Vasoactive intestinal peptide VIP Abcam Ltd. (UK) po Rabbit 1 : 200
Dopamine-beta-hydroxylase DBH Chemicon (USA) po Rabbit 1 : 2000
5-Hydroxytryptamine 5-HT Abcam Ltd. (UK) po Rabbit 1 : 200
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Quantification of the density of nerve fibers in the testis

Ten non-sequential sections per testis were randomly chosen for analysis under an Olympus microscope. To quantitate the density of nerve fibers in the testis, a gridded eyepiece was used at ×200 microscopic magnification, which provided an area of 0.04 mm2. The grid was placed at 25 randomly selected non-overlapping observation fields per section, which amounted to a total area of 1 mm2. Each type of peptidergic and monoaminergic fiber was counted separately based on location: (1) in association with Leydig cells, (2) along the seminiferous tubules, (3) around or within vascular walls. The counts obtained from different measurements on each section were averaged to obtain a median value per testis. A statistical analysis of the data was then performed by using a one-way analysis of variance (anova) followed by Tukey test. All results were expressed as mean ± SEM. Values of P < 0.05 were considered to be statistically significant.

Ethics

This study was approved by the Human Ethics Committees of Nanjing Medical University, and all the donors gave their formal consent for use of their mortal remains in medical research when they were alive.

Results

Peptidergic nerve fibers in the young adult human testis

The frequency of all peptidergic nerves is shown in Fig. 1.

Fig. 1.

Fig. 1

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Frequency of peptidergic and monoaminergic nerve fibers recorded on basis of their location. (#P < 0.05 vs. NPY and CPON, *P < 0.05 vs. NPY, CPON and VIP, •P < 0.05 vs. NPY and CPON).

Abundant nerve fibers exhibiting PGP 9.5 immunoreactivity were present in the interstitial compartments of the testis. Most of them showed a close association with Leydig cells, surrounding or penetrating the Leydig cell nests (Fig. 2A), whereas others were adjacent to the seminiferous tubules (Fig. 2B). All intratesticular blood vessels (except the large-sized arterioles) were innervated by PGP 9.5 fibers that are present around and within the vascular walls (Fig. 2C,D). Leydig cells and spermatogonia showed an intense expression of PGP 9.5 (Fig. 2E,F).

Fig. 2.

Fig. 2

Fig. 2

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Immunohistochemistry of PGP 9.5, NPY and CPON in the young adult human testis. PGP 9.5-, NPY- and CPON-IR nerves (black arrow) were localized in contact with Leydig cells (empty arrow) (A,G,M), along seminiferous tubules (B,H,N), and around and within arterioles (C,I,O) and veins (D,J,P). Expression of PGP 9.5, NPY and CPON was detected in Leydig cells (empty arrow) (E,K,Q), PGP 9.5 in spermatogonia (open arrow) (F), and NPY and CPON in peritubular myoid cells (open arrow) (L,R). Asterisk, cross-section of a nerve bundle; T, seminiferous tubules; L, large-sized arterioles; M, medium-sized arterioles; V, veins.

A profuse network of NPY-labeled nerves occurred in the testis. Besides a small proportion localized in contact with Leydig cell nests (Fig. 2G) or in the vicinity of seminiferous tubules (Fig. 2H), the vast majority of these fibers were confined to intratesticular blood vessels, including all types of arterioles and veins (Fig. 2I,J). NPY staining of moderate intensity was seen in both Leydig cells (Fig. 2K) and peritubular myoid cells (Fig. 2L).

CPON-positive nerves exhibited the same distribution pattern as NPY fibers, with similar frequency and location (Fig. 2M–P). Leydig cells and the peritubular myoid cells also showed a moderate staining of CPON (Fig. 2Q,R).

A small number of VIP-IR fibers were observed in some intratesticular arterioles (Fig. 3A,B). A moderate staining for this protein was detected in Leydig cells as well (Fig. 3C).

Fig. 3.

Fig. 3

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VIP-IR nerves (black arrow) were observed in arterioles (L, large-sized arterioles; M, medium-sized arterioles) (A), but not in veins (V) (B). Leydig cells (empty arrow) displayed an intense staining of VIP (C). Monoaninergic nerves immunoreactive to DBH and 5-HT were absent from testicular tissues including the arterioles (D,G) and veins (E,H), whereas Leydig cells (empty arrow) showed an expression of DBH and 5-HT (F,I). In positive controls, immunoreactivity against DBH and 5-HT was shown in medullary chromaffin cells (empty arrow) of rat adrenal glands (J) and EC cells (empty arrow) of human antral gastric mucosae (K). No positive staining was detected in the negative control (L). To test whether some non-specific staining occurred in our immunohistochemical procedures, PGP 9.5 antibody and NPY antibody were chosen to stain sections without haematoxylin counterstaining, in which no significant background staining was observed (black arrow, nerves; empty arrow, Leydig cells; open arrow, peritubular myoid cells; asterisk, cross-section of a nerve bundle; L, large-sized arterioles; M, medium-sized arterioles (M,N).

Monoaminergic immunohistochemistry in the young adult human testis

DBH-IR fibers were not observed in any of the 30 testes (Fig. 3D,E), but a moderate expression of this protein was demonstrated in the cytoplasm of Leydig cells (Fig. 3F). Nerve fibers containing 5-HT were also not detected in the testis of this age (Fig. 3G,H), while Leydig cells displayed a staining for this marker of the same intensity (Fig. 3I). In positive controls, immunoreactivity against DBH and 5-HT was shown in medullary chromaffin cells of rat adrenal glands (Fig. 3J) and EC cells of human antral gastric mucosae (Fig. 3K), respectively.

The cell types with positive staining for the six neuronal makers and their intensity are summarized in Table 2.

Table 2.

Summary of the cell types with positive staining for peptidergic and monoaninergic neuronal makers and their intensity

Positive cell types
Markers Leydig cells Spermatogonia Myoid cells
PGP 9.5 +++ ++ −
NPY ++ − ++
CPON ++ − ++
VIP ++ − −
DBH ++ − −
5-HT ++ − −
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The relative intensity of each neuronal marker in certain cell types is recorded using the following scoring system: −, no staining; +, weak staining; ++, moderate staining; +++, intense staining.

In immunohistochemistry, non-specific staining may be produced in some structures, e.g. collagen bundles, due to fixation with glutaraldehyde and/or the suboptimal preservation of tissues. To test whether this occurred in our study, we chose PGP 9.5 antibody and NPY antibody to stain sections without haematoxylin counterstaining. In these sections, there was no significant background staining (Fig. 3M,N).

Additionally, Mayerhofer et al. (1999) reported a dramatic increase of catecholaminergic neuron-like cells in the adult human testis with Sertoli cell only (SCO) syndrome or severe hypospermatogenesis and germ cell arrest (GA). Here, we examined all the sections and found no peptidergic and monoaminergic neuron-like cells in the gonads of young adult men with normal reproduction (not discussed further).

Discussion

This is the first study, to our knowledge, exploring the occurrence and distribution of peptidergic and monoaminergic nerve fibers within the gonads of young adult men. The novel finding of our study is that the testis of this age is richly innervated by peptidergic nerves, but devoid of monoaminergic nerves.

Monoaminergic peripheral nerves are made up of two types of fibers, adrenergic fibers and serotoninergic fibers. Intratesticular adrenergic nerves have been shown to exert regulatory effects on Leydig cell steroidogenesis (Mayerhofer et al. 1989,1993; Chiocchio et al. 1999; Frungieri et al. 2002), testicular blood supply (Chiocchio et al. 1999), and contractility of tubules (Miyake et al. 1986a,b; Yamamoto et al. 1987). 5-HT, the neurotransmitter of serotoninergic nerves, also serves as a modulator for testosterone production by Leydig cells (Csaba et al. 1998).

In past decades, the study of monoaminergic innervation of human testis has been limited, only adrenergic nerves being investigated. Using electron microscopy, Prince (1984,1992,1996) examined autonomic nerves in human testis from the neonatal period, childhood and puberty, and revealed an age-dependent change. He found that newborn gonads exhibited no adrenergic innervation. During childhood (3–10 years of age), however, many adrenergic nerves and terminals, which associated with immature Leydig cells and seminiferous tubules, appeared in the children's testes. With the beginning of puberty, their number fell to a low level. Tainio (1994) utilized antibody against tyrosine hydroxylase (TH), a marker for adrenergic fibers, to perform immunohistochemical detection of adrenergic nerves in testes from old men aged 58–74 years. Within the male gonads of this age group, some TH-positive fibers were observed localized between seminiferous tubules and around blood vessels. Based on these studies, an age-related variance of testicular adrenergic innervation also seems to occur to human beings. However, a definite conclusion on this point cannot yet be reached, as testicular innervation of young adult men remains unknown. In the present study, therefore, we collected testes from men aged 21–32 years and a specific antibody against DBH, an ideal marker recognizing adrenergic fibers, was selected to investigate adrenergic innervation in them. Surprisingly, within the testis of this age, no fiber displaying DBH immunoreactivity could be detected, although DBH staining was clearly shown in Leydig cells. As antibody against DBH not TH was employed in our study, one may attribute this to an inappropriate choice of the antiserum. However, this is impossible because, unlike TH expressed by both intratesticular nerves and germ cells, DBH is exclusively present in adrenergic nerve fibers and thus its immunohistochemistry enables visualization of even the finest axons between the seminiferous tubules due to complete absence of any background staining (Wrobel & Moustafa, 2000). Furthermore, the expression of DBH detected in medullary chromaffin cells of rat adrenal glands as well as human Leydig cells suggested that the DBH antibody used in this study worked well. Similarly, we did not observe serotoninergic fibers with 5-HT antibody in these testes, but 5-HT staining was displayed in EC cells of human antral gastric mucosae and also in human Leydig cells using the same antibody. Based on these findings, it can be concluded that the young adult human testis is devoid of monoaminergic innervation. Taken together, the results of the present study and those reported by Prince and Tainio reveal a significant age-dependent change of adrenergic innervation of human testis. Although the mechanisms underlying such a change remain to be explored, there are several lines of evidence indicating that testicular adrenergic innervation is more important for the developing testes than for the fully functional adult ones (Zieher et al. 1971; Nagai et al. 1982; Anakwe & Moger, 1984; Bergh et al. 1987; Mayerhofer et al. 1989, 1992). We postulate that the age-dependent variance of adrenergic innervation of human testis may be related to its differential functions in different age periods.

Due to limited evidence on peptidergic innervation of human testis, it is only known that human testes of newborns, adults and the aged are innervated by NPY fibers, which are located in the interstitial compartments and around blood vessels (Adrian et al. 1984; Vaalasti et al. 1986; Tainio, 1994; Jørgensen et al. 1996). Here, a panel of antibodies was used to detect peptidergic nerves in the testes from men aged 21–32 years. Using immunohistochemistry, we observed numerous PGP 9.5-, NPY- and CPON-IR fibers as well as a small number of VIP fibers present in the testes of this age, demonstrating that the young adult human testis is profusely innervated by peptidergic nerves despite the absence of monoaminergic innervation.

In the interstitial compartments, the peptidergic fibers were predominantly localized in contact with Leydig cells, around and within vascular walls, and along seminiferous tubules; the different locations may reflect their differential functions.

Many neuropeptides released from the peptidergic nerves are well known to act as modulators for steroidogenesis in Leydig cells. Pituitary adenylate cyclase-activating peptide (PACAP), for instance, stimulates testosterone secretion from Leydig cells through activation of pertussis toxin-sensitive G protein coupled to its receptors (Rossato et al. 1997), whereas substance P (SP) reduces the number of luteinizing hormone-binding sites in Leydig cells and inhibits their testosterone production (Kanchev et al. 1995). NPY could also have a role in testicular steroidogenesis, as has been suggested in the ovary (Barreca et al. 1998). Our results showed that many PGP 9.5-, NPY- and CPON-IR nerves had a close contact with Leydig cells. The intimate anatomical association enables quick and specific access and action of their neuropeptides on Leydig cells. The gap junctions between Leydig cells further make themselves able to respond promptly to the neurotransmitters as a group (Pérez-Armendariz et al. 1994). Due to lack of monoaminergic innervation, these peptidergic nerves may serve as major neuronal regulators for Leydig cell steroidogenesis in young adult men.

Blood vessels in the aged human testis have a relatively rich supply of NPY fibers (Tainio, 1994). In young adult men, we detected a high density of NPY-, CPON-, and PGP 9.5-positive nerves as well as a small number of VIP-containing fibers around and within the vascular walls of the testes. These peptidergic nerves may act as neuronal modulators for intratesticular vascular systems in young adult men, as their neurotransmitters, such as NPY and VIP, serve as potent vasomotors (Lissbrant & Bergh, 1997;Collin et al. 1998). In the male gonads, NPY binds to postjunctional Y1 receptors located primarily in the muscular layer of arteries (Kopp et al. 1997) and causes vasoconstriction through a direct effect (Hökfelt et al. 1998; Lang & Maron, 1998) or by counterbalancing VIP-induced vasodilation (Larsen et al. 1981; Polak & Bloom, 1984; Kopp et al. 1997). VIP is a neuropeptide with 28 amino acids (Zhu et al. 1995; Suburo et al. 2002) and is known to function as a vasodilator through binding to VIP-1 receptors (Lissbrant & Bergh, 1997).

Close association of testicular nerve fibers with myoid cells of the seminiferous tubules has been shown in rats and humans (Miyake et al. 1986a,b; Yamamoto et al. 1987; Prince, 1996). Moreover, direct effects of noradrenaline and acetylcholine on the contractility of seminiferous tubules have also been evidenced in vitro (Miyake et al. 1986a,b). The testicular nerves close to tubules are, therefore, thought to be involved in contraction of seminiferous tubules. In the young adult human testis, although we observed no adrenergic nerves, many peptidergic nerves immunoreactive to PGP 9.5, NPY and CPON were found running along the tubules. It is known that some neuropeptides, such as NPY and calcitonin gene-related peptide (CGRP), act as modulators for non-vascular smooth musculature motion (Iravani & Zar, 1994; Anouar et al. 1998; Yousufzai & Abdel-Latif, 1998). Furthermore, an evident expression of NPY and CPON in the peritubular myoid cells was for the first time detected in our study. It may then be speculated that these peptidergic nerves running along tubules could be implicated in contraction of seminiferous tubules in the young adult human testis. Also, the possibility of these peptidergic nerves to participate in paracrine control of spermatogenesis can not yet be ruled out.

In previous studies, expression of PGP-9.5, NPY, DBH and 5-HT were reported in human Leydig cells (Davidoff et al. 1993, 2005; Middendorff et al. 1993; Kanzaki et al. 1996; Suburo et al. 2002; Romeo et al. 2004), which was confirmed by our study. Furthermore, we found VIP and CPON also present in Leydig cells for the first time. Although the physiological significance of these proteins in Leydig cells remains unclear, our findings lend further support to the notion that Leydig cells of the human testis belong to the diffuse neuroendocrine system. Interestingly, we found some Leydig cells staining negative for these proteins, as described by Romeo et al. (2004). Whether these Leydig cells are in a different functional state from the positive ones remains to be explored. PGP 9.5 expression was also detected in spermatogonia of young adult men. This protein is considered to function as a regulator for the cell cycle of spermatogonia (Tokunaga et al. 1999) and is also used as an ideal marker for identifying and isolating this cell type (Luo et al. 2006; Rodriguez-Sosa et al. 2006).

In conclusion, our study has demonstrated that the young adult human is devoid of monoaminergic nerves but profusely innervated by peptidergic fibers, which may serve as major neuronal regulators for testicular functions at this age. The contribution of peptidergic fibers should, therefore, be considered while interpreting physiological or pathological events occurring in the young adult human testis.

Acknowledgments

The authors thank Professor K. H. Wrobel and J. H. Sha for technical support.

This study was supported by grants from National Natural Science Foundation of China 30400161, and in part by NIH HD050570 to Ren-shan Ge.

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