Inferior Spermatic Nerve Research Articles

Involvement of the mesenteric ganglia on androstenedione, noradrenaline and nitrite release using a testis ex vivo system

The autonomic nerve fibres converge to the testis along two major pathways, the superior spermatic nerve (SSN) and the inferior spermatic nerve (ISN). The object of this work was to evaluate whether the addition of noradrenaline (NA) in the ganglionic compartment of two ex vivo systems: superior mesenteric ganglion (SMG)-SSN-testis, inferior mesenteric ganglion (IMG)-ISN-testis modulate androstenedione (A₂), NA and nitrite release and to determine whether there are secretory differences between the right and the left testis. Each gonad with its respective ganglion was transferred into a cuvette with two compartments and incubated in a Dubnoff metabolic shaker. The testis incubation liquids were collected and analysed for NA by HPLC, A₂ by RIA and nitrites by the Griess method. When NA is added to the IMG, A₂ and NA release diminishes and nitrite increases in the left testis, while in the right gonad, A₂ and NA increase and nitrite decreases. When NA was administered to the SMG, A₂ and NA increase and nitrite diminishes in the left gonad, but they show opposite fluctuations in the right testis. These ex vivo systems appear to be excellent models for studying the sympathetic ganglionic control of the testis though A₂, NA and nitrite release from the male gonad. It is evident that a better knowledge about the role of catecholamines and nitric oxide in the testis physiology may facilitate the understanding of some reproductive diseases.

Testicular Denervation in Prepuberty Rat Inhibits Seminiferous Tubule Development and Spermatogenesis

In the adult rat, the superior spermatic nerve (SSN) and inferior spermatic nerve (ISN) are involved in regulating testosterone secretion and spermatogenesis, in addition to endocrine control mechanisms. However, there are currently few data on how the testis nerve supply regulates testicular development and related mechanisms. The present study was thus designed to investigate the regulating effects of testis nerve supply to testicular maturation, spermatogenesis and the involved mechanisms from prepuberty to adulthood in rats. We transected the SSNs and ISNs of rats on postnatal day (PD) 30 and then analyzed changes in testicular morphology and cauda epididymal sperm content, cell proliferation and apoptosis and primary spermatocyte meiosis on PD60 and PD90. The results demonstrated that testicular denervation significantly reduced testis mass, cauda epididymal sperm counts and serum testosterone concentrations. Proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 immunohistochemistry staining proved that the denervation had no influence on the proliferation of spermatogonia and primary spermatocytes, but obviously promoted the apoptosis of round spermatids and Leydig cells. It is novel that denervation reduced the meiotic activation of zygotene and pachytene spermatocytes through the expression of synaptonemal complex protein 3 (SCP3)-a marker of meiosis. In addition, RT-PCR showed that testis denervation significantly decreased testis 3beta-hydroxysteroid dehydrogenase 1 (3beta-HSD1) and luteinizing hormone receptor (LHR) mRNA levels, but had no obvious influence on testis follicle stimulating hormone receptor (FSHR) mRNA expression. These results suggest that the testicular nerve supply plays an important role in supporting seminiferous tubule development and spermatogenesis from prepuberty to adulthood.

Administration of noradrenaline in the autonomic ganglia modifies the testosterone release from the testis using an ex vivo system

The male gonad receives nerve fibres from the autonomic ganglionic system. These fibres converge on the testis along two pathways, the superior and the inferior spermatic nerves. The superior spermatic nerve runs from the superior mesenteric ganglion alongside the testicular artery, whereas the inferior spermatic nerve originates in inferior mesenteric ganglion, accompanies the vas deferens and penetrates the inferior pole of the testis. The aim of this work was to evaluate androgen release after the addition of noradrenaline or adrenoreceptor antagonists (propranolol or phentolamine) to the ganglionic compartment. An ex vivo system used in a previous work was incubated in two separate containers, one for the testis and the other for the ganglion. Both organs remain interconnected (as in vivo) by the respective spermatic nerve. When noradrenaline was added to the inferior mesenteric ganglion, testosterone release in the gonad container underwent a progressive and significant increment. Propranolol diminishes and phentolamine increases the androgen release. When using the superior mesenteric ganglion, no changes were observed. These results indicate that the ganglionic stimulation of the autonomic system clearly participates in testosterone release from the testis. This effect depends on the ganglion involved. These results make it evident that not only the classical and well-known hypothalamus-hypophysial axis, but also the peripheral nervous system, via the autonomic ganglia, are directly involved in the endocrine control of the testis.

Deprival of testicular innervation induces apoptosis of Leydig cells via caspase-8-dependent signaling: A novel survival pathway revealed

Leydig cells are the primary source of testosterone in adult males. Recently, a growing body of evidence has shown that testicular innervation functions as a major regulator in Leydig cell steroidogenesis. The question then arises whether this novel regulatory pathway also plays an important role in other biological behaviors of this cell type. In the present study, we selectively resected the superior spermatic nerves (SSNs) or the inferior spermatic nerves (ISNs) to investigate the effects of testicular denervation on survival of Leydig cells. After testicular denervation, Leydig cells displayed morphological characteristics of apoptosis, such as chromatin condensation, cell shrinkage and apoptotic body formation. Flow cytometry combined with TUNEL labeling demonstrated dramatic and persistent apoptosis of Leydig cells in the denervated testes 14 and 21 days after operation. Meanwhile, serum T concentrations in the SSN- or ISN-denervated rats dramatically decreased on day 14 and declined further on day 21. Plasma LH levels underwent a remarkable rise, while serum FSH levels remained unchanged. Immunofluorescent staining and flow cytometry further demonstrated that testicular denervation activated caspase-3 and caspase-8, but not caspase-9 in Leydig cells. Our data indicate that testicular innervation functions as an important survival factor for Leydig cells in vivo.

Innervation and serotoninergic receptors of the testis interact with local action of interleukin-1beta on steroidogenesis

Testosterone secretion by Leydig cells is affected by interleukin-1β (IL-1β). The aim of the present study was to investigate whether partial denervation of the testis or local administration of a serotonin (5-HT) receptor antagonist could alter the changes in testicular steoidogenesis induced by IL-1β. Intratesticular administration of IL-1β was combined with vasectomy or local injection of ketanserin (5-HT type 2 receptor antagonist) in immature hemicastrated rats and the effect of the interventions on testicular steroidogenesis was studied. One day after treatment with local injection of IL-1β induced a significant rise in testosterone secretion that could be prevented by vasectomy (that also means transection of the inferior spermatic nerve). In a model in which neither IL-1β nor ketanserin interfered with steroidogenesis, administration of the receptor antagonist just prior to IL-1β treatment significantly reduced testosterone secretion. Data indicate interaction between testicular nerves and IL-1β action and interaction between testicular 5-HT2 receptors and local effect of IL-1β on testosterone secretion.

Immunohistochemical investigations of the autonomous innervation of the cervine testis

The innervation of the cervine testis was studied in 6 roe deers, 7 red deers and 14 fallow deers. The results for the three species are rather similar. With anti-sera to neurofilament (NF) and neuron specific enolase (NSE), all small and large nerve fascicles can be demonstrated, but single fibers are incompletely stained. Immunoreactions against protein gene product-9.5 (PGP-9.5) and GAP-43 (growth-associated protein-43) are better suited to depict the complete innervation pattern. Bundles of the superior spermatic and inferior spermatic nerves reach the testis via three access routes as funicular, mesorchial and caudal nerve contributions. We found no morphological evidence that the nerves in the cervine testis are directly involved in regulating Leydig cell function or seminiferous tubular motility. The majority of the testicular nerves are associated with the testicular arteries, but the musculature in the walls of the venous plexus pampiniformis is also innervated. All vascular nerve fibers represent postjunctional sympathetic axons displaying a strong dopamine-beta-hydroxylase (DBH) activity, mostly co-expressed with neuropeptide Y (NPY). The presence of cholinergic fibers in the testis of the deer is only sporadic and probably of no functional importance. In all three species of deer, a small quantity of myelinated nerve fibers is encountered in spermatic cord and tunica albuginea and regarded as afferent. The viscerosensory quality in the testicular intrinsic innervation is very likely mediated by the CGRP (calcitonin gene-related peptide)-positive fibers that run independently from the testicular vessels and end in the connective tissue of spermatic cord and tunica albuginea. The testis of the red deer contains significantly more VIP (vasoactive intestinal polypeptide)-positive axons than that of roe and fallow deer. The nerve density in the interior of the testicular lobules shows no regional differences, but there are age- and season-related changes that correlate with the developmental and functional state of the seminiferous tubules. Small testes with solid and narrow tubules, as seen in the prepuberal phase and during seasonal reproductive quiescence, are better innervated than large testes with expanded and spermatogenetically active seminiferous tubules.

Administration of acetylcholine to the spermatic nerve plexus inhibits testosterone secretion in an in vitro isolated rat testis-nerve plexus system.

Strong evidence indicated that spermatic nerves are involved in the regulation of testosterone secretion. Our previous work showed that the inferior spermatic nerves play a more significant role than the superior ones in the regulation of testosterone secretion. However, it is unknown whether traditional neurotransmitters are involved in this regulation. In order to evaluate this point, the present experiments were carried out in an in vitro system where an isolated testis-spermatic nerve plexus preparation was incubated in two separate containers, one for the testis and the other for the nerve plexus and both interconnected by the inferior spermatic nerves. Both tissues were maintained in the same environmental conditions except for the neurotransmitter treatment, applied only to the nerve plexus. Acetylcholine can significantly inhibit the secretion of testosterone until the end of incubation. The present experiments suggest that the secretion of testosterone could be regulated, at least in part, by acetylcholine through the inferior spermatic nerves.

Peptidergic innervation of blood vessels and interstitial cells in the testis of the cat.

We studied the innervation of the cat testis using a panel of antisera against the following neuronal markers: protein gene product 9.5 (PGP), neuropeptide Y, C-terminal peptide of neuropeptide Y, galanin, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide, and substance P. Immunoreactivity against PGP, a general neuronal label, demonstrated the arrangement of fibers from the superior spermatic nerve (SSN) in the testicular pedicle and the cephalic testicular pole, and those of the inferior spermatic nerve (ISN) along the vas deferens and the inferior testicular ligament. The testicular parenchyma exhibited a very rich innervation, mainly distributed to blood vessels and Leydig cell nests, but also in close association with seminiferous tubules. Numerous peptidergic fibers were present in the SSN and ISN, albeit in different proportions. Thus, VIP-immunoreactive fibers were almost absent in the SSN, but were the most abundant subpopulation of the ISN. The testicular interstitium contained numerous peptidergic fibers, associated with blood vessels, interstitial Leydig cells, and seminiferous tubules. Similar fibers were related to the rete testis. Parenchymatous VIP-immunoreactive nerves disappeared after bilateral vasectomy. Stimulation of the ISN under experimental conditions was associated with an increase of blood flow, and induced a large release of VIP into the spermatic vein. The extensive and selective distribution of nerve fibers within the cat testicular parenchyma supports the importance of spermatic nerves for testicular function. Furthermore, the differences in the fiber composition of the SSN and ISN can be correlated with their opposing effects on testosterone secretion and testicular blood flow.

The Effects of Testicular Denervation on Spermatogenesis in the Sprague- Dawley Rat

In the rat, regression of spermatogenesis during the chronic stages of spinal cord injury (SCI) occurs in the presence of normal function of the pituitary-testis hormone axis, thus suggesting that nonendocrine mechanisms might be involved. The current study examined whether disruption of neural input to the testis contributes to the cascade that leads to the regression of spermatogenesis. Four weeks after denervation of the superior spermatic nerve (SSN), testis weight was 25% lower (p < 0.01) than that of the contralateral sham-operated testis. Defects in spermatogenesis including phagocytosis of mature spermatids, vacuolization of spermatid nuclei, delayed spermiation and incomplete cellular associations were observed in >60% of the tubules. In the remaining 30–40% of tubules, the seminiferous epithelium was severely regressed. While cutting the inferior spermatic nerve (ISN) alone did not affect spermatogenesis significantly, it enhanced the effect of SSN denervation on both spermatogenesis and testis weight (p < 0.01). Spermatogenesis was totally regressed in the SSN/ISN-denervated testes. At this time, quantitatively normal spermatogonial proliferation was maintained in SSN- or ISN-denervated testes. Twelve weeks after surgery, regression of the seminiferous epithelium characterized by absence of proliferating spermatogonia, while undifferentiating spermatogonia were present, was observed in all SSN-denervated testes. At this time, regression of the seminiferous epithelia also occurred in >30% of the tubules in ISN-denervated testes. At both times, serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were normal and >60% of normal testicular testosterone concentrations were maintained in the denervated testes. These results indicate that disruption of neural input to the testis is not a cause for the decrease in spermatogonial proliferation during the acute phase of SCI, but may contribute to the chronic effects of SCI on spermatogenesis.

Differential Effects of Superior and Inferior Spermatic Nerves on Testosterone Secretion and Spermatic Blood Flow in Cats

Differential Effects of Superior and Inferior Spermatic Nerves on Testosterone Secretion and Spermatic Blood Flow in Cats

Testicular denervation-induced nuclear changes in Leydig cell of hemicastrated adult rats

In the adult male rat, hemicastration (HC) induces a remarkable enhancement of testosterone secretion from the Leydig cells of the remaining testis. We have observed previously that the denervation of inferior spermatic nerves inhibits this enhancement. The present experiments were designed to assess morphometrically whether or not this change had a cytological correlate in Leydig cells. At least five testes from each group (denervation plus HC, sham denervation plus HC and intact rats) were prepared for both optical and electron microscopy studies. The results showed that after 24 h of denervation, the nuclear volume of the Leydig cells in denervation plus HC rats was smaller ( p < 0.01) than those of sham denervation plus HC (196.56 ± 16.53 vs. 280.71 ± 13.37), whereas both the volume density of heterochromatin (19.84 ± 3.14 vs. 10.03 ± 2.47%) and the heterochromatin index (expressed as periphery heterochromatin area divided by nuclear perimeter, 0.149 ± 0.046 vs. 0.094 ± 0.026) were significantly ( p < 0.01) higher in denervation plus HC rats than in its sham groups. No changes in Leydig cell numbers or cytoplasmic organelles were detected. The results suggest that some nuclear and heterochromatin-associated cellular activity might be inhibited by testicular denervation in hemicastrated rats.

Segmental distribution of afferent neurons innervating the canine testis

To clarify the afferent innervation of the canine scrotal contents, retrograde labeling of neurons in the dorsal root ganglia (DRG) has been carried out using two methods: (1) horseradish peroxidase (HRP) injection into the surface of the testis and epididymis; and (2) exposure of the superior spermatic nerve to a fluorescent dye (Fast blue; FB). Injections of HRP resulted in labeling of DRG cells located predominantly from T10 to L4 (87%) and, to a lesser extent, at S1–S3 (13%). Transection of the vas deferens previous to testicular injections eliminated labeling in the S1-S3 DRG, but not at thoracolumbar levels. These findings indicated that primary afferent fibers of the testis and epididymis project mainly to the DRG at higher than L4 through the superior spermatic nerve, but an additional population of the fibers also projects the sacral level through the inferior spermatic nerve. Exposure of the superior spermatic nerve to FB resulted in a similar distribution of labeled cells as compared with testicular injections of HRP after vasectomy. Labeled cells (8.1%) were also observed in the contralateral T13-L3 DRG. In both FB and HRP groups, the major part of the labeled cells was located in Ll and L2. The sizes of HRP- and FB-labeled cells were smaller than those of unlabeled cells in the Ll and L2 DRG. The cumulative frequency distribution histogram for the diameter of HRP- and FB-labeled cells could be fitted by a normal distribution.

Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat.

Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

Vasectomy modulates the local effect of intratesticular opioids on the gonad of immature rats.

Previous studies indicate that 7 days after testicular administration of naloxone, serum testosterone (T) concentration and basal T secretion in vitro decrease significantly. In neonatal rats, short-term (2 h) intratesticular treatment with 0.3 micrograms (D-Met2-Pro5)-enkephalinamide suppresses steroidogenesis. In the present study, testicular treatment with naloxone or enkephalinamide was combined with hemivasectomy (which also includes transection of the inferior spermatic nerve). When local treatment with naloxone was combined with vasectomy in 15-day-old rats, the opioid antagonist-induced increase in testicular weight, the decrease in basal T secretion of the treated gonad and the drop in serum T level could not be observed 7 days postsurgery, despite the fact that in immature rats, hemivasectomy, by itself, also reduces steroid secretion. Similarly, in 5-day-old animals, the short-term (2 h) effect of testicular enkephalinamide on T secretion was prevented by vasectomy. These data suggest that local testicular actions exerted by endogenous opioid peptides might be modulated by the inferior spermatic nerve.

Hypophysectomy and hemivasectomy can inhibit the testicular hemicastration response of the mature rat.

Three questions were asked in an attempt to understand how testosterone (T) concentration in the veins of the remaining testis can double within 24 h after hemicastration in the mature rat without a change in plasma luteinizing hormone (LH) levels. These three questions (and their answers) were: 1) Can the testicular hemicastration response occur in hypophysectomized rats? Answer, No. 2) Does LH binding to the testis increase after hemicastration? Answer, No. 3) Is there a neural route to the testis alternate to the superior spermatic plexi? Answer, Yes, apparently there is, since hemivasectomy contralateral to the excised testis partially suppressed the testicular hemicastration response (150.4 +/- 13.2 ng/ml in hemicastrated, sham- hemivasectomized rats [n = 18] vs. 109.4 +/- 11.6 ng/ml in hemicastrated, hemivasectomized rats [n = 18], P less than 0.026). It was concluded that LH was probably necessary to the testicular hemicastration response but that its presence did not provide a mechanism. The response was mediated at least partly through the inferior spermatic nerves associated with the vas deferens. A possible reason, although highly speculative, for failure to previously block the testicular hemicastration response by bilateral denervation of the superior spermatic plexi (Mock and Frankel , 1982) was that during the 12-wk interval between denervation and hemicastration, testicular innervation functionally transferred from the superior spermatic to the inferior spermatic nerves.

Effect of imidocarb dipropionate and hemicastration on spermatogenesis in pony stallions: Frerichs, W.M.: Am. J. Vet. Res. [formula omitted], 139–141, 1977. Animal Parasitology Institute, Agricultural Research Service, Beltsville, Md 20705

In the adult male rat, hemicastration (HC) induces a remarkable enhancement of testosterone secretion from the Leydig cells of the remaining testis. We have observed previously that the denervation of inferior spermatic nerves inhibits this enhancement. The present experiments were designed to assess morphometrically whether or not this change had a cytological correlate in Leydig cells. At least five testes from each group (denervation plus HC, sham denervation plus HC and intact rats) were prepared for both optical and electron microscopy studies. The results showed that after 24 h of denervation, the nuclear volume of the Leydig cells in denervation plus HC rats was smaller (p < 0.01) than those of sham denervation plus HC (196.56 ± 16.53 vs. 280.71 ± 13.37), whereas both the volume density of heterochromatin (19.84 ± 3.14 vs. 10.03 ± 2.47%) and the heterochromatin index (expressed as periphery heterochromatin area divided by nuclear perimeter, 0.149 ± 0.046 vs. 0.094 ± 0.026) were significantly (p < 0.01) higher in denervation plus HC rats than in its sham groups. No changes in Leydig cell numbers or cytoplasmic organelles were detected. The results suggest that some nuclear and heterochromatin-associated cellular activity might be inhibited by testicular denervation in hemicastrated rats.