Physiopharmacological properties of the testicular capsule: A concise review.

The testicular capsules of different mammalian species exhibit spontaneous motor activity. In addition, contractions can be mediated by neuronal stimulation or exogenous drug administration. However, the physiological role of testicular capsule motor activity is still not well understood. Nevertheless, there is evidence for putative roles in spermatozoa transport from the testis to the caput epididymis, control of interstitial/intratesticular pressure and testicular blood flow. In this review, we have collated information about the agents that regulate testicular capsule motor activity, their receptors and second messengers as well as the impact of altered testicular capsule function on the male reproductive system. Furthermore, we highlight the knowledge gaps in the physiology and pharmacology of the testicular capsule as indicators of future research directions that may lead to a better understanding of the physiological role of testicular capsule motor activity and its importance in male fertility.

Human cremaster muscle and cremasteric reflex: A comprehensive review.

OBJECTIVE: Human research on the cremaster muscle (CM), cremasteric reflex (CMR) and genitofemoral nerve (GFN) and reports on their clinical applications using electrophysiological and histological techniques are rare. We aimed to present a detailed review of the human CM and CMR based on our earlier publications and relevant literature. METHODS: Electromyography (EMG) of the CM was recorded using disposable needle electrodes. CMR was obtained with tactile and/or electrical stimulation of the inner thigh. Transcranial magnetic stimulation (TMS) and magnetic stimulation of the upper lumbar roots were applied; GFN was stimulated using a surface electrode at the anterior superior iliac spine. RESULTS: CM striated fibers comprised multiple motor end plates. CM needle EMG results were similar to those of the limb muscles in chronic neurogenic disorders. TMS produced clear-cut evoked motor responses from CM. GFN motor conduction time to CM was absent or delayed in patients with inguinal hernia. EMG of CM was abnormal in 40% of patients with premature ejaculation. CONCLUSION: CM is different from other skeletal muscles both morphologically and physiologically. Intersegmental sacrolumbar reflexes are useful for evaluating ejaculatory dysfunction. SIGNIFICANCE: CM is an important muscle for testis thermoregulation and sexual reflexes. Neurophysiological techniques are available for physiological and clinical studies.

A Randomized, Double-Blind, Controlled Trial Shows that Onabotulinum Toxin A Nerve Blocks do Not Provide Improved Pain Control in Men with Chronic Scrotal Pain.

PURPOSE: The use of onabotulinum toxin A to chemically denervate the testis has been studied as a minimally invasive therapy to treat chronic scrotal pain. To our knowledge no randomized, controlled trials of onabotulinum toxin A for chronic scrotal pain management have been reported to date. MATERIALS AND METHODS: In this double-blind, randomized, controlled trial men with chronic scrotal pain who achieved at least temporary pain relief following a cord block with local anesthesia were randomly assigned to a block using local anesthesia alone vs local anesthesia plus 200 IU onabotulinum toxin A. Standardized assessments of pain levels using a visual analogue score, disease impact, quality of life and mood were performed 1, 2, 3, 4, 12 and 18 weeks after injection. The study primary outcome was the change in the visual analogue score at 1 month. After study completion the men in the control group were given the option to receive onabotulinum toxin A as part of an open label trial. RESULTS: Of 64 men with a mean ± SD age of 45.9 ± 11 years and a mean 5.7 ± 5.7-year history of pain 32 received local anesthesia plus onabotulinum toxin A and 32 received local anesthesia alone. There was no statistically significant difference in any measured outcome when comparing those who received onabotulinum toxin A to controls. Nine of the 13 men (69.2%) in the open label trial achieved an improvement in the visual analogue score (mean group score 6.1 ± 1.66 to 4.5 ± 2.36, Student t-test p=0.022) with a reduction in persistent pain at 3 months in 6 of the 9 (66.7%). CONCLUSIONS: This randomized, double-blind, controlled trial showed no superiority of onabotulinum toxin A plus local anesthesia over local anesthesia alone for pain control in men with chronic scrotal pain. Interestingly, significant pain improvement was noted in our open label onabotulinum toxin A trial, suggesting a potential placebo effect.

Distribution and chemical coding of urinary bladder apex-projecting neurons in aorticorenal and testicular ganglia of the male pig.

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in aorticoerenal (ARG) and testicular (TG) ganglia supplying the urinary bladder apex (UBA) in the juvenile male pig (n=4, 12 kg. body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder apex under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG were collected and processed for double-labelling immunofluorescence. The presence of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were investigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. The TG and ARG were found to contain many FB-positive neurons projecting to the UBA (UBA-PN). The UBA-PN were distributed in both TG and ARG. The majority were found in the left ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBA-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons also contained immunoreactivity to NPY, SOM or GAL. Most of the UBA-PN were supplied with varicose VAChT-, or NOS- IR (immunoreactive) nerve fibres. This study has revealed a relatively large population of differently coded ARG and TG neurons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

Neural crest-derived neurons invade the ovary but not the testis during mouse gonad development.

Testes and ovaries undergo sex-specific morphogenetic changes and adopt strikingly different morphologies, despite the fact that both arise from a common precursor, the bipotential gonad. Previous studies showed that recruitment of vasculature is critical for testis patterning. However, vasculature is not recruited into the early ovary. Peripheral innervation is involved in patterning development of many organs but has been given little attention in gonad development. In this study, we show that while innervation in the male reproductive complex is restricted to the epididymis and vas deferens and never invades the interior of the testis, neural crest-derived innervation invades the interior of the ovary around E16.5. Individual neural crest cells colonize the ovary, differentiate into neurons and glia, and form a dense neural network within the ovarian medulla. Using a sex-reversing mutant mouse line, we show that innervation is specific to ovary development, is not dependent on the genetic sex of gonadal or neural crest cells, and may be blocked by repressive guidance signals elevated in the male pathway. This study reveals another aspect of sexually dimorphic gonad development, establishes a precise timeline and structure of ovarian innervation, and raises many questions for future research.

Accessory right spermatic ganglion: possible embryological basis and clinical significance.

The spermatic ganglia are collections of sympathetic neuron cell bodies located within the cords of the infrarenal aortic plexus, positioned at the origin of the testicular arteries in males. During routine dissection of the aortic plexus at our institution, one specimen exhibited a second (accessory) testicular artery on the right side that coursed retrocaval. Histology was used to confirm the presence of an accessory right spermatic ganglion at the base of the accessory retrocaval testicular artery. Interestingly, the accessory spermatic ganglion was also supplied by its own right lumbar splanchnic nerve. This is the first case to describe the anatomy of an accessory spermatic ganglion in a specimen that exhibits an accessory testicular artery on the right side. This neurovascular variation is of interest to surgeons who aim to perform nerve-sparing retroperitoneal lymph node dissections for malignancy.

The distribution of nerves supplying the testis, epididymis and accessory sex glands of Suncus murinus.

Chronic testicular pain remains an important challenge for urologists. Investigation of the innervation of male gonads thus becomes essential for deepening our understanding of their regulatory roles in male reproductive physiology and pathophysiology. Studies of testicular innervation are mainly limited to the intratesticular peptidergic nerves of the testis by immunohistochemical and acetylcholinesterase histochemical investigations in some animals. Little is known about the detailed, overall distribution in general experimental animal testis. In this study, the distribution of nerves supplying the testis, epididymis and accessory sex glands of Suncus murinus was investigated by whole mount immunohistochemistry staining using a neurofilament protein antibody. Testicular nerves arose through three routes: nerves deriving from the mesenteric and renal plexuses accompanied the testicular artery, entering into the testicular hilum through the superior ligament of the testis. The nerves originating from the hypogastric plexus then ran along the internal iliac artery, deferential artery, and passed through the mesoductus deferens or mesoepididymis, innervating the cauda and corpus of the epididymis, the vas deferens and the inferior pole of the testis. The third route arose from the pelvic plexus, distributed in the seminal vesicle and the prostate. The density of nerve fibers was higher in the cauda epididymidis than in the testis, and more abundant in the vas deferens. The different origins and distribution densities of testicular nerves in S. murinus may serve different neuronal regulatory functions, and, therefore, S. murinus may be an important model animal for understanding the different characteristics of testicular pain.

Induction of Bladder Overactivity by Nerve Growth Factor in Testes in Rats: Possible Neural Crosstalk Between the Testes and Urinary Bladder.

OBJECTIVES: To clarify the pathophysiological factor underlying neural crosstalk among pelvic organs, we investigated the possible role of nerve growth factor (NGF) in the neural crosstalk between the testes and urinary bladder. METHODS: Nerve growth factor (10, 30, and 100 µg/mL) or saline was injected into the testes of male Wistar rats. The change in bladder capacity via cystometry and duration of spontaneous scratching behavior induced by NGF in conscious rats was measured. The effects of pretreatment with capsaicin on NGF-induced changes in bladder capacity and behavior were examined. Further, we evaluated the effect of analgesics, indomethacin and morphine, and pretreatment with compound 48/80 on NGF-induced scratching behavior to elucidate the mechanism of the behavior. RESULTS: Injection of saline into the testes had no effect on bladder capacity or behavior. However, an injection of NGF (30 and 100 µg/mL) reduced bladder capacity, which was regarded as bladder overactivity, and evoked scratching behavior in a dose-dependent manner. Pretreatment with capsaicin inhibited NGF-induced bladder overactivity and scratching behavior. Neither indomethacin nor pretreatment with compound 48/80 affected the scratching behavior, but morphine inhibited the behavior. CONCLUSIONS: The present study provides evidence of a possible new role of NGF in the testes regarding the activation of testicular primary afferent neurons mediated by capsaicin-sensitive C-fibers, which evokes bladder overactivity via neural crosstalk between the testes and the urinary bladder as well as testicular pain.

Neurotrophin signaling in a genitofemoral nerve target organ during testicular descent in mice.

BACKGROUND/AIM: It has been proposed that androgens control inguinoscrotal testicular descent via release of calcitonin gene-related peptide (CGRP) from a masculinised genitofemoral nerve (GFN). As there are androgen receptors in the inguinoscrotal fat pad (IFP) during the window of androgen sensitivity (E14-17 in mouse embryos), we tested the hypothesis that neurotrophins in the IFP may masculinise the sensory fibers of the GFN supplying the gubernaculum and IFP prior to gubernacular migration. METHODS: Androgen-receptor knockout (ARKO) and wild-type (WT) mouse embryos were collected at E17, with ethical approval (AEC 734). Sagittal sections of IFP, mammary area and bulbocavernosus (BC) muscle were processed for standard histology and fluorescent immunohistochemistry for ciliary neurotrophic factor (CNTF), ciliary neurotrophic factor receptor (CNTFR) and cell nuclei (DAPI). RESULTS: In the ARKO mouse CNTFR immunoreactivity (CNTFR-IR) was increased in the IFP but decreased in BC. Perinuclear staining of CNTF-IR was seen in mouse sciatic nerve but only weakly in IFP. In the mammary area, also supplied by GFN, there were no differences in IR staining. CONCLUSION: This study found CNTFR-IR in the IFP was negatively regulated by androgen, suggesting that CNTF signaling may be suppressed in GFN sensory nerves to enable CGRP expression for regulating gubernacular migration in the male, but not the female. The indirect action of androgen via the GFN required for testicular descent may be one of the sites of anomalies in the putative multifactorial cause of cryptorchidism.

Post-chemotherapy laparoscopic retroperitoneal lymph node dissection is feasible for stage IIA/B non-seminoma germ cell tumors.

OBJECTIVE: To assess the efficacy, outcome and complications of post-chemotherapy laparoscopic retroperitoneal lymph node dissection (L-RPLND) for stage IIA/B testicular germ cell tumor (GCT) patients in comparison with open RPLND (O-RPLND). METHODS: L-RPLND was performed in 14 patients with stage IIA/B non-seminoma GCTs among 154 non-seminoma patients who received RPLND after completion of chemotherapy with tumor marker normalization at our institution between 1998 and 2013. Their outcomes were compared with those of 14 patients with stage IIA/B non-seminoma GCTs treated with O-RPLND during the same period. Clinical parameters were compared between L-RPLND and O-RPLND. RESULTS: There were no significant differences in the background characteristics of the two groups except for follow-up duration (36 months for L-RPLND, 70 months for O-RPLND; p = 0.02). Blood loss during surgery was significantly less for the L-RPLND group than for the O-RPLND group (155 mL for L-RPLND, 700 mL for O-RPLND; p < 0.001). Parameters related to post-operative recovery were significantly better for the L-RPLND group than for the O-RPLND group. Histopathological examination showed no difference between the two groups. Neither group had disease recurrence. CONCLUSION: Post-chemotherapy L-RPLND with a bilateral template and nerve-sparing method was safe, effective, and showed a high preservation rate of antegrade ejaculation with no deterioration of outcomes compared to O-RPLND.

Infertility in rats subjected to genitofemoral nerve section is not associated with testicular damage.

This work was aimed at assessing the relationship between testicular ascent and infertility induced by genitofemoral nerve (GFN) section in rats. Eighteen male rats were assigned to three experimental groups as follows: (i) Group SGFN was subjected to surgical section of genitofemoral nerve; (ii) Group Sham; (iii) Control group. The GFN was cut at puberty (28D), and the contralateral testis removed at 90D, with fertility tests at 120D. At 150D, maturity index, epithelial area and histopathological index of seminiferous tubules of all rats were determined and statistically compared between superior and inferior testicle poles, and between groups. There were no differences in testicular parameters, sperm morphology or sperm concentrations (P > 0.05). Section of NGF interfered with fertility (58.3 ± 15.4 in SGFN versus 83.3 ± 10.5 in Sham) and litter size (6.2 ± 1.1 in SGFN versus 10.7 ± 1.4 in Sham). Cremaster of SGFN group showed early neuropathy. The GFN section induced partial testicular ascent and diminished fertility without damage on testicular morphology or spermatic parameters, because, cremaster could affect the contractibility and ejaculation mechanisms in which it participates. The study of the damage on cremaster induced by an injury on GFN could have an overview of the mechanisms inherent in the testicular ascent induced by this iatrogenic alteration and their potential risks on fertility.

Building the mammalian testis: origins, differentiation, and assembly of the component cell populations.

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial cells arise from bipotential precursors present in the precursor tissue, the genital ridge. These cell types do not differentiate independently but depend on signals from Sertoli cells that differentiate under the influence of transcription factors SRY and SOX9. While these steps are becoming better understood, the origins and roles of many testicular cell types and structures-including peritubular myoid cells, the tunica albuginea, the arterial and venous blood vasculature, lymphatic vessels, macrophages, and nerve cells-have remained unclear. This review synthesizes current knowledge of how the architecture of the testis unfolds and highlights the questions that remain to be explored, thus providing a roadmap for future studies that may help illuminate the causes of XY disorders of sex development, infertility, and testicular cancers.

An immunohistochemical study of the presence and distribution of neuronal and glial markers in simple testicular cysts in the ostrich (Struthio camelus).

Simple testicular cysts are rare in birds and mammals. However, the condition has recently been reported in the ostrich (Struthio camelus), an economically important farmed bird. The innervation of normal and cryptorchid testes, unlike the simple testicular cysts of birds and mammals, has received considerable attention. This study, therefore, immunohistochemically demonstrates the presence and the general distribution pattern of neuronal and glial markers in the simple intratesticular cyst, and its associated structures, of fourteen adult ostriches using antibodies to neurofilament protein, S-100, neuron-specific enolase and protein gene product 9.5. The LSAB+ Kit(DakoCytomation, Denmark) immunostaining protocol was used in this study. The normal seminiferous peritubular tissue showed few or no immunoreactive nerve fibres. A greater density of neurofilament protein, S-100, neuron-specific enolase and protein gene product 9.5 immunopositive nerve fibres were observed in the tunica albuginea adjacent to the cyst, as well as in the peritubular connective tissue of cystic seminiferous tubules. In addition, the tunica adventitia of blood vessels within the interstitial space of the cystic seminiferous tubules displayed neurofilament protein, S-100 and protein gene product 9.5 immunoreactive nerve fibres of varying intensity and pattern. Protein gene product 9.5 immunostaining was also observed in the multinucleated giant cells of both the normal and the cystic seminiferous tubules. The cystic portion of the testis appears to have a richer innervation than the normal portion of the same testis. The richer innervation of simple testicular cysts in the ostrich is similar to that observed in the cryptorchid testis of mammals.

Journal of Pediatric Surgery-Sponsored Fred McLoed Lecture. Undescended testis: the underlying mechanisms and the effects on germ cells that cause infertility and cancer.

Testicular descent is a complex morphological process that occurs in at least 2 stages, with different hormonal control. Insl3 controls the first step of gubernacular enlargement, although the abnormality long gubernacular cord in persistent Mullerian duct syndrome remains unexplained. Androgens control inguinoscrotal migration, which may be triggered by local signalling from the mammary line, and which requires the genitofemoral nerve. However, there is still much to learn about this phase, which when abnormal frequently leads to cryptorchidism. Orchidopexy is being recommended in the first year of age, because increasing research suggests that the stem cells for spermatogenesis form between 3 and 9 months, with surgery aiming to permit this normally, although this is not yet proven. Acquired cryptorchidism is now becoming accepted and is likely to be caused by inadequate elongation of the postnatal spermatic cord. It is not yet known whether orchidopexy is always needed, as this remains controversial.

Intravital microscopic evaluation of cremasteric microcirculation in experimental testicular torsion.

AIM: Although absent cremasteric reflex is a significant clinical finding for testicular torsion (TT), there is limited information about microcirculation of the cremasteric muscle (CM) after TT. This experimental study was performed to evaluate CM microcirculation by intravital microscopy after TT. MATERIALS AND METHODS: Twelve Wistar rats were allocated into two equal groups: control (CG) and torsion (TG). After anesthetization of the CG rats, the CM flap was dissected through a left ventral inguinal incision with its vascular pedicle. In TG rats, TT was performed by rotating left testicles 720(°) in clockwise direction for 1 h. Then, the CM flap was dissected as in CG, and was placed under an intravital microscope. Vessel diameters, functional capillary perfusion and leukocyte activation in post-capillary venules were measured and evaluated statistically. RESULTS: There was a significant decrease in vessel diameter in TG compared to CG (p < 0.05). The median of perfused capillaries in CG and TG was 13 (11.75-14.30) and 5.5 (4.75-7.25), respectively (p < 0.05). Number of granulocytes (rolling, sticking, transmigrated) was greater in TG than CG (p < 0.05). CONCLUSION: Intravital microscopic evaluation of CM after TT showed decrease in vessel diameter and number of perfused capillaries, and increase in granulocyte activation. Clinical, electrophysiological alterations in CM after TT can be explained by deterioration of microcirculation of CM.

Effects of reserpine on reproduction and serotonin immunoreactivity in the stable fly Stomoxys calcitrans (L.).

Biogenic amines are known to play critical roles in key insect behaviors such as feeding and reproduction. This study documents the effects of reserpine on mating and egg-laying behaviors of the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), which is one of the most significant biting fly pests affecting cattle. Two sperm staining techniques were adapted successfully to reveal the morphology of stable fly sperm, for the first time, and determine successful mating in females through the assessment of sperm transfer. This approach was also applied to assess sperm transfer by males treated with different doses of reserpine. Mating or sperm transfer did not occur in flies during the first 3 days after emergence. Thereafter, the percentage of females that mated increased with age. Reserpine treatment of males reduced sperm transfer in a dose-dependent manner. Older males were more sensitive to reserpine treatment than younger flies. Reserpine treatment of 5 days old females reduced the number of eggs laid, but had no effect on egg-hatching rates. Results of immunoreactivity (IR) experiments indicated that serotonin in the neuronal processes innervating male testes was completely depleted by reserpine within 5h after treatment. This effect was transient as the serotonin immunoreactive signal was recovered in 33.3% of the males at 1 day post-treatment and in 94.4% of the flies at 3 days post-treatment. The results of this study concur with previous findings in other insect species and extend our knowledge of the critical roles biogenic amines play in mating and oviposition behaviors of the stable fly. The work could provide a foundation to further characterize the specific roles of individual biogenic amines and their receptors in stable fly reproduction.

Regression of the mammary branch of the genitofemoral nerve may be necessary for testicular descent in rats.

PURPOSE: Inguinoscrotal testicular descent has been proposed to occur via sensory fibers of the sexually dimorphic genitofemoral nerve, which release a neurotransmitter, calcitonin gene related peptide, to guide the migrating gubernaculum into the scrotum. We hypothesize that androgen mediated regression of the genitofemoral nerve mammary branch is necessary for inguinoscrotal descent in rats. We compared the spatiotemporal development of the genitofemoral nerve in control and antiandrogen treated rats. MATERIALS AND METHODS: A total of 29 Sprague-Dawley® rats were collected (animal ethics committee approval A644) in control and antiandrogen treated groups (flutamide, embryonic days 16 to 19, 75 mg/kg body weight/5% ethanol + oil) on embryonic days 17 and 19, and on postnatal day 2. Sagittal sections of the gubernaculum and its surrounding structures were processed for standard histology and immunohistochemistry for androgen receptor, nerves (Tuj1), calcitonin gene related peptide (marker for genitofemoral nerve) and cell nuclei (DAPI). RESULTS: The inguinal mammary bud, its adjacent androgen receptor and genitofemoral nerve mammary branch (containing calcitonin gene related peptide) persisted from embryonic day 17 to postnatal day 2 in all antiandrogen treated males, yet regressed in all control males by postnatal day 2. CONCLUSIONS: Antiandrogens resulted in the persistence of the mammary branch and inguinal mammary bud. Persistent genitofemoral nerve mammary branches may arrest or slow down gubernacular migration by releasing calcitonin gene related peptide in the mammary inguinal fat pad, thus reducing the chemotactic gradient to calcitonin gene related peptide from genitofemoral nerve branches in the distal scrotum. We hypothesize that this process may be related to antiandrogen induced cryptorchidism in the rodent.