The cellular localization and redistribution of multiple aquaporin paralogs in the spermatic duct epithelium of a maturing marine teleost.

Aquaporin-mediated fluid transport in the mammalian efferent duct and epididymis is believed to play a role in sperm maturation and concentration. In fish, such as the marine teleost gilthead seabream (Sparus aurata), the control of fluid homeostasis in the spermatic duct seems also to be crucial for male fertility, but no information exists on the expression and distribution of aquaporins. In this study, reverse transcriptase-polymerase chain reaction and immunoblotting analyses, employing available and newly raised paralog-specific antibodies for seabream aquaporins, indicate that up to nine functional aquaporins, Aqp0a, -1aa, -1ab, -3a, -4a, -7, -8bb, -9b and -10b, are expressed in the spermatic duct. Immunolocalization of the channels in the resting spermatic duct reveals that Aqp0a, -1aa, -4a, -7 and -10b are expressed in the monolayered luminal epithelium, Aqp8b and -9b in smooth muscle fibers, and Aqp1ab and -3a in different interstitial lamina cells. In the epithelial cells, Aqp0a and -1aa are localized in the short apical microvilli, and Aqp4a and -10b show apical and basolateral staining, whereas Aqp7 is solely detected in vesicular compartments. Upon spermiation, an elongation of the epithelial cells sterocilia, as well as the folding of the epithelium, is observed. At this stage, single- and double-immunostaining, using two aquaporin paralogs or the Na+ /K+ -ATPase membrane marker, indicate that Aqp1ab, -3a, -7, -8bb and -9b staining remains unchanged, whereas in epithelial cells Aqp1aa translation is supressed, Aqp4a internalizes, and Aqp0a and -10b accumulate in the apical, lateral and basal plasma membrane. These findings uncover a cell type- and region-specific distribution of multiple aquaporins in the piscine spermatic duct, which shares conserved features of the mammalian system. The data therefore suggest that aquaporins may play different roles in the regulation of fluid homeostasis and sperm maturation in the male reproductive tract of fish.

Testis Cord Maintenance in Mouse Embryos: Genes and Signaling.

Testis cords, embryonic precursors of the seminiferous tubules, are fundamental for testis structure and function. Delay or disruption of testis cord formation could result in gonadal dysgenesis. Although mechanisms regulating testis cord formation during sex determination have been well-studied, the genes and signaling pathways involving in testis cord maintenance after the cords have formed are not well characterized. It is now clear that the maintenance of cord structure is an active process. In this review, we summarize the recent findings regarding the regulation of testis cord integrity by a series of Sertoli cell transcription factors, including the WT1-SOX8/SOX9-beta-CATENIN-DHH network, GPR56, STIM1, and NR0B1 (also known as DAX1). In particularly, we emphasize the underappreciated role of peritubular myoid cells in testis cord maintenance and their cooperation with Sertoli cells. The regulation of the size, shape, and number of testis cords by Sertoli cell proliferation (e.g., SMAD4, GATA4, and TGF-beta signaling), Leydig cell products (e.g., ACTIVIN A), vascular development (a lesson learned from PDGF signaling), and available gonad space (as observed in Ift144 mutant mice) is also addressed. Further efforts and new genetic models are needed to unveil the gene networks and underlying mechanisms regulating testis cord integrity and morphology after sex determination.

Microsurgical Anatomy of the Spermatic Cord and Spermatic Fascia: Distribution of Lymphatics, and Sensory and Autonomic Nerves.

PURPOSE: An understanding of the microsurgical anatomy of the spermatic cord and spermatic fascia is important for surgeons during microsurgical varicocelectomy and denervation. We examined the distribution of the lymphatics, and the sensory and autonomic nerves of the spermatic cord. MATERIALS AND METHODS: We collected spermatic cords from 11 men undergoing orchiectomy for localized testicular tumors and we biopsied a third of the spermatic fascia from 36 men undergoing microsurgical varicocelectomy. Immunohistochemical staining of the pan-neuronal marker PGP 9.5 (protein gene product 9.5), the sensory nociceptor marker CPRP (calcitonin gene-related peptide), the sympathetic marker TH (tyrosine hydroxylase), the parasympathetic marker VIP (vasoactive intestinal polypeptide) and the lymphatic marker D2-40 was performed. We counted the number of nerves and lymphatics. RESULTS: PGP 9.5 staining revealed dense nerve distributions in the spermatic cord and fascia. Sensory and autonomic nerve fibers were basically co-localized in the same nerve. Of the nerves 50% were identified near the vas deferens and 20% were identified in the spermatic fascia. Sensory and sympathetic nerve fibers represented most of the nerves but a few parasympathetic nerve fibers were observed. Of the lymphatics 36 per patient were identified in the spermatic cord but only a few were identified in the spermatic fascia. CONCLUSIONS: Sensory and sympathetic nerves accounted for the majority of the nerves. Although the functional aspects of the nerves remain undetermined, information on the distribution of nerves and lymphatics is useful when dealing with nerves and preserving lymphatics during microsurgical varicocelectomy or denervation.

Twisting of the spermatic cord: ischemia and reperfusion, toxicogenetic evaluation, and the effects of phosphatidylcholine in pre-clinical trials.

Phosphatidylcholine is the main phospholipid present in cell membranes and in lipoproteins, and can interfere with various biological processes. This lipid also has antioxidant activity, and protects against damage caused by free radicals under conditions of ischemia/reperfusion. Therefore, the present study was designed to evaluate toxicogenetic damage caused by twisting of the spermatic cord in ischemia/reperfusion, and whether phosphatidylcholine plays a role in conditions of ischemia/reperfusion in preclinical trials. The results indicate that spermatic cord torsion does not cause genotoxic damage or mutagenesis. A dose of 300 mg/kg of phosphatidylcholine is toxic and is thus not recommended. However, a dose of 150 mg/kg does not promote toxicogenetic damage, and though it does not statistically prevent tissue damage occurring from lack of oxygenation and nutrition of testicular cells, it has a tendency to reduce this damage. Therefore, this research suggests that further studies should be conducted to clarify this tendency and to provide a better explanation of the possible therapeutic effects of phosphatidylcholine in cytoprotection of germ cells affected by ischemia/reperfusion.

[Liposarcoma of the spermatic cord].

The paper reviews the literature on primary paratesticular tumors. It describes the classification, brief characteristics of liposarcomas and the morphological pattern of the tumor with an immunohistochemical profile. A clinical case of differentiated liposarcoma of the spermatic cord with myxoid and rhabdomyoblastic differentiation is depicted in a 61-year-old man.

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii.

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

Histological development of human testicular cords from 70 to 90 days of gestation.

The development of the testicular cord structure was investigated in 4 human fetuses between 70 and 90 days of gestation, in which the testicular cords are differentiating into the seminiferous tubules. Histological examinations were performed using stains with haematoxylin-eosin (HE), Masson's trichrome (MT), periodic acid schiff (PAS), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibodies, and TUNEL methods. It was found that the testicular cords structures were indefinitely observed in HE-stained sections of four fetuses. However, the basement membranes of the testicular cord were clearly stained with MT, showing the tubular structure. Furthermore, cells in the testicular cords were positive with PAS, but the interstitial tissues outside the testicular cords were negative. PCNA-positive cells were detected not only inside but also outside the testicular cords, however, TUNEL positive cells are not detected throughout all testicular tissues.

The effect of experimental varicocele on the apelin and APJ expressions in rat testis tissue.

Varicocele, which is one of the causes of infertility in men, can be defined as the expansion of spermatic cord veins. The presence of apelin and apelin receptor (APJ) in many tissues and the effects of apelin have been reported in several studies. There is no study showing apelin and APJ protein expressions in normal and varicocele-induced testicular tissues. In this study, we aimed to demonstrate varicocele-induced changes in apelin and APJ expressions in testicular tissue by immunohistochemical and western blotting techniques. In our study, Wistar male rats were randomly divided into three groups as control, varicocele, and sham. While the control group rats were not subjected to any treatment, the unilateral varicocele model was created under anesthesia in the varicocele group. In the sham group, the left abdominal region was opened and closed to exclude the effect of the surgical procedure. At the 13th postoperative week, the left testes were obtained under anesthesia in all groups, and the immunohistochemistry and Western blotting techniques were used to detect apelin and APJ expressions. In our study; apelin and APJ were significantly expressed in control group's testicular tissue; apelin in testicular tissues of varicocele groups increased compared to the control group, whereas APJ expression decreased. In conclusion, the presence of apelin/APJ system in normal testis and the increased expression of apelin in varicocele-induced testicular tissue suggested that apelin may have a role in the varicocele etiopathogenesis.

DDB1 Regulates Sertoli Cell Proliferation and Testis Cord Remodeling by TGFß Pathway.

Testis cords are the embryonic precursors of the seminiferous tubules. Development of testis cords is a key event during embryonic testicular morphogenesis and is regulated by multiple signaling molecules produced by Sertoli cells. However, the exact nature and the cascade of molecular events underlying testis cord development remain to be uncovered. In the current study, we explored the role of DNA damage binding protein 1 (DDB1) in Sertoli cells during mouse testis cord development. The genetic ablation of Ddb1 specifically in Sertoli cells resulted in the compromised Sertoli cell proliferation and disruption of testis cord remodeling in neonatal mice. This testicular dysgenesis persisted through adulthood, resulting in smaller testis and low sperm production. Mechanistically, we observed that the DDB1 degradation can stabilize SET domain-containing lysine methyltransferase 8 (SET8), which subsequently decreases the phosphorylation of SMAD2, an essential intracellular component of transforming growth factor beta (TGFß) signaling. Taken together, our results suggest an essential role of Ddb1 in Sertoli cell proliferation and normal remodeling of testis cords via TGFß pathway. To our knowledge, this is the first upstream regulators of TGFß pathway in Sertoli cells, and therefore it furthers our understanding of testis cord development.

Mesothelial glandular structures within pseudosarcomatous proliferative funiculitis--a diagnostic pitfall: report of 17 cases.

We describe 17 cases of distinct benign pseudomalignant mesothelial proliferations, involving the spermatic cord. All cases revealed necrosis. The areas adjacent to the necrotic tissue comprised a cellular spindle cell proliferation with a haphazard arrangement of the myofibroblasts that in many areas revealed transitions into plump oval epithelioid cells and into cells with genuine epithelial appearances arranged in linear cords and often luminized into small microcysts. These epithelial cells formed isolated groups with glandular structures arising on the myofibroblastic background. Glandular structures were often situated deeply in the stroma of the spermatic cords. All cellular elements were strongly positive with AE1/AE3 antibody. All myofibroblasts stained with SM-actin antibody. Ultrastructurally, the spindle cells displayed features of myofibroblasts including actin microfilaments, as did the plump epithelioid cells that, additionally, had desmosomes, and the cords of the epithelial cells including those forming glandular structures had characteristics of mesothelias including the characteristic microvilli.

Influence of hernioplastic implants on male fertility in rats.

This study explored the vulnerability of the ductus deferens due to mesh induced inflammation and shrinkage after hernia repair in the rodent model. Two commonly used types of hernioplastic implants (Prolene and Vypro II) were surgically wrapped around the ductus deferentes on both sides in 20 juvenile and 20 adult Sprague-Dawley rats. Twenty male rats underwent sham surgeries as controls. After 3 months, each male was mated with 2-3 adult females, which were subsequently sacrificed and oocytes or embryos were flushed and counted. Histochemical investigations of the implants and the ductus recovered surgically 4 weeks after implantation (one side) and after the fertility test (second side) were conducted. All groups exhibited 1-3 males with decreased or restricted fertility but there was no difference between groups. Histochemical analysis of the implants and the ductus recovered 4 weeks and 4 months after implantation revealed some sperm granulomes due to lesions of the spermatic cord caused by the implant in the Prolene group. There was no inflammatory reaction in the mucosa or blockage of the spermatic cord visible. Both types of hernioplastic implants tested in this investigation do not give an indication of a negative influence on male fertility in juvenile or adult rats.

New insight on the role of melatonin receptors in reproductive processes of seasonal breeders on the example of mature male European bison (Bison bonasus, Linnaeus 1758).

Melatonin receptors (MT1 and MT2) were shown to regulate proper functioning of reproductive system, especially in seasonally breeding animals. European bison is a unique endangered seasonal breeder and knowledge of the molecular mechanisms of its reproduction is crucial for the survival of the species. The aim of this study was to assess gene expression, protein synthesis and immunohistochemical localization of MT1 and MT2 receptors in testicular and spermatic cord vessels tissues collected in pre-rut (June) and post-rut (December) seasons from adult male European bisons in Bialowieza National Park. We confirmed the highest expression of MT1 and MT2 mRNA and protein levels in testis in December, while in spermatic cord gene expression was also highest in December, but protein amounts were comparable in both analyzed periods. Furthermore, immunohistochemical staining revealed the same amount of both receptors in arteries and veins of spermatic cord in both periods and increased amounts in December in Leydig, Sertoli and germ cells. The high level of testicular melatonin in December confirms the inhibition of spermatogenesis and increased anti-oxidant and anti-inflammatory protection. In spermatic cord vessels, it may prevent from age-related changes due to the overexploitation and ensure a constant temperature regardless of changing environmental conditions. This knowledge can contribute to finding a solution of problems associated with male infertility in general and also further explore the mechanisms regulating the proper functions of the male reproductive system.

Expression and Subcellular Localization of Retinoic Acid Receptor-α (RARα) in Healthy and Varicocele Human Spermatozoa: Its Possible Regulatory Role in Capacitation and Survival.

Varicocele, an abnormal tortuosity and dilation of veins of the pampiniform plexus, is the most common identifiable and correctable cause of male infertility. It is now becoming apparent that signaling through vitamin A metabolites, such as all-trans retinoic acid (ATRA), is indispensable for spermatogenesis and disruption of retinoic acid receptor-α (RARα) function may result in male sterility and aberrant spermatogenesis. Herein, we investigated by Western blot and immunogold electron microscopy the expression profiles and subcellular localization of RARα in healthy and varicocele human sperm; in addition, we analyzed the effects of ATRA on cholesterol efflux and sperm survival utilizing enzymatic colorimetric CHOD-PAP method and Eosin Y technique, respectively. In varicocele samples, a strong reduction of RARα expression was observed. Immunogold labeling evidenced cellular location of RARα also confirming its reduced expression in "varicocele" samples. Sperm responsiveness to ATRA treatment was reduced in varicocele sperm. Our study showed that RARα is expressed in human sperm probably with a dual role in promoting both cholesterol efflux and survival. RARα might be involved in the pathogenesis of varicocele as its expression is reduced in pathologic samples. Thus, ATRA administration in procedures for artificial insemination or dietary vitamin A supplementation might represent a promising therapeutic approach for the management of male infertility.

Prorenin secretion from human testis: no evidence for secretion of active renin or angiotensinogen.

To determine if the testis secretes active renin and prorenin, we collected internal spermatic venous blood from 29 young men undergoing varicocelectomy and measured plasma prorenin and active renin together with angiotensinogen and testosterone. Prorenin was higher in internal spermatic venous plasma than in peripheral plasma (+5.3 +/- 1.2 (+/- SE) ng/mL.h [+1.21 ng/(L.s)]; P less than 0.001) as was testosterone [+344 +/- 32 ng/mL [(+1193 nmol/L; P less than 0.001], but there was no significant difference in either active renin (-0.74 +/- 0.45 ng/mL.h [-0.17 ng/(L.s)] or angiotensinogen [+12 +/- 24 ng/mL (+0.01 mumol/L)]. These results demonstrate that the testis secretes prorenin, but not active renin or angiotensinogen, into the general circulation. They support the hypothesis that extrarenal renin systems cannot process prorenin to renin.

Measurement of inhibin concentrations in men: study of changes after castration and comparison with androgen levels in testicular tissue, spermatic venous blood, and peripheral venous blood.

We measured serum inhibin levels in eight untreated patients with prostatic cancer undergoing castration by RIA using an antiserum against 31-kDa bovine follicular fluid inhibin. The inhibin concentrations in testicular tissue and spermatic venous blood were also measured in six of these patients. Serum inhibin levels (mean +/- SD, 377.8 +/- 212.1 U/L), declined rapidly after castration (15 min after, 233 +/- 171.4; 30 min, 224.6 +/- 156.6; 1 h, 181.5 +/- 95.9; 2 h, 174.3 +/- 69.4; 4 h, 122 +/- 6.4; 6 h, less than 120). High concentrations of inhibin were detected in testicular tissue (31,360 +/- 15,180 U/kg), and the levels in spermatic venous blood (3,178.3 +/- 1,386.8 U/L) were approximately 10 times greater than those in peripheral blood (385.5 +/- 233.1 U/L). Testosterone levels were 1,968.2 +/- 992.3 nmol/kg in testicular tissue and approximately 100 times greater in spermatic venous blood (1,631.6 +/- 389.7 nmol/L) than in peripheral blood (18.0 +/- 4.4 nmol/L). These results suggest that circulating inhibin in men mainly originates from testis and that one of the routes of secretion is via the bloodstream.

Aggressive fibromatosis of the spermatic cord. A typical lesion in a "new" location.

The authors describe a 31-year-old man with a 7 cm aggressive fibromatosis (desmoid tumor) of the spermatic cord presenting as a swelling in the left inguinal area that was excised along with the testis and cord. The desmoid tumor is histologically typical, but such tumors arising primarily from the paratesticular structures have apparently not been previously reported and the diagnosis would not be questioned if it not for the unusual site. This tumor is histologically and immunohistochemically indistinguishable from abdominal wall desmoid tumor, with or without Gardner's syndrome. Desmoid tumors at this location should be distinguished from reactive processes, such as pseudosarcomatous myofibroblastic proliferation (so-called "proliferative funiculitis") and inflammatory fibrous pseudotumor, all of which exhibit fibroblastic/myofibroblastic differentiation. Paratesticular fibrosarcoma and leiomyosarcoma should also be differentiated from desmoid tumor that does not have the metastatic potential of sarcomas. Thirty-four months post-operatively, an 8 cm local recurrence in the remaining portion of the left vas deferens causing left hydroureter and hydronephrosis was detected.

Aggressive angiomyxoma of the spermatic cord. Two unusual cases occurring in childhood.

We report on two cases of aggressive angiomyxoma (AAM) of the spermatic cord occurring in two 13-year-old children. Clinically, the tumor simulated a mass of the spermatic cord. Histologically, it represented a poorly circumscribed, benign myxoid tumor, with a sparse population of stromal cells immunoreactive for vimentin and, focally, for smooth muscle actin. No immunostaining for desmin, S-100, p53, p21waf-1, c-Erb-B2 and estrogen-progesterone receptors was found. High proliferating cell nuclear antigen (PCNA) immunoexpression found in most of the tumor cells may explain the high risk of recurrence. AAM should be considered in the differential diagnosis of a spermatic cord mass occurring during infancy.

Leiomyosarcoma of the spermatic cord: a light and ultrastructural description of one case.

A case of leiomyosarcoma arising in the spermatic cord is described. A 83-year-old man required medical care for an irreducible inguinal hernia. The patient underwent herniorraphy and transinguinal radical orchiectomy. Macroscopically, the spermatic cord was enlarged by a gray-tan and ill-defined neoplasm measuring 4 x 4 x 3 cm. Histologically, this proliferation was composed of atypical spindle cells with blunted end nuclei and eosinophilic cytoplasm. Immunohistochemistry and ultrastructure confirmed the smooth muscle nature of the neoplastic cells. The diagnosis of leiomyosarcoma of the spermatic cord was made. To improve the assignment of this rare lesion to its specific anatomic location, we analyzed the immunohistochemical and ultrastructural characteristics of the smooth muscle tumoral cells and in particular those of the intracellular filament aggregates.

Primary paratesticular lymphoma: a report of 2 cases and review of literature.

Non-Hodgkin lymphoma arising in the paratesticular organs without testicular involvement is rare. In most previously reported cases, the classification systems that were used are now outdated and/or immunologic studies were not done. We report the clinical and pathologic features of 2 cases of non-Hodgkin lymphoma arising in the epididymis and the spermatic cord. Patient 1 was a 35-year-old man who presented with a painless scrotal mass. Patient 2 was a 61-year-old man who presented with a right inguinal mass. Orchiectomy performed in both patients revealed a mass confined to the epididymis in patient 1 and to the spermatic cord in patient 2. Histologic examination in both cases revealed diffuse large cell lymphoma, and immunohistochemical studies supported B-cell lineage. Subsequent staging studies showed no other site of disease in patient 1 and an isolated mass anterior to the right psoas muscle in patient 2. Malignant lymphoma involving testicular adnexal structures without involvement of the testis is extremely uncommon. To our knowledge, only 6 cases confined to the epididymis and 12 cases confined to the spermatic cord have been reported previously.

[A study on the intratubular androgen receptor in male infertility].

In order to investigate the presence of androgen insensitivity in patients with male infertility, intratubular androgen receptor (AR) was measured in patients with idiopathic oligozoospermia and azoospermia. The specimens were obtained by testicular biopsy or orchiectomy from 56 patients with oligozoospermia and 5 with azoospermia for clinical study, and 17 with varicocele, 22 with vas disorders and prostatic cancer, which had a mean germinal epithelium score count of 8.5 or greater by the method of Johnsen (JSC) for deciding the cut-off levels, as the control group. Intratubular AR was measured by a 5-point micro-receptor assay, an exchange assay with the DCC method, using 40 microliters of each sample extract and 3H-methyltrienolone as the ligand. The genital skin AR assay was also conducted simultaneously in 34 patients. The results were as follows: 1) No significant correlation was noted between intratubular ARs and genital skin ARs. 2) The maximum binding (Bmax) of AR in the total intratubular extract was intermediate between that of the cytosol fraction and the nuclear extract. 3) Significant correlation was noted between the Bmax of ARs by the micro-receptor assay and those by the conventional assay. 4) The Bmax of AR in the control group (n = 22) was 30.38 +/- 9.89 fmol/mg protein (mean +/- S.D.) and was over 11 fmol/mg protein in all cases. Therefore, 11 fmol/mg protein was decided as the cut-off level for androgen insensitivity. 5) Comparative studies were undertaken between two groups, i.e., low AR group and normal AR group, with AR as a parameter for male infertility.(ABSTRACT TRUNCATED AT 250 WORDS)