Epididymal Duct Research Articles

Seasonal immunohistochemical reactivity of S-100 and α-smooth muscle actin proteins in the epididymis of dromedary camel, Camelus dromedarius.

The S-100 and alpha smooth muscle actin (α-SMA) proteins have been localised in epididymal tissue of several mammalian species, but there have been no data for a seasonal work in camel. The aim of this study was to investigate the immunoreactivities of S-100 and α-SMA proteins in the epididymis of dromedary camel during breeding and nonbreeding seasons. The immunopositive signals for both proteins were observed in different regions of camel epididymis. S-100-immunopositive signals were noted in both the epididymal epithelium and the intertubular connective tissue, while α-SMA signals were confined to the intertubular connective tissue, especially in the peritubular smooth muscle coat and the blood vessels. This study showed an increase in the intensity of S-100 and α-SMA immunoreactions during the breeding season in different regions of camel epididymis than that seen in the nonbreeding season. In conclusion, epididymis might be considered as a source of S-100 and α-SMA proteins in the camel and the secretion of these proteins showed distinct seasonal variations. Further, S-100 and α-SMA may affect the structural and physiological states of the epididymal duct.

G9a-mediated histone methylation regulates cadmium-induced male fertility damage in pubertal mice.

Increasing evidence suggests that cadmium (Cd) is associated with male fertility damage. However, the effects of histone modification on Cd-induced male fertility damage remain obscure. This study aims to evaluate the roles of histone methylation mediated by euchromatin histone methyltransferase (EHMT2/G9a) in regulating Cd-induced male fertility damage. We exposed 4-week-old male C57BL/6J mice to Cd by intraperitoneal injection at 2mg/kg for 1, 3 and 5days. Our data showed that Cd exposure decreased the numbers of impregnated females and litter sizes, which was concomitant with sperm count reduction, histological changes in the cauda epididymal ducts and seminiferous epithelium, and testicular cell apoptosis as evaluated by terminal dUTP nick-end labeling (TUNEL) assay and immunoblotting with increased levels of cleaved caspase 3, PARP and Bax and a decreased level of Bcl-2. Cd-induced male fertility damage was accompanied by enhanced G9a activity followed by increased histone H3 lysine 9 monomethylation (H3K9me1) and dimethylation (H3K9me2) in testes. Furthermore, inhibition of G9a by BIX-01294 normalized H3K9me1 and H3K9me2 to basal levels and prevented Cd-induced male fertility damage and testicular cell apoptosis. Our present study revealed that G9a-mediated histone methylation plays a critical role in Cd-induced male fertility damage and testicular cell apoptosis.

Phospholipase Cγ2 Is Required for Luminal Expansion of the Epididymal Duct during Postnatal Development in Mice.

Phospholipase Cγ2 (PLCγ2)-deficient mice exhibit misconnections of blood and lymphatic vessels, and male infertility. However, the cell type responsible for vascular partitioning and the mechanism for male infertility remain unknown. Accordingly, we generated a mouse line that conditionally expresses endogenous Plcg2 in a Cre/loxP recombination-dependent manner, and found that Tie2-Cre- or Pf4-Cre-driven reactivation of Plcg2 rescues PLCγ2-deficient mice from the vascular phenotype. By contrast, male mice rescued from the vascular phenotype exhibited epididymal sperm granulomas. As judged from immunostaining, PLCγ2 was expressed in clear cells in the epididymis. PLCγ2 deficiency did not compromise differentiation of epididymal epithelial cells, including clear cells, and tube formation at postnatal week 2. However, luminal expansion of the epididymal duct was impaired during the prepubertal period, regardless of epithelial cell polarity and tube architecture. These results suggest that PLCγ2-deficient clear cells cause impaired luminal expansion, stenosis of the epididymal duct, attenuation of luminal flow, and subsequent sperm granulomas. Clear cell-mediated luminal expansion is also supported by the observation that PLCγ2-deficient males were rescued from infertility by epididymal epithelium-specific reactivation of Plcg2, although the edematous and hemorrhagic phenotype associated with PLCγ2 deficiency also caused spontaneous epididymal sperm granulomas in aging males. Collectively, our findings demonstrate that PLCγ2 in clear cells plays an essential role in luminal expansion of the epididymis during the prepubertal period in mice, and reveal an unexpected link between PLCγ2, clear cells, and epididymal development.

Region-specific localization of NOS isoforms and NADPH-diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus).

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage-dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)-NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH-d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH-d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell-specific localization in the efferent ductules and region- and cell-specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation.

Comparison of Toxicity of CdSe: ZnS Quantum Dots on Male Reproductive System in Different Stages of Development in Mice.

Quantum dots (QDs) are new types of fluorescent materials for biological labeling. QDs toxicity study is an essential requirement for future clinical applications. Therefore, this study aimed to evaluate cytotoxic effects of CdSe: ZnS QDs on male reproductive system. In this experimental study, the different concentrations of CdSe: ZnS QDs (10, 20 and 40 mg/kg) were injected to 32 male mice (adult group) and 24 pregnant mice (embryo group) on day 8 of gestation. The histological changes of testis and epididymis were studied by a light microscopy, and the number of seminiferous tubules between two groups was compared. One-way analysis of variance (one-way Anova) using the Statistical Package for the Social Sciences (SPSS, SPSS Inc., USA) version 16 were performed for statistical analysis. In adult group, histological studies of testis tissues showed a high toxicity of CdSe: ZnS in 40 mg/kg dose followed by a decrease in lamina propria; destruction in interstitial tissue; deformation of seminiferous tubules; and a reduction in number of spermatogonia, spermatocytes, and spermatids. However, there was an interesting result in fetal testis development, meaning there was no significant effect on morphology and structure of the seminiferous tubules and number of sperm stem cells. Also histological study of epididymis tissues in both groups (adult and embryo groups) showed no significant effect on morphology and structure of tubule and epithelial cells, but there was a considerable reduction in number of spermatozoa in the lumen of the epididymal duct in 40 mg/kg dose of adult group. The toxicity of QDs on testicular tissue of the mice embryo and adult are different before and after puberty. Due to lack of research in this field, this study can be an introduction to evaluate the toxicity of QDs on male reproduction system in different stages of development.

Morfologia do escroto, do testículo e das vias espermáticas de Metachirus nudicaudatus (Geoffroy, 1803), Didelphidae-Marsupialia

Resumo: Foram utilizadas as gônadas e vias espermáticas de cinco animais machos, adultos em fase reprodutiva, da espécie Metachirus nudicaudatus Geoffroy 1803, única espécie do gênero, para descrever a morfologia do escroto, do testículo e das vias espermáticas. O Metachirus possui escroto pré-peniano e que contém os testículos permanentemente. A pele escrotal é não pigmentada e com poucos pelos e glândulas. A lâmina parietal da túnica vaginal apresenta-se pouco pigmentada. Os testículos são ovais e ligados ao epidídimo através do pedículo testículo-epididimário. Eles são envolvidos, externamente, pela cápsula testicular e sustentados por um estroma de natureza conjuntiva. As células intersticiais são os elementos predominantes no abundante tecido intertubular. Os túbulos seminíferos são largos, enovelados e envolvidos por uma túnica própria fibroelástica, contendo células mióides. O epitélio seminífero é formado pelas células espermatogênicas e de Sertoli intercaladas. Os túbulos seminíferos convergem em direção à extremidade capitata do testículo, ficando revestidos por apenas células de sustentação, caracterizando uma região de transição entre túbulos seminíferos e túbulos retos, ocupada por uma estrutura tipo "válvula" que obstrui parcialmente o lume tubular. Os túbulos retos reúnem-se para formar um único dúctulo eferente, que percorre uma pequena extensão intratesticular, atravessa a albugínea e penetra no pedículo testículo-epididimário. A parte flexuosa do dúctulo eferente forma um lóbulo separado na parte medial do corpo do epidídimo. O epidídimo é envolvido pela cápsula epididimária e constituído pelo ducto epididimário, que se encontra bastante enovelado. O ducto epididimário é revestido por epitélio simples colunar pseudoestratificado apresentando células principais, basais, apicais e de "halo claro". As células principais são predominantes e apresentam características morfológicas e histoquímicas que diferem ao longo do ducto, possibilitando a caracterização de nove diferentes zonas epididimárias. É no lume da zona sete (início da cauda) que começa o pareamento de espermatozoides. Esse fenômeno coincide com alterações morfológicas bem evidentes e uma maior quantidade de mucossubstâncias neutras é secretada nessa zona.O ducto deferente apresenta-se dividido em três partes: justa-epididimária, funicular e abdominal, baseando nas variações histológicas e histoquímicas de seu epitélio e componentes envolventes. O ducto deferente não apresenta ampola e nem cruza o ureter antes de desembocar na uretra. O funículo espermático contém o ducto deferente, artéria e veias testiculares, vasos linfáticos, nervos e um desenvolvido músculo cremáster. Seus componentes apresentam modificações estruturais nas regiões proximal, média e distal, sendo notável a peculiar rede admirável.

Acrosin inhibitor detection along the boar epididymis

Epididymal sperm maturation represents a key step in the reproduction process. Spermatozoa are exposed to epididymal fluid components representing the natural environment essential for their post-testicular maturation. Changes in sperm membrane proteins are influenced by proteolytic, glycosylation and deglycosylation enzymes present in the epididymal fluid. Accordingly, the occurrence of inhibitors of these enzymes in the epididymis is very important for the regulation of sperm membrane protein processing. In the present study, we monitored acrosin inhibitor distribution in boar epididymal fluid and in spermatozoa from different segments of the organ. Using specific polyclonal antibody we registered increasing signal of the acrosin inhibitor (AI) from caput to cauda epididymis. Mass spectroscopy examination of the immunoprecipitated acrosin inhibitor (12kDa) unequivocally identified sperm-associated acrosin inhibitor (SAAI) in the epididymal tissue. Lectin staining showed N-glycosylation in AI from boar epididymis. Protein detection of AI was supported by the results of semi-quantitative RT-PCR showing the presence of mRNA specifically coding for SAAI and similarly increasing throughout the epididymal duct, from its proximal to distal part. Additionally, the immunofluorescence technique showed the AI localization in the secretory tissue of caput, corpus and cauda epididymis, and in the acrosome region and midpiece of the sperm.

Epididymitis: ascending infection restricted by segmental boundaries

Is the regionalization of epididymitis related to epididymal segmentation? We show for the first time that luminal ascent of bacteria is strictly gated by epididymal segment boundaries, involving ductal constriction adjacent to the infected area. The epididymal duct is a continuous, unbranched tube, coiled into segments that are divided by connective tissue septa. Sonographic analysis indicates that swelling associated with epididymitis is predominant in the cauda region. Epididymal segmentation has never been investigated in the context of pathological alterations. We analyzed segment-specific changes in the epididymal duct in a mouse model and in men. In the mouse epididymitis model (3 days post-infection, injection of bacteria into the lumen of the vas deferens), two Escherichia coli strains were tested: a uropathogenic strain CFT073 (UPEC, n = 7) and a fecal non-pathogenic strain NPEC470 (NPEC, n = 5). Two control groups: phosphate-buffered saline, sham-treated animals (n = 4) and untreated mice (n = 8). In addition, segmentation was verified by ex vivo injection of dye into the interstitial spaces of untreated mouse epididymides. Histological findings were compared with specimens from epididymitis patients (n = 10, age range 14-78, median 60 years) who underwent surgical intervention; control: samples from patients without epididymitis (n = 16, age range 38-87, median 73 years). We investigated the ascending infections by detailed histological analysis in correlation with local infection status in a mouse epididymitis model. As a proof of concept, rare patient material from two archives was analyzed: epididymides from patients who underwent surgical intervention for persisting epididymitis, and for control, histologically normal epididymides from men who underwent orchiectomy for therapy of prostatic carcinoma. Luminal ascent of E. coli in mice was strictly gated by epididymal segment boundaries. In the mouse model, both strains of E. coli were detected exclusively in the distal cauda segment associated with damage of the epithelium and muscle layer. Ductal constriction occurred in the non-infected upstream segments of infected area, putatively blocking further luminal ascent of bacteria in UPEC-infected animals. Corresponding histological and morphological changes were found in epididymitis patients. The caput region was found to be unaffected in patients and the mouse model. Patient samples represented advanced cases of epididymitis that made surgical intervention necessary. Our data demonstrate the impact of epididymal segmentation, presumably a protective response mechanism against infectious invasion and bacterial ascent, during epididymitis and affirm the importance of rapid intervention. This work was supported by grants from the State of Hessen (LOEWE-MIBIE) and the DFG (KFO 181). The authors have no conflicts of interest to declare. No clinical trial involved.

Effects of CAII/CAIV double knockout on fertility

Bicarbonate (HCO3‐) is an essential factor to modulate sperm motility. Female genital tract fluids are rich in HCO3‐ and contribute to an increase of sperm beat frequency thus supporting sperm to reach the site of fertilization. Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to HCO3‐ and are important candidates in the regulation of both the HCO3‐ homeostasis in sperm and the composition of the male and female genital tract fluids. We show that CAII and CAIV are localized in epithelial cells lining the epididymal duct and that they appear with the onset of puberty. Gene expression analyses with tissue from respective knockout animals reveal an up‐regulation of CAII and CAIV in the different epididymal sections, indicating a compensatory mechanism for the gene defect. In sperm, the presence of CAII is restricted to the principal piece, whereas CAIV is detectable in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an impaired HCO3‐ homeostasis resulting in significantly diminished sperm motility, velocity and response to HCO3‐. Enzyme activity tests show that CAII and CAIV account for nearly 95% of the total CA activity in sperm. We generated CAII/CAIV double knockout animals which display a significant reduction in sperm motility resulting in sub‐fertility. Interestingly, biochemically created CAIV deficient sperm of CAII knockout animals show a lower response to HCO3‐ as compared to sperm from double knockout animals. From these results we conclude that both CAII and CAIV play a key role for a successful fertilization.

Different locations of RANTES and its receptors on mouse epididymal spermatozoa.

Our previous study showed that the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) originating from the mouse epididymis bound to the midpiece of luminal spermatozoa. The present study was undertaken to investigate the association between RANTES and epididymal spermatozoa and to determine whether the association is mediated by the RANTES receptors CCR1, CCR3 or CCR5. The use of reverse transcription polymerase chain reaction (RT-PCR), immunohistochemical staining and immunofluorescent staining demonstrated that RANTES secreted by apical and narrow cells of mouse epididymal ducts was associated with luminal spermatozoa. Flow cytometric analysis and immunofluorescent labelling revealed that the association between RANTES and spermatozoa of different regions weakened gradually as the spermatozoa moved along the epididymis. Moreover, CCR1, CCR3 and CCR5 were expressed in epididymal spermatozoa and located on the head of epididymal spermatozoa, while RANTES was generally located at the midpiece. In conclusion, RANTES and its receptors were not in the same sperm location, suggesting that RANTES binding to mouse epididymal spermatozoa is independent of CCR1, CCR3 and CCR5.

Chemical sterilisation of Bos indicus bull calves following intratesticular injection of zinc acetate: Effects on semen quality and testicular changes

The aim of this study was to determine the effects in Bos indicus bull calves of intratesticular administration of 1mL of either saline (n=9) or one of the two doses of zinc acetate (ZA1, 57.75mg, n=10 or ZA2, 71.75mg, n=10) on semen quality and testicular changes. Semen was collected by electroejaculation on Days 343, 524 and 783 and animals were slaughtered on Day 860. Treatment reduced median maximum number of progressively motile and morphologically normal sperm collected (P=0.001) and the percentage of animals in which sperm were recovered (saline: 100%, 9/9; ZA1: 44.9%, 4/9 and ZA2: 40.0%, 4/10; P=0.013). Compared to saline treated controls, treatment with ZA reduced the mean diameter of the testes after Day 34 of treatment (treatment×time, P=0.013) and total testicular weight at slaughter (treatment: mean±SEM; saline: 569.4±59.0g, ZA1: 249.3±72.9g, ZA2: 247.5±68.1g; P=0.004). Histological changes in testes of bulls treated with ZA were characterized by germ cell depletion, vacuolation of Sertoli cells, interstitial fibrosis, epididymal duct atrophy with variable remnants of testicular tissue and degeneration. We conclude that intratesticular administration of two doses of ZA in B. indicus calves is able to severely impair spermatogenesis and cause varying degrees of testicular degeneration and a reduction in testicular diameter and mass. Further investigation is required to determine ways of obtaining more consistent results from treatment.

Bupropion treatment increases epididymal contractility and impairs sperm quality with no effects on the epididymal sperm transit time of male rats.

Bupropion is a dopamine (DA) and norepinephrine (NE) reuptake inhibitor used as smoking cessation and antidepressant drug with a lower incidence of male sexual dysfunction. We showed previously that sibutramine, a norepinephrine/serotonine reuptake inhibitor, reduced male rat fertility. As there are no studies evaluating the impact of bupropion treatment on spermatic parameters and male fertility, we evaluated the effects of bupropion treatment (15 and 30 mg kg(-1), 30 days) on sexual behavior, spermatic parameters and fertility of male Wistar rats and on the epididymal duct in vitro contractility. Bupropion 15 mg kg(-1) increased the serum luteinizing hormone level and the epididymal duct contractility, but the sperm quality was not affected. At 30 mg kg(-1) bupropion impaired sperm quality increasing the incidence of non-progressive sperm. The male sexual behavior and fertility were not modified at both bupropion doses. These results, in rats, suggest the importance of studies evaluating the effects of bupropion on the human male sperm quality.

Immunohistochemical Characterization of Spontaneous Sertoli Cell Clusters in the Seminiferous Tubules of C57BL/6J Mice.

Cell clusters were observed in the seminiferous tubules of C57BL/6J mice as a spontaneous lesion in a 2-week toxicity study, and they were demonstrated to be basically composed of Sertoli cells by immunohistochemistry for claudin-11 and GATA-4 (GATA-binding protein 4), which are both Sertoli cell markers. The clusters were composed of about 5 to 50 cells, which had eosinophilic and occasionally vacuolated cytoplasm with an unclear cell boundary. The cell clusters involved some sperm. No mitotic figures were observed and no immunoreactivity for proliferating cell nuclear antigen (PCNA) was detected in the clusters. In most cases, the cell clusters were observed in seminiferous tubules that also showed degenerative changes. In rare instances, cell aggregates immunohistochemically positive for claudin-11 were observed in the lumen of the epididymis, suggesting that some of the Sertoli cell clusters were sloughed off from the seminiferous epithelium into the epididymal ducts. To our knowledge, this is the first report of Sertoli cell clusters in any animal species except for transgenic or surgically altered animals.

Effect of neonatal or adult heat acclimation on testicular and epididymal morphometry and sperm production in rats

The accessory gland weight, testicular and epididymal morphometry and sperm production were analyzed in four groups of rats housed at 20 or 34°C: (1) control rats (CR) kept at 20°C from birth to day 90; (2) adult heat-acclimated rats (AHA) kept at 20°C from birth to day 45 followed by 34°C to day 90; (3) neonatal heat-acclimated rats (NHA) kept at 34°C from birth to day 90 and (4) de-acclimated rats (DA) kept at 34°C from birth to day 45 followed by 20°C to day 90. In NHA and DA rats, accessory gland weight was higher than in controls. Despite the lack of differences in testicular and epididymal morphometry, curvilinear velocity of spermatozoa was lower in the NHA group compared to controls. Areas of seminiferous tubules were lower in the DA than in CR and NHA groups, however, sperm concentration and motility were not affected by the treatment in this group. In AHA rats, epithelium of approximately 20% of seminiferous tubules was degenerated and Sertoli cell number was lower in the remaining tubules. In contrast to sperm motility, epididymal duct area, area of the duct occupied by spermatozoa and cauda epididymis sperm concentration were lower in AHA rats than in the other groups. In conclusion, neonatal heat acclimation did not affect the testicular morphometry and epididymal sperm concentration, suggesting adjustment to high ambient temperature. On the contrary, adult heat acclimation of rats affected the examined parameters, leading to decreased sperm concentration.

Epididymitis: revelations at the convergence of clinical and basic sciences.

Acute epididymitis represents a common medical condition in the urological outpatient clinic. Mostly, epididymitis is caused by bacterial ascent through the urogenital tract, with pathogens originating either from sexually transmitted diseases or urinary tract infections. Although conservative antimicrobial therapy is possible in the majority of patients and is usually sufficient to eradicate the pathogen, studies have shown persistent oligozoospermia and azoospermia in up to 40% of these patients. Animal models of epididymitis are created to delineate the underlying reasons for this observation and the additional impairment of sperm function that is often associated with the disease. Accumulated data provide evidence of a differential expression of immune cells, immunoregulatory genes and pathogen-sensing molecules along the length of the epididymal duct. The evidence suggests that a tolerogenic environment exists in the caput epididymidis, but that inflammatory responses are most intense toward the cauda epididymidis. This is consistent with the need to provide protection for the neo-antigens of spermatozoa emerging from the testis, without compromising the ability to respond to ascending infections. However, severe inflammatory responses, particularly in the cauda, may lead to collateral damage to the structure and function of the epididymis. Convergence of the clinical observations with appropriate animal studies should lead to better understanding of the immunological environment throughout the epididymis, the parameters underlying susceptibility to epididymitis, and to therapeutic approaches that can mitigate epididymal damage and subsequent fertility problems.

Data in support of peptidomic analysis of spermatozoa during epididymal maturation.

The final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several epididymal maturation states could be an informative approach to finding sperm modifications related to maturation and fertility. Here we show the data from differential peptidomic/proteomic analyses on spermatozoa isolated from 4 epididymal regions (immature to mature stage) using a profiling approach based on MALDI-TOF mass spectrometry and, combined to top-down MS in order to identify peptidoforms and proteoforms. By this way, 172m/z peaks ranging between 2 and 20 kDa were found to be modified during maturation of sperm. A total of 62m/z were identified corresponding to 32 different molecular species. The interpretation of these data can be found in the research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1].

AB61. Advances of surgical treatment in male infertility

Approximately 15% of couples cannot conceive a child after 1 year of regular, unprotected intercourse. Male factor infertility is contributory in another 30% to 40%. Most causes of male infertility are treatable and the goal of many treatments is to restore the ability to conceive naturally. The dramatic recent improvements in the management of male infertility are largely contributable to improved surgical techniques and assisted reproductive technology (ART). Specifically in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) allow us to overcome even the most severe defects in spermatogenesis in which only a few are available.Varicocele repair may be considered as the primary treatment option when a man with a varicocele has suboptimal semen quality and the female partner is normal. varicocele repair can reverse a pathologic condition and halt further damage to testicular function, and improve spermatogenesis. Preferred approaches of most experts are microsurgical inguinal and subinguinal operations. Studies have shown that varicocele repair can improve semen parameters, testicular function and pregnancy rates in couples with male-factor infertility associated with varicocele. Also varicocele repair can result in sperm in the ejaculate of azoospermia men when severe hypospermatogenesis or maturation arrest spermatid stage is present.Obstructive azoospermia may result from epididymal, vasal or ejaculatory duct abnormalities. Microsurgical reconstruction remains the safest and most cost-effective treatment option for these patients (vasovasostomy, vasoepididymostomy). It is controversial that the technique of sperm retrieval (open or percutaneous) or the source of sperm (testicular, epididymal, vasal or seminal vesicular) affects pregnancy rate. Sperm extraction or aspiration for IVF via ICSI is needed in cure of surgically uncorrectable azoospermia or failed microsurgical reconstruction and the majority of patients with congenital bilateral absence of the vas deferens (CBAVD). Also sperm retrieval with IVF/ICSI is preferred to surgical treatment when the advanced female partner age, female infertility requiring IVF.Nonobstructive azoospermia (NOA) is the most challenging type, but no specific treatment was available previously. With advent of ICSI in conjunction with sperm retrieval via testicular sperm extraction (TESE), many of nonobstructive azoospermic patients are able to father own babies. However, 20-50% of NOA patients are not able to have sperm retrieved for ART. Microsurgical TESE is an advanced type of TESE that applies microsurgical techniques. This microsurgical TESE is an effective sperm retrieval from men with NOA for ICSI. The advantages of this technique are minimally invasive technique, removal of minimal amount of testicular tissue and minimalizing negative impact on testicular function. Microsurgical TESE is more effective in men with NOA than conventional TESE.Treatment strategies for male infertility have changed as dramatically over the past decade. These advances are largely contributable to microsurgical varicocele repair, microsurgical reconstructive techniques, and microsurgical techniques for surgical sperm retrieval and ART specifically ICSI.

Epithelial Basal Cells Are Distinct from Dendritic Cells and Macrophages in the Mouse Epididymis1

The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.

NHE8’s role in male fertility involves LHR protein trafficking (1097.8)

We reported last year that NHE8, the newest member of the sodium/hydrogen exchanger family, plays an important role in male fertility. NHE8 has abundant expression in the male reproductive organs, particularly in Leydig cells and epididymal duct cells. Male mice lacking NHE8 are infertile. Loss of NHE8 expression also resulted in reduced luteinizing hormone receptor (LHR) expression in male mice. Furthermore, luteinizing hormone (LH)‐stimulated cAMP production was reduced in isolated NHE8KO Leydig cells. After NHE8 expression was silenced by NHE8 siRNA in Leydig cells, reduced LHR expression and dramatically reduced cAMP production after LH stimulation were noted. Confocal microscopic imaging showed that LHR protein was targeted to early endosome in Leydig cells in the absence of NHE8 function instead of the plasma membrane. Therefore, male infertility in NHE8KO mice is due to the disruption of LHR protein trafficking, which results in an interruption in the precise hormone regulation that governs normal sperm development. This study is supported by NIH grant R01DK073638.Grant Funding Source: Supported by NIH grant R01DK073638

Time-Lapse Imaging as a Tool to Investigate Contractility of the Epididymal Duct – Effects of Cgmp Signaling

The well orchestrated function of epididymal smooth muscle cells ensures transit of spermatozoa through the epididymal duct during which spermatozoa acquire motility and fertilizing capacity. Relaxation of smooth muscle cells is mediated by cGMP signaling and components of this pathway are found within the male reproductive tract. Whereas contractile function of caudal parts of the rat epididymal duct can be examined in organ bath studies, caput and corpus regions are fragile and make it difficult to mount them in an organ bath. We developed an ex vivo time-lapse imaging-based approach to investigate the contractile pattern in these parts of the epididymal duct. Collagen-embedding allowed immobilization without impeding contractility or diffusion of drugs towards the duct and therefore facilitated subsequent movie analyses. The contractile pattern was made visible by placing virtual sections through the acquired image stack to track wall movements over time. By this, simultaneous evaluation of contractile activity at different positions of the observed duct segment was possible. With each contraction translating into a spike, drug-induced alterations in contraction frequency could be assessed easily. Peristaltic contractions were also detectable and throughout all regions in the proximal epididymis we found regular spontaneous contractile activity that elicited movement of intraluminal contents. Stimulating cGMP production by natriuretic peptide ANP or inhibiting degradation of cGMP by the phosphodiesterase 5 inhibitor sildenafil significantly reduced contractile frequency in isolated duct segments from caput and corpus. RT-PCR analysis after laser-capture microdissection localized the corresponding molecules to the smooth muscle layer of the duct. Our time-lapse imaging approach proved to be feasible to assess contractile function in all regions of the epididymal duct under near physiological conditions and provides a tool to evaluate acute (side) effects of drugs and to investigate various signaling pathways.