Oncogenic Wnt/ß-catenin signalling pathways in the cancer-resistant epididymis have implications for cancer research.

Aberrant activation of the Wnt/ß-catenin pathway occurs in cancers. This review presents several important cancer-related aspects of Wnt/ß-catenin signalling relevant to the epididymis, provides evidence of such epididymal gene expression and suggests a new direction for further research. The data presented here indicate that besides containing many Wnt/ß-catenin-pathway components, the normal adult human epididymis expresses much more ß-catenin than the colorectal carcinoma cell line HCT116, which possesses elevated ß-catenin expression. The low cancer incidence in the epididymis may be due to factors present in the human epididymis that regulate this oncogenic Wnt/ß-catenin pathway, including (i) 14 of 17 secreted pathway inhibitors, (ii) the majority of the micro-RNAs known to target this pathway, (iii) plasma membrane-associated E-cadherin and CEACAM1 that anchor ß-catenin, preventing its availability for nuclear entry and oncogenic transcriptional activity, (iv) the recently identified membrane-located tumourigenesis inhibitors RNF43 and ZNRF3 that mediate the degradation of the Wnt receptor components Fzds and Lrp5/6 and (v) nuclear KLF4, which competes with TCF for ß-catenin, limiting its transcriptional activity and stabilizing telomeres, thereby reducing mutation incidence. The above regulatory factors expressed by the human epididymis, and the absence of androgen receptor translocation known to promote nuclear translocation of ß-catenin in tumourigenesis in an animal model, may act synergistically to provide hostility in different cell compartments towards tumour formation. The lack of evidence for ß-catenin in epididymal nuclei is noteworthy. Studying this phenomenon may help reveal the mechanisms underlying oncogenic Wnt/ß-catenin signalling and shed new light on cancer therapy and prevention.

Clinical experience with azoospermia: aetiology and chances for spermatozoa detection upon biopsy.

The clinical workup of the infertile male with azoospermia aims at determining the aetiology and estimating the chances of finding spermatozoa by testicular sperm extraction (TESE). To establish prognostic criteria, 1583 consecutive patients with azoospermia consulting the Centre of Reproductive Medicine and Andrology, Münster, a tertiary referral centre, between 1976 and 2009 comprising 9.8% of all patients providing a semen sample were included in this retrospective analysis. The frequencies of diagnoses were as follows: 21% genetic causes (14% Klinefelter syndrome, 1% other chromosomal aberrations, 2% Y-chromosomal microdeletions, 1% hypogonadotropic hypogonadism, 3% congenital bilateral absence of the vas deferens), 31% current or former maldescended testes, varicocele, urogenital infections, 15% malignancies, 11% obstructions, 7% endocrine or other chronic diseases and 12% idiopathic azoospermia. Receiver-operating characteristic curves for chances of finding spermatozoa by testicular biopsy were calculated for testicular volume, serum follicle-stimulating hormone (FSH) and the seminal markers α-glucosidase, fructose and zinc where these data were available (N=283). Histograms of the seminal markers comparing data from men with obstructive azoospermia and normozoospermia visualize their discriminating power. Evidence-based threshold values for high chances of positive testicular biopsy serving as surrogate marker for TESE were derived from the subgroup of men with obstructive azoospermia for testicular volume (≥21mL), FSH (≤10U/L) and seminal α-glucosidase (≤18mU/ejaculate). Fructose and zinc could not predict the chances of finding spermatozoa upon biopsy. Based on these three parameters, positive biopsy and presumably TESE success can be quickly and reliably estimated in everyday practice with the colour-coded figures constructed from these data. As a seminal α-glucosidase reference limit of 18mU/ejaculate can also be used to diagnose congenital bilateral absence of the vas deferens, α-glucosidase (rather than seminal fructose) should be determined as part of the clinical routine when counselling patients before testicular biopsy.

The epididymis as a target for male contraceptive development.

The epididymis is an excellent target for the development of a male contraceptive. This is because the process of sperm maturation occurs in this organ; spermatozoa become motile and are able to recognise and fertilise an egg once they have traversed the epididymal duct. However, a number of attempts to interfere in sperm maturation and epididymal function or both have not been successful. The use of transgenic animals has proved useful in identifying a few epididymal targets but has yet to open the doors for drug development. Continuous focus on identifying additional epididymal targets and sperm-specific and epididymal-specific drugs is key to bringing a male contraceptive acting on the epididymis to the public.

Aquaporin AQP11 in the testis: molecular identity and association with the processing of residual cytoplasm of elongated spermatids.

AQP11 is one of the latest aquaporin (AQP) family members found, which differs from the other AQPs by its intracellular localisation and unusual water pore nucleotides with unclear function. Despite the highest mRNA expression among organs having been reported in the testis, the testicular molecule has not been studied in detail. Immunohistochemistry of rat adult testis localised AQP11 to the elongated spermatids (ES) and no other cell types except residual bodies inside Sertoli cells. It was absent from early ES at least until stage 13, and after a first diffuse appearance in the caudal cytoplasm became concentrated in intracellular organelles by stage 17, was strongest in vesicles in the anterior cytoplasm at the final ES stages and appeared in residual bodies. Staining was detected on the distal quarter of the sperm tail only immediately before spermiation. A similar localisation was found in the mouse and developmental profiles for both the open reading frame mRNA and protein expression in 8-50 dpp testis pinpointed its first appearance coinciding with late stage ES. Sequencing of PCR products of testicular Aqp11 containing the open reading frames confirmed a full match with GenBank databases for rat, mouse and human. Western blotting revealed two or more molecular forms with the 26/27 kDa species dominating in the rat/mouse testis and the 33/34 kDa form selectively allocated to the spermatozoa. In view of intracellular vacuolation leading to polycystic kidney in Aqp11-null mice, a possible role of testicular AQP11 in the recycling of surplus cytoplasmic components of the ES and sustaining Sertoli cell capacity in the support of spermatogenesis was discussed.

Aquaporins in the human testis and spermatozoa - identification, involvement in sperm volume regulation and clinical relevance.

Despite the high water-permeability of human spermatozoa, little is known about the identity and the role of aquaporins (AQP) in them or germ cells. Using ejaculates from donors, sperm AQPs were identified by western blotting followed by the analysis of mRNA with RT-PCR. Protein expression in the testis and spermatozoa was localized by immunocytochemistry. Inhibitors were used to investigate the involvement of aquaporins in water transport when ejaculated spermatozoa were swollen in medium mimicking uterine hypo-osmolality by quinine that blocks volume regulation. Sperm AQP7 and AQP8 in 39 infertile patients and 11 healthy donors were quantified by flow cytometry. AQP1 was absent from spermatozoa. Sperm and testicular AQP7-9 had nucleotide sequences identical to those of somatic cells but AQP8 mRNA also showed shorter variants. AQP7 was expressed abundantly by round and elongated spermatids and ejaculated spermatozoa, AQP8 by all germ cells and spermatozoa, and AQP9 rarely by spermatocytes or Sertoli cells. Protein bands showed specificity by western blotting for AQP7 and AQP8 but not AQP9. The absence of sperm AQP9 was further suggested by the ineffectiveness of its inhibitor phloretin in blocking quinine-induced swelling, but HgCl(2,) which inhibits AQP8, was effective. Sperm AQP7 expression was correlated with progressive motility and was lower in patients than in donors. Sperm AQP8 expression was inversely correlated with the extent of sperm coiling, which is a swelling phenomenon, but showed no difference between patients and donors. In conclusion, AQP7 and AQP8 were identified in human spermatozoa and could play a role in glycerol metabolism and water transport respectively.

[Semen analysis: spermiogram according to WHO criteria]. / Die Ejakulatdiagnostik : Spermiogramm nach WHO-Kriterien.

Semen analysis plays a key role in diagnostics of male infertility. Semen analysis should be done according to WHO guidelines (1999), which are currently under revision. Standard procedures of semen analysis include evaluation of sperm concentration, motility, morphology, and vitality as well as determination of round cells, leukocytes, and sperm antibodies. Analysis of biochemical markers is optional. Several methods of sperm preparation are available for assisted reproduction procedures. Patients should be asked to remain abstinent for at least 48 h and a maximum of 7 days prior to ejaculation.Special focus has to be on quality control programs in andrological laboratories based on WHO guidelines. Only by means of standardized semen analysis the andrologist is capable of counseling infertile couples and diagnosing andrological deficits to finally determine therapeutic strategies including techniques of assisted reproduction.

Channels for water efflux and influx involved in volume regulation of murine spermatozoa.

The nature of the membrane channels mediating water transport in murine spermatozoa adjusting to anisotonic conditions was investigated. The volume of spermatozoa subjected to physiologically relevant hypotonic conditions either simultaneously, or after isotonic pre-incubation, with putative water transport inhibitors was monitored. Experiments in which quinine prevented osmolyte efflux, and thus regulatory volume decrease (RVD), revealed whether water influx or efflux was being inhibited. There was no evidence that sodium-dependent solute transporters or facilitative glucose transporters were involved in water transport during RVD of murine spermatozoa since phloretin, cytochalasin B and phloridzin had no effect on volume regulation. However, there was evidence that Hg(2+)- and Ag(+)-sensitive channels were involved in water transport and the possibility that they include aquaporin 8 is discussed. Toxic effects of these heavy metals were ruled out by evidence that mitochondrial poisons had no such effect on volume regulation.

The tonicity of murine epididymal spermatozoa and their permeability towards common cryoprotectants and epididymal osmolytes.

The permeability of murine cauda epididymidal spermatozoa was determined from the swelling caused by penetrating agents at isotonicity, which lies between 422 and 530 mmol/kg. Spermatozoa were permeable to a range of solutes with size <200 Da. Relative entry rates of cryoprotective agents (CPAs) were ethylene glycol approximately DMSO>propane-1,2-diol>glycerol>propane-1,3-diol. More polar compounds including major epididymal secretions were impermeant. None of the compounds entered spermatozoa through quinine-sensitive channels; rather, quinine increased the size of solute-swollen spermatozoa, suggesting that regulatory volume decrease and osmolyte loss occurred under these conditions. Volume responses to lowered osmolality revealed a greater volume-regulating ability of spermatozoa from the B6D2F1 strain than the C57BL6 strain. As the former strain displays better post-thaw fertility, their spermatozoa may have greater osmolyte loads enabling them to cope better with osmotic stress. Inadequate volume regulation, due to CPA-induced osmolyte loss, may affect post-thaw fertility. Knowing the permeability towards cryoprotectants will help to make a better choice of CPAs that are less damaging to sperm during cryopreservation.

Association of knee bone bruise frequency with time postinjury and type of soft tissue injury.

This study correlated the frequency of bone bruises with soft tissue injuries in the knee and examined bruise frequency as a function of time postinjury. Magnetic resonance imaging of 1546 patients revealed bone bruises in 19% of patients without an associated meniscal or ligamentous injury. For those patients presenting with at least one meniscoligamentous injury, the frequency of bruising was 60% at 0 to 4 weeks, 42% at 4 to 10 weeks, and 31% at 10 to 26 weeks postinjury. The frequency of bruising varied with the presence of concomitant injuries, with the greatest frequency of bruises (78%) observed in patients with anterior cruciate ligament injuries.

Potassium channels involved in human sperm volume regulation--quantitative studies at the protein and mRNA levels.

KCNE1, KCNA5 and KCNK5 have been identified, by using specific blockers, as K(+)-channels involved in sperm volume regulation under physiological conditions. All three channels were localised on the cytoplasmic droplets and tail of human ejaculated spermatozoa by fluorescence microscopy. Using flow cytometric quantification, KCNE1 was found to be present in 80% or more spermatozoa and KCNK5 in only about 20%, with KCNA5 expressed by 20-90% of cells. Whereas the extents of such protein expression did not differ statistically between semen donors and subfertile patients, the former group exhibited higher capacities for sperm volume regulation which were correlated with other sperm qualities including normal morphology and motile sperm number in the ejaculate. Channel identification was further confirmed at the protein level using Western blotting. RT-PCR analysis of testicular and sperm RNA of proven quality indicated the presence of Kcne1, Kcna5 and Kcnk5 transcripts. Subsequent sequencing of PCR products demonstrated that the nucleotide sequences of the entire encoding regions of Kcne1 and Kcnk5 were identical to those published in the database, whereas that of Kcna5 mRNA showed a single nucleotide synonymous deviation that agrees with the published genomic sequence. Quantitative real-time PCR analysis of sperm RNA revealed the amounts of Kcne1 > Kcna5 > Kcnk5, in the same order as for protein expression. Thus, KCNE1 is probably the major K(+)-channel involved in regulatory volume decrease in human spermatozoa, and channel activity is regulated beyond the extent of protein expression.

Involvement of potassium and chloride channels and other transporters in volume regulation by spermatozoa.

Spermatozoa produced in the testis undergo maturation in the epididymis which secretes an osmolyte-rich fluid that bathes the sperm cells. These cells need to maintain their volume after ejaculation when they first encounter hypo-osmolal environments of accessory gland fluids and later within the female tract. If they do not, they experience swelling that is manifested in flagellar angulation that prevents their passage through cervical mucus or the uterotubal junction and they never reach the oocytes. This is a cause of male infertility in domestic species and certain infertile transgenic mice in which flagellar angulation has been shown to indicate cell swelling as a consequence of reduced epididymal provision of osmolytes. The reduced volume regulating ability of spermatozoa from subfertile boars and bulls has prompted study of volume regulation of spermatozoa as a possible cause of human male infertility. Understanding this process may make its manipulation possible and could suggest better sperm handling and storage techniques and thus provide therapy for infertile patients. On the other hand, volume regulation is a potential target for contraception if mimicking the conditions expressed by the "sterile studs" were possible. The evidence for the presence of ion channels probably responsible for regulatory volume decreases in spermatozoa is reviewed here that implicate voltage-gated potassium channels (especially Kv1.5 (KCNA5), minK (KCNE1) and TASK2 (KCNK5)) and the chloride channels CLCN3 and CLNS1A. The involvement of ion co-transporters in volume regulation of spermatozoa is also discussed.

The role of potassium chloride cotransporters in murine and human sperm volume regulation.

Spermatozoa need to undergo regulatory volume decrease (RVD) upon ejaculation to counteract swelling due to the hypo-osmolality of female tract fluids. Defects in sperm RVD lead to failure in both cervical mucus penetration in humans and utero-tubal junction passage in mice. The role of K/Cl cotransporters (KCCs) in RVD was investigated by incubation of spermatozoa from the murine cauda epididymidis and from human ejaculates in media mimicking female tract fluid osmolalities in the presence of KCC inhibitors. Furosemide at 100 microM or more caused swelling of murine spermatozoa as detected with a flow cytometer by increased laser forward scatter over 30 to 75 min of incubation. Bumetanide, known to have low affinity for KCCs, was effective at 1 mM, whereas 10 microM and 20 microM of the specific inhibitor DIOA (dihydroindenyl-oxy alkanoic acid) increased cell volume. These drug doses were ineffective in human spermatozoa, which, however, responded to quinine, confirming the occurrence of RVD under control conditions. The molecular identity of the murine KCC isoform involved was determined at both mRNA and protein levels. Conventional RT-PCR indicated the presence of transcripts from Slc12a4 (KCC1), Slc12a6 (KCC3), and Slc12a7 (KCC4) in the testis, whereas RT-nested PCR revealed the latter two isoforms in sperm mRNA. Of these three isoforms, only SLC12A7 (KCC4) was detected in murine sperm protein by Western blotting. Therefore, besides organic osmolyte efflux and KCl release through separate K(+) and Cl(-) ion channels, SLC12A7 also is involved in murine but not human sperm RVD mechanisms.

Public exposure to radio waves near GSM microcell and picocell base stations.

Exposures of the general public to radio waves at locations near 20 randomly selected GSM microcell and picocell base stations in the UK have been assessed in the context of the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Compliance distances were calculated for the antennas of the base stations from their reported radiated powers. Under pessimistic assumptions that would maximise exposures, the minimum height at which the general public reference level could potentially be exceeded near any of the base station antennas was calculated to be 2.4 m above ground level. The power densities of the broadcast carriers transmitted by the base stations have been measured and scaled to include all other possible carriers. Exposures were generally in the range 0.002-2% of the ICNIRP general public reference level, and the greatest exposure quotient near any of the base stations was 8.6%. Exposures close to microcell base stations were found to be generally greater than those close to macrocell base stations.

Physiological volume regulation by spermatozoa.

Maturing spermatozoa passing through the epididymis experience increasing osmolality in the luminal environment and mature cells are stored in fluids hyper-osmotic to serum. When ejaculated into the female tract, they encounter a hypo-osmotic challenge which initiates the process of regulatory volume decrease (RVD). Defects in RVD result in hindrance of mucus penetration in man and failure of utero-tubal passage in mice. Epididymal sperm from the mouse and cynomolgus monkey and ejaculated sperm from man and monkey have been isolated and dispersed in media with osmolalities mimicking those of uterine fluid or cervical mucus. The effects of specific and broad-spectrum ion channel blockers indicate the involvement of separate K+ and Cl- channels as well as organic osmolytes in physiological sperm RVD, with mechanisms developed during epididymal maturation. Western blotting and immuno-cytochemistry identify and localise some of these channels which play a crucial role in fertilisation in vivo and could be targets for post-testicular contraception.

Regionalization of epididymal duct and epithelium in rats and mice by automatic computer-aided morphometric analysis.

AIM: To establish a rat and mouse epididymal map based on the use of the Epiquatre automatic software for histologic image analysis. METHODS: Epididymides from five adult rats and five adult mice were fixed in alcoholic Bouin's fixative and embedded in paraffin. Serial longitudinal sections through the medial aspect of the organ were cut at 10 microm and stained with hematoxylin and eosin. As determined from major connective tissue septa, nine subdivisions of the rat epididymis and seven for the mouse were determined, consisting of five sub-regions in the caput (rat and mouse), one (mouse) or three (rat) in the corpus and one in the cauda (rat and mouse). Using the Epiquatre software, several tubular, luminal and epithelial morphometric parameters were evaluated. RESULTS: Statistical comparison of the quantitative parameters revealed regional differences (2-5 in the rat, 3-6 in the mouse, dependent on parameters) with caput regions 1 and 2 being largely distinguishable from the similar remaining caput and corpus, which were in turn recognizable from the cauda regions in both species. CONCLUSION: The use of the Epiquatre software allowed us to establish regression curves for different morphometric parameters that can permit the detection of changes in their values under different pathological or experimental conditions.

Chloride channels in physiological volume regulation of human spermatozoa.

As with other mammalian species, human spermatozoa experience a decrease in extracellular osmolarity in cervical mucus upon ejaculation, which requires the efflux of osmolytes and water to counteract swelling that hinders mucus penetration. Recent evidence for the operation of K+ channels in the process of volume regulation suggests parallel involvement of Cl-/anion channels for electro-neutrality as in somatic cells. This was studied using ejaculated spermatozoa washed at seminal osmolality and incubated for 30 min in a medium of mucus osmolality in the presence of Cl- channel blockers. Increases in cell size measured as laser forward-scatter by flow cytometry were detected in the presence of 100 microM 5-nitro-2(3-phenylpropylamino) benzoic acid, 400 microM diisothiocyanato-stilbene-2,2'-disulphonic acid, and 20 microM tamoxifen. No volume changes were found with 400 microM 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid, 200 microM verapamil, or niflumic acid, whereas 1 mM niflumic acid induced shrinkage. Among the candidate channel proteins, Western blotting revealed the presence of ClC-3 (CLCN3) at 87 kDa, but the absence of ClC-2 (CLCN2) from sperm proteins in all samples tested. ICln (CLNS1A) was found in only one of eight samples. Immunocytochemistry localized CLCN3 to the sperm tail. To confirm molecular identities, sperm mRNA was extracted and checked for quality by the presence of protamine 2 transcripts and the absence of sperm DNA and leukocyte mRNA using reverse transcription-polymerase chain reaction. Transcripts of Clcn3 were found in all samples and that of Clns1a in some but not all samples. Clcn3 was therefore considered the most likely candidate of Cl- channel involved in volume regulation of human sperm.

The effects of putative K+ channel blockers on volume regulation of murine spermatozoa.

Volume regulation is a necessary task for spermatozoa as the osmolarity of female tract fluids is lower than that in the epididymis and because the disruption of it in transgenic mice results in infertility. As the specific mechanisms behind this phenomenon are unknown, spermatozoa from mice were screened for sensitivities to inhibitors known to affect specific channels involved in volume regulation of somatic cells. Spermatozoa from the cauda epididymidis were exposed to physiological hypotonic conditions with and without inhibitor. Flow cytometric forward scatter measurements were taken to indicate relative sperm size at 5 and 75 min of incubation. The presence of quinine (0.8 mM), cadmium (0.2 mM), flecainide (100 microM), 4-aminopyridine (4 mM), barium (1 mM), clofilium (10 microM), and phrixotoxin (100 nM) for 75 min resulted in significantly higher forward scatter values than sperm incubated in medium without an inhibitor. These results imply that channels potentially involved in volume regulation of murine spermatozoa include the voltage-dependent Kv1.4 (also known as KCNA1), Kv1.5 (KCNA5), Kv4.1 (KCND1), Kv4.2 (KCND2), Kv4.3 (KCND3), mink (KCNE1), and acid-sensitive TASK2 (KCNK5) and TASK3 (KCNK9). Western blots confirmed the presence of Kv1.5 and TASK2 proteins in sperm plasma membranes at similar (Kv1.5) or higher (TASK2) molecular weight than in somatic cells. Incubation in a different pH did not reveal acid sensitivity of volume regulation. Volume regulation of spermatozoa may involve novel voltage-gated and pH-sensitive potassium channels, which could be valuable targets for the development of a posttesticular male contraceptive.

Characterization of potassium channels involved in volume regulation of human spermatozoa.

Fertility depends in part on the ability of the spermatozoon to respond to osmotic challenges by regulating its volume, which may rely on the movement of K+. These experiments were designed to characterize the K+ channels possibly involved in volume regulation of human ejaculated spermatozoa by simultaneously exposing them to a physiological hypo-osmotic challenge and a wide range of K+ channel inhibitors. Regulation of cellular volume, as measured by flow cytometry, was inhibited when spermatozoa were exposed to quinine (QUI; 0.3 mM), 4-aminopyridine (4AP; 4 mM) and clofilium (CLO; 10 microM) which suggests the involvement of voltage-gated K+ channels Kv1.4, Kv1.5 and Kv1.7, acid-sensitive channel TASK2 and the beta-subunit minK (IsK) in regulatory volume decrease (RVD). QUI and 4AP and, to some extent, CLO also induced hyper activation-like motility. A sensitivity of RVD to pH could not be demonstrated in spermatozoa to support the involvement of TASK2 channels. Western blotting indicated the presence of Kv1.5, TASK2, TASK3 and minK channel proteins, but not Kv1.4. Furthermore, Kv1.5, minK and TASK2 were localized to various regions of the spermatozoa. Although Kv1.4, Kv1.7, TASK2 and TASK3 channels may have important roles in human spermatozoa, Kv1.5 and minK appear to be the most likely candidates for human sperm RVD, serving as targets for non-hormonal contraception.