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Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

The epididymis is composed of a pseudostratified epithelium that is comprised of various cell types. Studies have shown that rat basal cells share common properties with adult stem cells and begin to differentiate in vitro in response to fibroblast growth factor and 5α-dihydrotestosterone. The characterization of rat basal cells is therefore necessary to fully understand the role of these cells. The objectives of this study were to assess the ability of single basal cells to develop organoids and to assess their ability to self-renew and differentiate in vitro. We isolated basal cells from the rat epididymis and established three-dimensional cell cultures from the basal and nonbasal cell fractions. Organoids were formed by single adult epididymal basal cells. Organoids were dissociated into single basal cells, which were able to reform new organoids, and were maintained over 10 generations. Long-term culture of organoids revealed that these cells could be differentiated into cells expressing the principal cell markers aquaporin 9 and cystic fibrosis transmembrane conductance regulator. Electron microscopy demonstrated that organoids were composed of several polarized cell types displaying microvilli and the ability to form tight junctions. Additionally, organoids could be formed by basal cells from either the proximal or distal region of the epididymis and are able to secrete clusterin, a protein implicated in the maturation of spermatozoa. These data indicate that rat basal cells can be used to derive epididymal organoids and further support that notion that these may represent a stem cell population in the epididymis.

The importance of the epididymis on sperm maturation and consequently male fertility has been well documented. The pseudostratified epithelium of the epididymis is comprised of multiple cell types, including principal cells, which are the most abundant, and basal cells. The role of basal cells has been unclear and has been a source of discussion in the literature. However, the recent demonstration that these cells are multipotent or adult stem cells has opened new areas of research in epididymal biology. One such avenue is to understand the regulation of these stem cells, and to exploit their properties to develop tools for toxicological studies to elucidate the effects of chemicals on cell differentiation and epididymal function in vitro. Studies in both rat and mouse have shown that purified single epididymal basal cells cultured under 3D conditions can proliferate and differentiate to form organoids, or mini organs. Furthermore, these epididymal basal stem cells can self-renew and differentiate into other epididymal cell types. It is known that during epididymal development, basal cells are derived from undifferentiated columnar cells, which have been reported to share common properties to stem cells. Like basal cells, these undifferentiated columnar cells can also form organoids under 3D culture conditions and can differentiate into basal, principal and clear cells. Organoids derived from either basal cells or columnar cells offer unique models for toxicology studies and represent an exciting and emerging approach to understand the epididymis.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Epididymal development can be subdivided into three phases: undifferentiated, a period of expansion, and a period of differentiation. The objectives of this study were: (1) to assess gene expression profiles in epididymides; (2) predict signalling pathways and (3) develop a novel 3D cell culture method to assess the regulation of epididymal development in vitro. Microarray analyses indicate that the largest changes in differential gene expression occurred between the 7 to 18-day period, in which 1452 genes were differentially expressed; while 671 differentially expressed genes were noted between day 18 and 28, and there were 560 differentially expressed genes between days 28 and 60. Multiple signalling pathways were predicted at different phases of development. Pathway associations indicated that in epididymides of 7 to 18-day old rats. there was a significant association of regulated genes implicated in stem cells, estrogens, thyroid hormones, and kidney development, while androgen- and estrogen-related pathways were enriched at other phases of development. Organoids were derived from CD49f+ columnar cells from 7-day old rats, while no organoids developed from CD49f- cells. Cells cultured in an epididymal basal cell organoid medium versus a commercial kidney differentiation medium supplemented with DHT revealed that irrespective of the culture medium, cells within differentiating organoids expressed p63, AQP9 and V-ATPase after 14 days of culture. The commercial kidney medium resulted in an increase in the number of organoids positive for p63, AQP9, and V-ATPase. Together, these data indicate that columnar cells represent an epididymal stem/progenitor cell population.

Cystinosis is an inherited metabolic disorder caused by recessive mutations in the CTNS gene. The disease primarily affects the kidneys followed by extra-renal organ involvement later in life. Azoospermia is one of the unclarified complications which is not improved by cysteamine treatment, the only available disease-modifying treatment. We aimed at unravelling the origin of azoospermia in cysteamine-treated cystinosis by confirming or excluding an obstructive factor, and investigating the effect of cysteamine on fertility in the Ctns-/- mice model compared to wild-type. Azoospermia was present in the vast majority of infantile type cystinosis patients. While spermatogenesis was intact either at testicular or epididymal level in cystinosis patients, an enlarged caput epididymis and reduced levels of seminal markers for obstruction Neutral α-Glucosidase (NAG) and extracellular matrix protein 1 (ECM1) pointed towards an epididymal obstruction. Transcriptomic analysis of CTNS KD in a fertile human caput epididymal cell line demonstrated upregulation of pathways related to inflammation and cell polarization. Interestingly, histopathological examination in human and mice revealed a disturbed blood-testis barrier (BTB) characterized by an altered zonula occludens-1 (ZO-1) protein expression. Animal studies ruled out a negative effect of cysteamine on fertility, and showed that cystine in the testes is irresponsive to regular cysteamine treatment. We conclude that the azoospermia in infantile cystinosis is due to an obstruction related to epididymal dysfunction, irrespective of the severity of an evolving primary hypogonadism as an additional non-obstructive process. Regular cysteamine treatment does not affect fertility but has subtherapeutic effects on cystine accumulation in testes. This article is protected by copyright. All rights reserved.

Background Studies on epididymal toxicology are scarce. Betamethasone (BM) is a glucocorticoid used in clinical practice for antenatal therapy. We previously reported changes to testicular morphology, altered sperm quality, and fertility in adult rats following intrauterine administration of BM. Objectives Given that high levels of corticosteroids during gestation lead to fetal androgen depletion, and the essential role of testosterone during epididymal development, here we investigated epididymal morphology and physiology in the F1 and F2 male offspring of female rats treated with BM during gestation. Materials and methods Pregnant rats were randomly divided into two experimental groups: control (saline vehicle, n = 11) and BM-treated group (0.1 mg/kg betamethasone 21-phosphate disodium, n = 13). Rats received an intramuscular injection of vehicle or BM on gestational days 12, 13, 18, and 19. This encompasses the beginning of the critical window of male rat reproductive tract development. A subset of three males from each litter (n = 5 litters/group) was used: One rat per litter was euthanized at puberty, one was euthanized at adulthood, while the others were mated with a non-treated female to obtain the F2 generation. The same protocol described for the F1 was applied for F2, except for the mating protocol. Results In both F1 and F2 generations, prenatal BM exposure resulted in delayed differentiation of the cauda epididymal epithelium, characterized by increased cribriform appearance on PND 45, and displayed weaker or non-detectable Cx43 immunostaining. Furthermore, in the F1 generation only, immunostaining of TP63, a transcription factor expressed in basal cells, appeared more intense with a greater number of TP63-positive cells observed in the cauda epididymis. In adults, the epithelial area was reduced in the F1 BM rats. The contractile activity of isolated epididymal ducts was comparable between groups. Discussion and conclusion Prenatal BM exposure leads to intergenerational impairment in the development and structure of the rat epididymis.

BACKGROUND: In most pseudostratified epithelia, basal cells represent a multipotent adult stem cell population. These cells generally remain in a quiescent state, until they are stimulated to respond to tissue damage by initiating epithelial regeneration. In the epididymis, cell proliferation occurs at a relatively slow rate under normal physiological conditions. Epididymal basal cells have been shown to share common properties with multipotent adult stem cells. The development of organoids from stem cells represents a novel approach for understanding cellular differentiation and characterization of stem cells. OBJECTIVE: To review the literature on tissue regeneration in the epididymis and demonstrate the presence of an epididymal stem cell population. METHODS: PubMed database was searched for studies reporting on cell proliferation, regeneration, and stem cells in the epididymis. Three-dimensional cell culture of epididymal cells was used to determine whether these can develop into organoids in a similar fashion to stem cells from other tissues. RESULTS: The epididymal epithelium can rapidly regenerate following orchidectomy or efferent duct ligation, in order to maintain epithelial integrity. Studies have isolated a highly purified fraction of rat epididymal basal cells and reported that these cells displayed properties similar to those of multipotent adult stem cells. In two-dimensional cell culture conditions, these cells differentiated into cells which expressed connexin 26, a marker of columnar cells, and cytokeratin 8. Furthermore, three-dimensional cell culture of epididymal cells resulted in the formation of organoids, a phenomenon associated with the proliferation and differentiation of stem cells in vitro. CONCLUSIONS: The rapid proliferation and tissue regeneration of the epididymal epithelium to preserve its integrity following tissue damage as well as the ability of cells to differentiate into organoids in vitro support the notion of a resident progenitor/stem cell population in the adult epididymis.

A crucial function of the epididymis is providing a surface glycocalyx that is important for sperm maturation and capacitation. Defensins are antimicrobial peptides expressed in the epididymis. In the macaque epididymis, defensin beta 126 (DEFB126) is important for sperm motility, however, it is not known whether this is the case in humans. The objectives were to determine: (1) if DEFB126 on human ejaculated sperm was correlated with sperm motility in fertile and infertile men, (2) that recombinant DEFB126 could induce immature sperm motility in vitro. Immunofluorescence staining indicated that the proportion of DEFB126-positive sperm was significantly higher in motile sperm. Furthermore, the proportion of DEFB126-labeled sperm was positively correlated with sperm motility and normal morphology. Additional studies indicated that the proportion of DEFB126-positive spermatozoa in fertile volunteers was significantly higher than in volunteers with varicocele, and in infertile volunteers with semen deficiencies. To determine the role of DEFB126 on sperm motility, the DEFB126 gene was cloned and used to generate recombinant DEFB126 in H9C2 cells (rat embryonic heart myoblast cells). Deletion mutations were created into two regions of the protein, which have been linked to male infertility. Immotile testicular spermatozoa were incubated with cells expressing the different forms of DEFB126. Full-length DEFB126 significantly increased motility of co-cultured spermatozoa. However, no increase in sperm motility was observed with the mutated forms of DEFB126. In conclusion, these results support the notion that DEFB126 is important in human sperm maturation and the potential use of DEFB126 for in vitro sperm maturation.

Seminiferous tubules of the testis and epididymal tubules in adult rodents are enveloped by contractile myoid cells, which move sperm and fluids along the male reproductive tract. Myoid cells in the testis influence Sertoli cells by paracrine signaling, but their role in the epididymis is unknown. Electron microscopy revealed that elongated myoid cells formed several concentric layers arranged in a loose configuration. The edges of some myoid cells in a given layer closely approximated one another, and extended small foot-like processes to cells of overlying layers. Gap junction proteins, connexins 32 and 43, were detected within the myoid cell layers by immunohistochemistry. These myoid cells also had caveolae that contained caveolin-1 and cavin-1 (also known as PTRF). The number of caveolae per unit area of plasma membrane was significantly reduced in caveolin-1-deficient mice (Cav1(-/-) ). Morphometric analyses of Cav1-null testes revealed an enlargement in whole-tubule and epithelial profile areas, whereas these parameters were slightly reduced in the epididymis. Although sperm are non-motile as they pass through the proximal epididymis, statistical analyses of cauda epididymidis sperm concentrations revealed no significant differences between wild-type and Cav1(-/-) mice. Motility analyses, however, indicated that sperm velocity parameters were reduced while beat cross frequency was higher in gametes of Cav1(-/-) mice. Thus while caveolae and their associated proteins are not necessary for myoid cell contractility, they appear to be crucial for signaling with the epididymal epithelium to regulate the proper acquisition of sperm motility. Mol. Reprod. Dev. 83: 526-540, 2016. © 2016 Wiley Periodicals, Inc.

To develop a better understanding for fixed biomass processes, the development of a nitrifying bacterial biofilm, as well as the performance of treatment during modifications to operational conditions of a full-scale submerged biological filter were examined. The development of the nitrifying biofilm was investigated at four depth levels (1, 2, 4 and 5 feet). The result of bacterial subpopulations analyzed by qPCR relative to the physico-chemical parameters of the wastewater during the various tests (sustained aeration, modified backwash parameters and inflow restriction) revealed an increase of the relative presence of nitrifying microorganisms throughout the biofilm (especially for nitrite oxidizing bacteria (NOB)), but this was not necessarily accompanied by a better nitrification rate. The highest observed nitrification rate was 49% of removal in the test cell during backwashing conditions, whereas the relative ammonia oxidizing bacteria (AOB) population was 0.032% and NOB was 0.008% of the total biomass collected. The highest percentage of nitrifying bacteria observed (0.034% AOB and 0.18% NOB) resulted in a nitrification rate of 21%. The treatment of organic matter determined by measuring the chemical and biochemical oxygen demand (COD, CBOD5) was improved.

Epididymal sperm maturation is a critical aspect of male reproduction in which sperm acquire motility and the ability to fertilize an ovum. Sperm maturation is dependent on the creation of a specific environment that changes along the epididymis and which enables the maturation process. The blood-epididymis barrier creates a unique luminal micro-environment, different from blood, by limiting paracellular transport and forcing receptor-mediated transport of macromolecules across the epididymal epithelium. Direct cellular communication between cells allows coordinated function of the epithelium. A limited number of studies have directly examined the effects of toxicants on junctional proteins and barrier function in the epididymis. Effects on the integrity of the blood-epididymis barrier have resulted in decreased fertility and, in some cases, the development of sperm granulomas. Studies have shown that in addition to tight junctions, proteins implicated in the maintenance of adherens junctions and gap junctions alter epididymal functions. This review will provide an overview of the types and roles of cellular junctions in the epididymis, and how these are targeted by different toxicants.

Gap junctions are responsible for intercellular communication. In the adult mammalian epididymis, gap junction protein alpha 1 (GJA1) is localized between basal and either principal or clear cells. GJA1 levels and localization change during the differentiation of basal cells. The present objective was to determine the role of basal cells and prostaglandin E2 (PGE2) on GJA1 in the rat epididymis. Prior to basal cell differentiation, GJA1 is colocalized with TJP1 at the apical lateral margins between adjacent epithelial cells. When basal cells are present, GJA1 becomes associated between basal and principal cells, where it is primarily immunolocalized until adulthood. Basal cells express TP63, differentiate from epithelial cells, and produce prostaglandin-endoperoxide synthase 1 by 21 days of age. Prior to day 21, GJA1and TP63 are not strongly associated at the apical region. However, by day 28, TP63-positive basal cells migrate to the base of the epithelium, and also express GJA1. To assess effects of PGE2 on GJA1, rat caput epididymal (RCE) cells were exposed to PGE2 (50 μM) for 3 h. PGE2 increased levels of Gja1 mRNA in RCE cells, while levels of Gjb1, Gjb2, Gjb4, and GjB5 were unaltered. Furthermore, PGE2 increased protein levels of GJA1, phospho-GJA1, phospho-AKT, CTNNB1, and phospho-CTNNB1. Total AKT and the tight junction protein claudin1 were also not altered by PGE2. Data suggest that development of the epididymal epithelium and differentiation of epididymal basal cells regulate the targeting of GJA1, and that this appears to be mediated by PGE2.

Made available in DSpace on 2018-12-11T17:11:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-01 Prenatal betamethasone (BM) exposure in rats negatively impacts sperm quality and male fertility. Studies have shown that BM can cause multi-generational effects on the pituitary-adrenal-axis of rats. The objective of this study was to assess the reproductive development and fertility of male rats (F2) whose fathers (F1) were exposed to BM (0.1 mg/kg) on gestational days 12, 13, 18 and 19. In F2 rats, there was a significant reduction in body weights of the BM-treated group at PND 1 as well as delayed onset of puberty, and decreases in FSH levels, Leydig cell volume, sperm number and motility, seminal vesicle contractility and ejaculated volume. Furthermore, increased serum LH levels, sperm DNA damage and abnormal morphology were observed, resulting in reduced fertility. In conclusion, prenatal BM-treatment leads to intergenerational long-term reproductive impairment in male rats, raising concern regarding the widespread use of BM in preterm births. Department of Morphology Institute of Biosciences of Botucatu UNESP – São Paulo State University, Distrito de Rubião Junior s/nº Department of Pharmacology Institute of Biosciences of Botucatu UNESP – São Paulo State University, Distrito de Rubião Junior s/nº Department of Pathology Botucatu Medical School UNESP – São Paulo State University, Distrito de Rubião Junior s/n° Laboratory for Reproductive Toxicology INRS-Institut Armand-Frappier University of Quebec, 531 Boulevard des Prairies Department of Morphology Institute of Biosciences of Botucatu UNESP – São Paulo State University, Distrito de Rubião Junior s/nº Department of Pharmacology Institute of Biosciences of Botucatu UNESP – São Paulo State University, Distrito de Rubião Junior s/nº Department of Pathology Botucatu Medical School UNESP – São Paulo State University, Distrito de Rubião Junior s/n°

International audience; Seminiferous tubules of the testis and epididymal tubules in adult rodents are enveloped by contractile myoid cells, which move sperm and fluids along the male reproductive tract. Myoid cells in the testis influence Sertoli cells by paracrine signaling, but their role in the epididymis is unknown. Electron microscopy revealed that elongated myoid cells formed several concentric layers arranged in a loose configuration. The edges of some myoid cells in a given layer closely approximated one another, and extended small foot-like processes to cells of overlying layers. Gap junction proteins, connexins 32 and 43, were detected within the myoid cell layers by immunohistochemistry. These myoid cells also had caveolae that contained caveolin-1 and cavin-1 (also known as PTRF). The number of caveolae per unit area of plasma membrane was significantly reduced in caveolin-1-deficient mice (Cav1(-/-) ). Morphometric analyses of Cav1-null testes revealed an enlargement in whole-tubule and epithelial profile areas, whereas these parameters were slightly reduced in the epididymis. Although sperm are non-motile as they pass through the proximal epididymis, statistical analyses of cauda epididymidis sperm concentrations revealed no significant differences between wild-type and Cav1(-/-) mice. Motility analyses, however, indicated that sperm velocity parameters were reduced while beat cross frequency was higher in gametes of Cav1(-/-) mice. Thus while caveolae and their associated proteins are not necessary for myoid cell contractility, they appear to be crucial for signaling with the epididymal epithelium to regulate the proper acquisition of sperm motility. Mol. Reprod. Dev. 83: 526-540, 2016. © 2016 Wiley Periodicals, Inc.

In prepubertal rats, connexin 26 (GJB2) is expressed between adjacent columnar cells of the epididymis. At 28 days of age, when columnar cells differentiate into adult epithelial cell types, Gjb2 mRNA levels decrease to barely detectable levels. There is no information on the regulation of GJB2 in the epididymis. The present study characterized regulation of the Gjb2 gene promoter in the epididymis. A single transcription start site at position −3829 bp relative to the ATG was identified. Computational analysis revealed several TFAP2A, SP1, and KLF4 putative binding sites. A 1.5-kb fragment of the Gjb2 promoter was cloned into a vector containing a luciferase reporter gene. Transfection of the construct into immortalized rat caput epididymal (RCE-1) cells indicated that the promoter contained sufficient information to drive expression of the reporter gene. Deletion constructs showed that the basal activity of the promoter resides in the first −230 bp of the transcriptional start site. Two response elements necessary for GJB2 expression were identified: an overlapping TFAP2A/SP1 site (−136 to −126 bp) and an SP1 site (−50 bp). Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays confirmed that SP1 and TFAP2A were bound to the promoter. ChIP analysis of chromatin from young and pubertal rats indicated that TFAP2A and SP1 binding decreased with age. SP1 and TFAP2A knockdown indicated that SP1 is necessary for Gjb2 expression. DNA methylation did not appear to be involved in the regulation of Gjb2 expression. Results indicate that SP1 and TFAP2A regulate Gjb2 promoter activity during epididymal differentiation in rat.

Made available in DSpace on 2018-12-11T17:03:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Antenatal betamethasone is used for accelerating fetal lung maturation for women at risk of preterm birth. Altered sperm parameters were reported in adult rats after intrauterine exposure to betamethasone. In this study, male rat offspring were assessed for reproductive development after dam exposure to betamethasone (0.1 mg/kg) or vehicle on Days 12, 13, 18 and 19 of pregnancy. The treatment resulted in reduction in the offspring body weight, delay in preputial separation, decreased seminal vesicle weight, testosterone levels and fertility, and increased testicular weight. In the testis, morphologically abnormal seminiferous tubules were observed, characterized by an irregular cell distribution with Sertoli cell that were displaced towards the tubular lumen. These cells expressed both Connexin 43 (Cx43) and Proliferative Nuclear Cell Antigen (PCNA). In conclusion, intrauterine betamethasone treatment appears to promote reproductive programming and impairment of rat sexual development and fertility due to, at least in part, unusual testicular disorders. Department of Morphology Institute of Biosciences Univ Estadual Paulista-UNESP, Distrito de Rubião Junior s/n Laboratory for Reproductive Toxicology INRS-Institut Armand-Frappier University of Quebec, 531 boulevard des Prairies Department of Morphology Institute of Biosciences Univ Estadual Paulista-UNESP, Distrito de Rubião Junior s/n CNPq: 308842/2013-8

In the past years with the advance of analytical instrumentation/methods, some chemicals are being discovered in water and other environmental matrices that previously had not been noticed or are being detected at levels that may be diverse more than expected. These are often generally referred to as “contaminants of emerging concern” because the potential risk to human health and the environment associated with their presence, frequency of occurrence, or source may not be known. Some of these compounds include household and industrial chemicals such as same retardants, plasticizers, detergent compounds, pharmaceutical and personal care products, fragrances, antimicrobial cleaning agents, nanomaterials, and some toxic elements [1–5]. This special issue affords the opportunity to bring together the results of 5 research articles and 1 review paper covering several aspects related to this topic. An overview of emerging contaminants and associated human health effects was presented by M. Lei et al. The authors have summarized the conclusions of the comprehensive epidemic literature and representative case reports relevant to emerging contaminants and the human body to address concerns about potential harmful health effects in the general population. They concluded that the current evidence is not conclusive and comprehensive, suggesting prospective cohort studies in the future to evaluate the associations between human health outcomes and emerging environmental contaminants. W.-H. Yu et al. evaluated the expression and function of Oat1 and Oat3 after administering arsenic and mercury containing traditional Chinese medicine (realgar and cinnabar) in vivo in mice. The authors have found that the traditional Chinese medicine investigated is probably related to kidney damage through inhibiting several members of the organic anion transporters (such as OAT1 and OAT3). Q. Liu et al. evaluated whether there were miRNA and mRNA aberrant expression profiles and potential role in malignant transformation of 16HBE induced by cadmium. Their results provided a link for the miRNA-mRNA integrated network and implied the role of novel miRNAs in malignant transformation of 16HBE induced by cadmium. N. Sarker et al. determined the levels of various metals in three different floral honeys from Bangladesh. Several physical parameters were also determined. According to the authors, honeys from Bangladesh are of good quality and the tested parameters met the requirements set by the International Honey Commission. The heavy metal concentrations were also within the limits, indicating their purity. Moreover, the higher concentrations of Fe in the investigated honeys signify that these honeys are a good source of these elements, which is very important for the human diet. Q. Hu et al. concluded that mycotoxins from mycoinsecticides have limited ways to enter environments. Moreover, according to the authors the risks of mycotoxins from mycoinsecticides contaminating foods are likely controllable. I. Macharia investigates the determinants of pesticide-related cost of illness (COI) and acute symptoms, using a balanced panel of 363 farmers interviewed from seven major vegetable producing districts of Kenya. The author observed that the personal protective equipment (PPE), education level, record keeping, and geographical location considerably determined health impairments. Moreover, encouraging the proper use of PPE and record keeping of pesticide use could greatly reduce poisoning cases and COI.

Oculodentodigital dysplasia (ODDD) is a dysmorphogenesis syndrome resulting from mutations in the GJA1 gene encoding the gap junction protein, connexin43 (CX43). In the testis this connexin localizes between Leydig cells, Sertoli cells and between Sertoli cells and germ cells. It is essential for Sertoli cell differentiation and spermatogenesis. This study explored male fertility in Gja1(Jrt) /+ mice which carry a dominant mutation that causes an amino acid substitution (G60S) in CX43. Gja1(Jrt) /+ mice mimic the phenotype of ODDD. Immunodetection methods revealed a reduction of both total CX43 and CX43 in membrane plaques in mutant testes. Correspondingly, intercellular coupling along the tubules was diminished as revealed by fluorescent dye transfer. Light and electron microscopy revealed loss of germ cells and sloughing of germ cells into the tubular lumen. There were also irregularities in size and shape of Sertoli cell nuclei. Analyses of cauda epididymal sperm indicated significant decreases in sperm count and sperm velocity parameters associated with sperm vigour, and significantly lower sperm head movement parameters associated with progressiveness. A significant decrease was also observed in total per cent motility. These results further confirm a critical role for CX43 in spermatogenesis and sperm maturation and support the possibility of subfertility in ODDD human males.

Gap junctions and connexins are critical for coordinating cellular functions in complex epithelia. In recent years there has been increased interest in understanding the regulation and function of gap junctions in both the testis and epididymis. Studies in transgenic mice in which connexin 43 (Cx43) is mutated or is knocked down only in Sertoli cells have demonstrated the essential role of Cx43 in spermatogenesis and differentiation of Sertoli cells. In the epididymis developmental studies have shown a role for numerous connexins in the differentiation of epithelial cells and communication between the basal cells and both principal and clear cells. In both tissues several factors, such thyroid hormones and androgens, are important in regulating expression and function of connexins. Pannexins, which form cellular channels but are structurally similar to gap junction proteins, have been identified in both testis and epididymis and, in the epididymis, are regulated by androgens. The objective of this review is to summarize the advances that have been made on the role and regulation of connexins and pannexins in the testis and epididymis and their implication in spermatogenesis and sperm maturation.

A single in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestation day 15 decreased epididymal sperm count in adult rats and thus was used to establish a tolerable daily intake for TCDD. However, several laboratories have been unable to replicate these findings. Moreover, conflicting reports of TCDD effects on daily sperm production suggest that spermatogenesis may not be as sensitive to the adverse effects of TCDD as previously thought. We performed a PubMed search using relevant search terms linking dioxin exposure with adverse effects on reproduction and spermatogenesis. Developmental exposure to TCDD is consistently linked with decreased cauda epididymal sperm counts in animal studies, although at higher dose levels than those used in some earlier studies. However, the evidence linking in utero TCDD exposure and spermatogenesis is not convincing. Animal studies provide clear evidence of an adverse effect of in utero TCDD exposure on epididymal sperm count but do not support the conclusion that spermatogenesis is adversely affected. The mechanisms underlying decreased epididymal sperm count are unknown; however, we postulate that epididymal function is the key target for the adverse effects of TCDD.Uma única exposição in utero a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) no 15º dia de gestação diminuiu a contagem de esperma epididimal em ratos adultos e por isso foi utilizada para estabelecer uma dosagem diária tolerável para TCDD. No entanto, diversos laboratórios não conseguiram reproduzir esses resultados. Além disso, relatórios conflitantes dos efeitos de TCDD na produção diária de esperma sugere que espermatogênese pode não ser tão sensível aos efeitos adversos do TCDD como antes se pensava. Foi feita uma pesquisa no PubMed usando termos de pesquisa relevantes, relacionados à exposição à dioxina com efeitos adversos na reprodução e na espermatogênese. Exposição em desenvolvimento ao TCDD é consistentemente relacionada à diminuição da contagem da cauda epididimal de esperma, mas não apoia a conclusão de que a espermatogênese é afetada. Os mecanismos por trás da diminuição da contagem de esperma epididimal são desconhecidos; no entanto, contestamos que a função epididimal é a chave para efeitos adversos do TCDD.

International audience; The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Pannexins (Panxs) are channel-forming proteins that have homology to the invertebrate gap junction proteins, the innexins. These proteins form membrane channels implicated in ATP release. To evaluate the role of Panxs in the male reproductive tract, we investigated the distribution and regulation of Panx1 and 3 in the testis, efferent ducts (ED), and epididymis of adult rats. In the testis, Panx1 localized to the basal compartment of the seminiferous epithelium, while Panx3 was expressed in Leydig cells. In the ED, both Panxs were expressed in the apical region of ciliated cells. In the epididymis, Panx1 was detected at the base of the epithelium, at times encompassing basal cells, while Panx3 was restricted to the apical plasma membrane of principal cells. Panx3 immunoreactions were high throughout the entire epididymis while Panx1 was high in all regions except the initial segment. Multiple transcripts for Panx1 were identified, and sequence analysis indicated that alternative splicing might account for them. Orchidectomy resulted in the expression of multiple immunoreactive Panx1 bands, and these appeared to be androgen-repressed throughout the epididymis. Panx3 levels in all epididymal regions were also androgen-repressed. Deglycosylation experiments indicated that some Panx1 species were due to glycosylation, but this did not account for all Panx1 immunoreactive species. In summary, Panxs expressed in the epididymis and regulated by both alternative splicing events and androgens. These proteins may play a role in ATP secretion into the epididymal lumen and basal extracellular spaces for functions involving sperm transport and maturation.

Post-testicular sperm maturation requires a specific luminal environment in the epididymis that is created, in part, by the blood-epididymis barrier. There is limited information on gene expression in the epididymis of infertile obstructive azoospermia (OA) patients due to the difficulty in obtaining tissues. The objectives of this study were to determine if epididymal tight junction proteins are altered in OA and to develop cell lines that could serve to elucidate alterations in the epididymis of infertile men. Epididymal claudin (CLDN) 1, CLDN4, and CLDN10 mRNA levels were altered in OA downstream from the obstruction site. Epithelial cell lines derived from the caput epididymidis of one OA patient were developed (infertile human caput epididymal cell line [IHCE]). IHCEs were composed of homogenous populations of diploid cells that ultrastructurally resembled in vivo principal cells. The cells expressed cytokeratin, SPAG11B, CLDN2, CLDN3, desmoplakin, and vimentin. However, the cells did not express several other epididymal markers (CRISP1, SPINLW1, NPC2, CD52, or DCXR) or junctional proteins (CDH1, CDH2, CLDN1, CLDN4, CLDN7, or CLDN8). Further studies using IHCE1 and transepithelial resistance indicated that the cells were unable to form tight junctions. Microarray analyses comparing gene expression in IHCE1 and a recently developed fertile human caput epididymal cell line revealed differential expression of genes encoding junctional proteins, cell junction regulators, and epididymal proteins. Together, these data indicate that epididymal cellular junctions appear to be altered in OA.

Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000-50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.

The epididymis is responsible for posttesticular sperm maturation. Sperm maturation is dependent on the luminal microenvironments along the epididymis. Though the role of the epididymis is well established, the molecular and cellular mechanisms responsible for sperm maturation remain to be elucidated, particularly in the human, as limited biological tools exist. We have established the first stable epithelial cell lines transformed with SV40 large T antigen (LTAg) from two regions of the human adult epididymis. The cell lines are composed of homogenous populations of diploid principal cells that possess ultrastructural characteristics similar to those of human principal cells in vivo. These cells express transcripts for adherens (cadherins CDH1 and CDH2) and tight (claudins CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN8) junctions as well as desmosomes (desmoplakin, DSP). Transepithelial resistance (TER) measurements in fertile human caput epididymal cell line 1 (FHCE1) as well as the immunolocalization of tight junctional protein 1 (TJP1), occludin, and CLDN1 indicate that these cells form functional tight junctions. Furthermore, knockdown of CLDN1, CLDN3, CLDN4, or CLDN7 using specific siRNAs resulted in significant decreases in TER, suggesting that these CLDNs are essential for the barrier function of the blood-epididymis barrier. Disruption of CLDN1, CLDN3, CLDN4, and CLDN7 could, therefore, lead to epididymal dysfunction, resulting in male infertility.

Dioxins are lipophilic chemicals that resist biological and environmental degradation, making them persistent in the environment. Seventy-five dioxin congeners and 135 furan congeners comprise the complex mixture of dioxins, of which 7 and 10 congeners, respectively, are capable of binding to and activating the aryl hydrocarbon receptor (AhR) (Van den Berg et al. 2006). Of the 209 polychlorinated biphenyl (PCB) congeners, 12 have the potential to activate the AhR (Van den Berg et al. 2006). Among these, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic environmental contaminant in animal studies (Denison and Nagy 2003) and thus is significant for human health (Birnbaum 1994; Larsen 2006; Schecter et al. 2006). Dioxins are by-products of industrial processes such as chlorine bleaching of pulp and paper, the manufacture of certain pesticides, and incineration of medical waste and plastics (Anderson and Fisher 2002; Hewitt et al. 2006; Lin et al. 2006; Thornton et al. 1996). Resistance to degradation leads to bioaccumulation and biomagnification of dioxins in the food chain. Inclusion of animal fat in animal feed is another route of dioxin entry to the food supply and a source of exposure (Hoogenboom et al. 2007). Human exposure is primarily through consumption of contaminated food, especially high-fat foods such as milk, cheese, meat, some fish, fast foods, and breast milk (Schecter and Li 1997; Schecter et al. 1997, 1998a, 1998b, 2001). Residue levels have been measured in the serum of pregnant Canadian women with a mean ± SE of 0.34 ± 0.01 pg TCDD/g lipid (Foster et al. 2005), lower than the serum concentrations measured in pregnant German women (range, 4.34–97.3 pg TCDD/g lipid; Wittsiepe et al. 2007) and women from central Taiwan (mean, 6.7 pg TCDD/g lipid; Wang et al. 2004). The different concentrations in these studies reflect differences in measurement techniques; a gene reporter assay [chemical-activated luciferase gene expression (CALUX) assay] and high- resolution mass spectrometry have been used to quantify World Health Organization (WHO) toxic equivalence quotient concentrations or dioxin-like activity. Regardless, these studies demonstrate that the fetus is exposed to dioxin-like chemicals during a critical window of development. The half-life of dioxin ranges from 5.8 to 14.1 years in humans and is influenced by body composition, with higher body fat associated with a longer half-life (Michalek et al. 1992, 1996, 2002). By comparison, the half-life ranges from 10 to 15 days in mice (Grassman et al. 1998) and is approximately 3 weeks in rats (Rose et al. 1976). Given the documented adverse effects on the adult rat reproductive tract after a single in utero exposure to TCDD, developmental exposure and differences in the half-life of TCDD could have important consequences for the relevance of results from animal studies for human health. Experimental evidence demonstrates that most toxic actions of TCDD are mediated through the AhR, which is a ligand-activated transcription factor (Robles et al. 2000) ubiquitously expressed in many human tissues and cell lines (Dolwick et al. 1993; Harper et al. 1991; Li et al. 1998). AhR is inactive and unbound in the cytoplasm. Upon ligand binding, the AhR binds to the aryl hydrocarbon nuclear translocator (ARNT) protein, resulting in translocation to the nucleus, where the ligand–AhR–ARNT complex binds to response elements in the promoter of AhR-regulated genes (dioxin response element). In humans, exposure to dioxins has been linked to a variety of adverse effects, including chloracne (Baccarelli et al. 2005a, 2005b), immune suppression (Weisglas-Kuperus et al. 2000), thyroid dysfunction (Koopman-Esseboom et al. 1994; Pavuk et al. 2003), increased risk for diabetes (Longnecker and Michalek 2000) and endometriosis (Eskenazi et al. 2000; Heilier et al. 2007), impaired neurodevelopment (Koopman-Esseboom et al. 1996; Vreugdenhil et al. 2002), and reproductive/developmental abnormalities (Dimich-Ward et al. 1996; Halldorsson et al. 2009; Leijs et al. 2008; Mocarelli et al. 1996, 2000, 2008). A number of studies attribute background dioxin exposure to adverse effects on development or pathophysiology in multiple organ systems. However, the results of these studies are controversial, and it is not possible to establish causal associations; thus, animal studies are essential. In animal studies, dioxin exposure has been shown to cause thymic atrophy (Chahoud et al. 1989), immune suppression (Hogaboam et al. 2008), hepatotoxicity (Chahoud et al. 1989), and impaired thyroid function (Fan and Rozman 1995; Henry and Gasiewicz 1987; Kohn 2000). Of these, TCDD effects on the reproductive tract are the most notable, owing to the sensitivity of this system. Developmental exposure to TCDD induces placental dysfunction (Ishimura et al. 2006; Kawakami et al. 2006); decreased offspring survival (Bell et al. 2007a, 2007b; Bjerke and Peterson 1994; Flaws et al. 1997; Gray and Ostby 1995; Gray et al. 1995, 1997b; Roman et al. 1995; Sommer et al. 1996); developmental defects of the palate, heart, and kidney (Aragon et al. 2008; Birnbaum et al. 1989; Theobald and Peterson 1997) and reproductive tract of males (Gray et al. 1997a; Vezina et al. 2008) and females (Flaws et al. 1997; Gray and Ostby 1995; Gray et al. 1997b; Heimler et al. 1998; Wolf et al. 1999); decreased weights of reproductive organs (Bjerke and Peterson 1994; Gray and Ostby 1995; Gray et al. 1995, 1997a; Loeffler and Peterson 1999; Mably et al. 1992a, 1992c; Moore et al. 1985; Ohsako et al. 2001, 2002; Wolf et al. 1999); delayed onset of sexual maturation (Bjerke et al. 1994; Faqi and Chahoud 1998; Flaws et al. 1997; Gray et al. 1997a, 1997b); feminization of males (Bjerke et al. 1994; Mably et al. 1992b); and decreased sperm counts (Faqi et al. 1998; Gray et al. 1995, 1997a; Mably et al. 1992a; Sommer et al. 1996). Of the reproductive/developmental effects of TCDD, decreased sperm counts are considered as the most sensitive outcome. Mably et al. (1992a) reported that epididymal sperm counts were significantly decreased in rats after a single exposure to 0.064 μg TCDD/kg on gestational day (GD) 15. Similarities in spermatogenesis between rats and humans, together with the marked apparent sensitivity of sperm production to the adverse effects of TCDD exposure, has yielded the establishment of a tolerable daily intake of approximately 2 pg/kg/day for TCDD and related compounds by the WHO [Joint FAO/WHO (Food and Agriculture Organization/WHO) Expert Committee on Food Additives 2001]. However, results of recent studies have been unable to reproduce the effect of in utero TCDD exposure on epididymal sperm counts (Bell et al. 2007a, 2007c; Ohsako et al. 2001; Yonemoto et al. 2005), although one study did report a decrease in epididymal sperm counts with a TCDD concentration of 1 μg/kg (Ohsako et al. 2001). Reasons for the divergent results are unclear, but differences in methodology used to quantify sperm count could account for the observed differences. In addition, disparities in dioxin toxicokinetics and species sensitivity to dioxins raise important questions concerning the use of animal models to estimate human risk (Adler 1996; Aylward et al. 2005; Simanainen et al. 2004a, 2004b). Therefore, the objective of this review is to evaluate the effects of TCDD exposure on spermatogenesis. We undertook a systematic review of the literature and performed a PubMed (National Center for Biotechnology Information 2009) search using the following search terms: dioxin, TCDD, reproductive, developmental, testis, spermatogenesis, sperm, semen quality, fertility, and fecundity. The search yielded 4,224 titles; duplicate papers, review articles, letters to the editor, and articles describing tissue culture or nonmammalian studies were excluded from further analysis. The abstracts of all remaining articles were read by two independent investigators and were included for further analysis if they fit the following criteria: a) the paper was published in English, b) the abstract described an epidemiologic or animal study in which TCDD or dioxin-like chemicals were either measured in human tissues or administered to experimental animals; and c) effects on reproductive organs, sperm count, and sperm characteristics were assessed. From the original data set, 33 articles described the reproductive toxicity of TCDD; of these, 9 specifically examined the effect of a single in utero TCDD exposure on spermatogenesis. For each paper, we extracted details of the experimental methods and the resulting data for further study.

International audience; p-tert-Octylphenol (OP) is a degradation product of alkylphenol ethoxylates. OP is an endocrine disruptor known to bind to the estrogen receptor; however, effects on males are controversial. The objective of this study was to evaluate the effects of chronic exposure to OP on male reproduction. Adult Sprague-Dawley rats were administered OP for 60 d, representing 1.5 cycles of spermatogenesis. Experimental groups included a vehicle control, and three doses of OP (25, 50, or 125 mg/kg body weight [bw]) administered daily by gavage. There was a significant decrease in body weight in the 125-mg/kg group after 60 d of treatment. Both testicular and epididymal weights and histology were not altered by treatment with OP at any of the doses administered. There were no marked differences in cauda epididymal sperm counts at any doses; however, total percent sperm motility was significantly lower in rats exposed to the intermediate dose (50 mg/kg bw). There was an increase in percent static sperm cells in all OP-treated groups, with the intermediate dose (50 mg/kg) displaying a significantly higher proportion of static cells relative to untreated controls. Caput epididymal sperm motility was unaltered by OP treatment. Gene expression profiles of testes from control and high-dose-exposed rats indicate that 14 genes were modulated by at least twofold, although these changes were not statistically significant. Taken together, results from this study indicate that OP treatment of adult rats does not appear to exert major effects on male reproductive endpoints at relevant environmental exposure doses.

Municipal sewage effluents are complex mixtures of contaminants known to disrupt both immune and endocrine functions in aquatic organisms. The present study sought to determine the impacts of municipal effluent on the immune systems of juvenile rainbow trout (Oncorhynchus mykiss), by exposing specimens to low concentrations (0.01%, 0.1%, 1% or 10%) of sewage effluent for periods of 28 or 90 days. The soluble and insoluble fractions of the effluent were also studied to assess the contribution of fractions rich in microorganisms and particles on fish immune systems. To this end, the trout were also exposed to soluble and insoluble fractions of the effluent for a period of 28 days. Immunocompetence was assessed by the following three parameters: phagocytosis, natural cytotoxic cells (NCC) and blastogenesis of lymphocytes under mitogen stimulation. Fish exposed to the 1% sewage effluent concentration for 28 days had enhanced phagocytic activity; at 90 days, phagocytic activity was reduced. T and B lymphocyte proliferation in fish from both groups was similarly stimulated. Phagocytosis and NCC activities were influenced more by the insoluble fraction than the soluble fraction of the effluent. Conversely, mitogen-stimulated T lymphocyte proliferation was enhanced in cells of fish exposed to the soluble fraction of the effluents, with a dampening effect on the insoluble (particulate) fraction of the effluent. In conclusion, the effects of the effluent and its fractions were higher at the cellular-mediated immunity level than at the acquired immunity level. Immunotoxicity data on the soluble fraction of the effluent were more closely associated to data on the unfractionated effluent, but the contribution of the particulate fraction could not be completely ignored for phagocytosis and B lymphocyte proliferation.

International audience; Municipal sewage effluents are complex mixtures that are known to compromise the health condition of aquatic organisms. The aim of this study was to evaluate the impacts of various wastewater disinfection processes on the immune system of juvenile rainbow trout (Oncorhynchus mykiss). The trout were exposed to a primary-treated effluent for 28 days before and after one of each of the following treatments: ultraviolet (UV) radiation, ozonation and peracetic acid. Immune function was characterized in leucocytes from the anterior head kidney by the following three parameters: phagocytosis activity, natural cytotoxic cells (NCC) function and lymphocyte (B and T) proliferation assays. The results show that the fish mass to length ratio was significantly decreased for the primary-treated and all three disinfection processes. Exposure to the primary-treated effluent led to a significant increase in macrophage-related phagocytosis; the addition of a disinfection step was effective in removing this effect. Both unstimulated and mitogen-stimulated T lymphocyte proliferation in fish decreased dramatically in fish exposed to the ozonated effluent compared to fish exposed to either the primary-treated effluent or to aquarium water. Stimulation of T lymphocytes proliferation was observed with the peracetic acid treatment group. In conclusion, the disinfection strategy used can modify the immune system in fish at the level of T lymphocyte proliferation but was effective to remove the effects on phagocytosis activity.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

Spermatozoal maturation in the epididymis is dependent on proteins secreted by the epithelium and those that create the proper ionic composition and pH of the lumen as well as the blood-epididymal barrier. For the human epididymis, little information exists about the regulation of these proteins in male infertility. Our objectives were to assess gene expression profiles in the caput epididymidis from men with normal spermatogenesis and men with nonobstructive azoospermia. With microarrays, we identified 414 genes in the caput epididymidis that were differentially regulated in infertile men by at least 2-fold compared with the fertile men. They were mostly involved in transcription, intracellular signaling, immunity, and fertility. Although the expression of genes encoding tight junctional proteins was not affected, the localization of CLDN10 and TJP1, but not CLDNs 1, 3, and 8, was altered in infertile patients, suggesting that there are changes in the paracellular functions of the blood-epididymal barrier. Differentially regulated genes included several encoding proteins involved in spermatozoal maturation, water and ion channels, and beta-defensins: CRISP1, SPINLW1, FAM12B, and DEFB129 were upregulated, whereas CFTR, AQP5, KCNK4, KCNK17, SLC6A20, SLC13A3, DEFB126, and DEFB106A were downregulated. Furthermore, the immunolocalization of AQP5, but not of CFTR or CRISP1, varied in infertile and fertile patients. The observation that the expression of genes involved in water and ion transport were repressed in infertile patients suggests that these genes are regulated by the presence of testicular products or spermatozoa in the epididymal lumen or are part of a broader syndrome associated with nonobstructive azoospermia.

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.

Gap junctions are essential for spermatogenesis. Exposure to municipal wastewater effluent can modify spermatogenesis in fish. The present aim was to determine if municipal wastewater effluent could alter the expression of testicular connexins (Cxs) in brook trout. Trout were exposed for 4 and 12 weeks to various concentrations of municipal effluent (0, 1, 10 and 20%, v/v). Hepatic vitellogenin (vtg) mRNA levels were not different between exposure groups after 4 weeks. At this time, testicular cx43 and cx31 mRNA levels increased in the 1% group, but cx30 and cx43.4 levels were not different at any concentration. Immunolocalization of each Cx did not differ between groups after 4 weeks. After 12 weeks, spermatogenesis in the 1% group was more advanced than in other groups, and hepatic vtg mRNA levels were significantly increased at the higher exposure concentrations. Testicular cx43 mRNA levels were higher than controls at all doses, while cx43.4 levels increased in a dose-dependent manner but remained lower than controls. cx31 mRNA levels were significantly lower in the 1 and 10% groups than in control and 20% group, while cx30 levels did not vary. Immunolocalization of Cxs did not differ between groups except for Cx43.4, which was expressed between spermatocytes in the 1% group. Furthermore, the Cx31 immunoreaction appeared to decrease in testicular blood vessels of fish exposed to the highest dose. Furthermore, vegf mRNA levels were unaltered by treatment at both time points. Thus, long-term exposure to environmental concentrations of wastewater effluent can alter the expression of testicular Cxs.

Thyroid hormones play a role in the initiation of ovarian maturation in fish. Thus, reports of delayed sexual maturation in female walleye (Sander vitreus) exposed to contaminants in the Ottawa River suggest the presence of endocrine disrupting chemicals. The objectives of this study were to assess the effects of environmental contaminants in the Ottawa River on thyroid hormones of immature walleye and to develop a molecular biomarker of thyroid status. Walleye were sampled in the Ottawa River at Deep River (reference site), at Rivière Blanche (downstream from the Ottawa and Gatineau municipal wastewater treatment plants outflows), and at Plaisance (downstream from a pulp and paper mill). Plasma thyroid hormone levels were measured by radioimmunoassay. Walleye at Plaisance had two-fold elevated levels of thyroxine (T(4)) and 1.5-fold elevated levels triiodothyronine (T(3)), whereas the molar ratio of T(3):T(4) was reduced by over 50% compared to Deep River. Plasma T(3) levels were also elevated by approximately 1.5-fold at Rivière Blanche. Three iodothyronine deiodinases, a family of enzymes responsible for converting the prohormone T(4) to biologically active T(3), as well as for inactivating these two hormones, were partially cloned in walleye. A real-time PCR assay of deiodinase expression indicated that hepatic mRNA levels of type I and type III deiodinase were not modified between sites, whereas they were increased for type II deiodinase at Rivière Blanche as compared to the other sites. The response of this novel molecular transcript indicates a divergence with that expected based on the effects of experimentally induced hyperthyroidism on fish deiodinase expression; additional endpoints are therefore necessary to interpret changes in thyroid hormones levels in fish exposed to environmental contaminants.

Although spermatozoa are formed during spermatogenesis in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical spermatozoal functions are acquired in the epididymis where a specific luminal environment is created by the blood-epididymal barrier; proteins secreted by epididymal principal cells bind to maturing spermatozoa and regulate the maturational process of the spermatozoa. In the epididymis, epithelial cell-cell interactions are mediated by adhering junctions, necessary for cell adhesion, and by tight junctions, which form the blood-epididymal barrier. The regulation of these cellular junctions is thought to represent a key determinant in the process of sperm maturation within the epididymis. Tight junctions between adjacent principal cells permit the formation of a specific microenvironment in the lumen of the epididymis that is essential for sperm maturation. Although we have made significant progress in understanding epididymal function and the blood-epididymal barrier, using animal models, there is limited information on the human epididymis. If we are to understand the normal and pathological conditions attributable to human epididymal function, we must clearly establish the physiological, cellular and molecular regulation of the human epididymis, develop tools to characterize these functions and develop clinical strategies that will use epididymal functions to improve treatment of infertility.

The luminal environment along the epididymal duct is important for spermatozoal maturation. This environment is unique and created by the blood-epididymal barrier, which is formed by tight and adhering junctions. For the human epididymis, little information exists on the proteins that comprise these junctions. Our objectives were to assess the gene expression profiles in the different segments of the human epididymis and to identify the proteins that make up the blood-epididymal barrier. Using microarrays, we identified 2980 genes that were differentially expressed by at least 2-fold between the various segments. Of the many genes involved in diverse functions, were those that encoded adhesion proteins (cadherins and catenins) and tight junctional proteins (claudins [CLDN] and others). PCR analyses confirmed the microarray data. Immunolocalization of CLDNs 1, 3, 4, 8, and 10 revealed that the localization of CLDNs differed along the epididymis. In all three segments, CLDNs 1, 3, and 4 were localized to tight junctions, along the lateral margins of adjacent principal cells, and at the interface between basal and principal cells. CLDN8 was localized to tight junctions in all three segments, in addition to being localized in the caput along the lateral margins of principal cells, and in the corpus, at the interface between principal and basal cells. CLDN10, tight junction protein 1, and occludin were localized exclusively to tight junctions in all three epididymal segments. These data indicate that the epididymis displays a complex pattern of gene expression, which includes genes that are implicated in the formation of the blood-epididymal barrier, which suggests complex regulation of this barrier.

Principal cells of the epididymis are the most prominent cell type and are noted for an apical cell surface studded with microvilli. The latter contain channel proteins that condition the microenvironment of epididymal lumen and promote sperm maturation; however, the regulation of the structure and integrity of microvilli is not well known. Espins are a family of proteins implicated in microvillar growth. The objectives of this study were to assess the regulation of espin in epididymal principal cells both in vitro and in vivo. Treatment of immortalized rat caput epididymal (RCE) cells with increasing doses of a homogenized testicular extract revealed a dose-dependent increase in the size of microvilli. Reverse transcriptase-polymerase chain reaction (RT-PCR) of adult rat epididymal RNA using espin-specific primers indicated the presence of a band at about 290 base pairs (bp) in all regions. Western blot analysis using affinity-purified espin antibody confirmed the presence of an approximately 110-kDa band in the epididymis, corresponding to espin isoform 1. In adult rats, immunocytochemistry revealed espin expression over principal cells. In orchidectomized rats, espin expression was significantly reduced, whereas ligation of the efferent ducts resulted in a decrease of espin expression but not to the extent of orchidectomy. The fact that espin expression was restored to control levels in orchidectomized rats supplemented with high levels of testosterone indicated that its expression was dependent on androgens and not on other lumicrine factors derived from the testis. Taken together, these data indicate that espin is expressed in the epididymis and is regulated by androgens.

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.

Claudin 1 (CLDN1) is a tight junctional protein present in the epididymis. Limited information exists regarding the regulation of Cldn1 transcription. In the epididymis, the regulation of the 5' flanking region of genes coding for tight junctional proteins is unknown. The present objectives were to investigate the transcriptional regulation of the Cldn1 gene in the rat epididymis. A 1.8-kb sequence of the 5' flanking region of the rat Cldn1 gene was cloned. The transcriptional start site is an adenine located at the -198 position relative to the first codon, and 26 bp downstream of the putative TATA box. It is the only start site for the Cldn1 gene transcription in the rat epididymis. The Cldn1 promoter was inserted into a luciferase gene expression vector and transfected into a rat caput epididymal cell line (RCE-1). Sequential deletion analysis revealed that minimal promoter activity was achieved with the construct containing -61 to +164 bp of the promoter. This sequence contained a TATA box and two consensus SP1 binding sites. Electrophoretic mobility shift and supershift assays confirmed that SP1 and SP3 were present in RCE-1 cells and epididymal nuclear extracts, and that they bind to the 5' SP1 binding motif of the promoter. Site-directed mutagenesis of the 5' SP1 binding site resulted in a 4-fold decrease in transactivation of the minimal promoter sequence. These findings indicate that SP1 and SP3 bind to the Cldn1 promoter region, and that this interaction influences the expression of Cldn1 in the rat epididymis.

GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development.

Certain fish, such as rainbow trout (Oncorhynchus mykiss), are seasonal breeders. Spermatogenesis in rainbow trout is synchronous; therefore, at any time point during this process, germ cells are predominantly at the same stage of development. As such, rainbow trout represent an excellent model in which to study spermatogenesis. Gap junctions are composed of connexons, which are themselves formed by six transmembrane proteins termed connexins (Cxs). The objectives of this study were to assess which Cxs are expressed in the rainbow trout testis, and if their expression was stage specific during gonadal maturation. Rainbow trout were killed at various stages of maturation, and total cellular RNA was isolated from the testes. RT-PCR using degenerate primers recognizing all vertebrate Cxs indicates that there are several different Cxs in trout testes. Amplicons were cloned and sequenced. Homology comparisons indicate that these were cx43, cx43.4, cx31, and cx30. Immunolocalization of these Cxs indicate that Cx43 was localized primarily to Sertoli cells, while Cx43.4 was localized along the lateral plasma membranes between adjacent spermatocytes. Cx30 was localized to the interstitial Leydig cells, and Cx31 was localized primarily to the endothelium of interstitial blood vessels. The expression of each Cx varied as a function of the stage of spermatogenesis, suggesting that the expression of these proteins is highly regulated. Together, these results indicate that intercellular communication in the testis is complex, involves several different Cxs, and is a highly regulated process.

Infertility affects between 10 and 15 % of couples worldwide and more than a quarter million couples in Canada. Approximately 25 % of these cases are related to problems with the male partner, and occur from idiopathic or unexplained causes. In addition to these, numerous studies have reported a generalized worldwide decline in male reproductive health, including decreasing sperm counts, increased malformations, such as hypospadias and cryptorchidism, as well as increasing rates of testicular cancer. Environmental toxicants and occupational exposure to toxics have been considered as significant contributors to these effects. The increased sensitivity of immunohistochemical approaches has facilitated the assessment of subtle effects in the testis and epididymis, and represents an important advancement for toxicology. The objective of this review is to demonstrate examples of specific targets that may be used for assessing the effects of environmental toxicants, and to provide examples of how some of these have been used to determine the effects of toxicants within complex epithelia such as the testis and epididymis.