Publications by authors named "Daniel G Cyr"

82 Publications

Self-renewal and differentiation of rat epididymal basal cells using a novel in vitro organoid model†.

Biol Reprod 2021 Oct;105(4):987-1001

Laboratory for Reproductive Toxicology, INRS-Centre Armand-Frappier Santé Biotechnologie, University of Quebec, Laval, QC, Canada.

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.
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http://dx.doi.org/10.1093/biolre/ioab113DOI Listing
October 2021

Severe Bilateral Vision Loss in 2 Patients With Coronavirus Disease 2019.

J Neuroophthalmol 2020 09;40(3):403-405

Department of Ophthalmology (DGC, CMV, NYS, SEE), Brookdale University Hospital Medical Center, Brooklyn, New York; and Department of Ophthalmology (DGC, CMV, NYS), St John's Episcopal Hospital, Far Rockaway, New York.

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http://dx.doi.org/10.1097/WNO.0000000000001039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382416PMC
September 2020

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

FASEB J 2020 06 13;34(6):7593-7609. Epub 2020 Apr 13.

Faculty of Medicine, Department of Obstetrics, Gynecology and Reproduction, Université Laval, CHU de Québec Research Center (CHUL), Quebec City, QC, Canada.

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.
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http://dx.doi.org/10.1096/fj.202000328RDOI Listing
June 2020

Beta-defensin126 is correlated with sperm motility in fertile and infertile men†.

Biol Reprod 2020 02;102(1):92-101

Laboratory for Reproductive Toxicology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Quebec, Canada.

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.
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http://dx.doi.org/10.1093/biolre/ioz171DOI Listing
February 2020

Operational modifications for the development of nitrifying bacteria in a large-scale biological aerated filter and its impact on wastewater treatment.

Water Sci Technol 2018 Nov;78(8):1704-1714

Laboratoire de toxicologie environnementale, INRS-Institut Armand-Frappier - Université du Québec, 531, boulevard des Prairies, Laval, Québec H7V 1B7, Canada.

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, CBOD) was improved.
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http://dx.doi.org/10.2166/wst.2018.447DOI Listing
November 2018

A special issue on the effects of toxicants on cellular junctions in development and reproduction.

Reprod Toxicol 2019 01 30;83:80-81. Epub 2018 Aug 30.

Laboratory for Reproductive Toxicology, INRS-Institut Armand-Frappier, 531 boulevard des Prairies, Laval, QC, H7V 1B7 Canada.

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http://dx.doi.org/10.1016/j.reprotox.2018.08.019DOI Listing
January 2019

Cellular junctions in the epididymis, a critical parameter for understanding male reproductive toxicology.

Reprod Toxicol 2018 10 18;81:207-219. Epub 2018 Aug 18.

Laboratory for Reproductive Toxicology, INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, Québec, H7V 1B7, Canada.

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.
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http://dx.doi.org/10.1016/j.reprotox.2018.08.013DOI Listing
October 2018

Effects of prostaglandin E2 on gap junction protein alpha 1 in the rat epididymis.

Biol Reprod 2019 01;100(1):123-132

Laboratory for Reproductive Toxicology, INRS-Institut Armand-Frappier, University of Quebec, Laval, Quebec, Canada.

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.
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http://dx.doi.org/10.1093/biolre/ioy171DOI Listing
January 2019

Betamethasone causes intergenerational reproductive impairment in male rats.

Reprod Toxicol 2017 08 10;71:108-117. Epub 2017 May 10.

Department of Morphology, Institute of Biosciences of Botucatu, UNESP - São Paulo State University, Distrito de Rubião Junior s/nº, Botucatu, SP 18618-689, Brazil.

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 (0.1mg/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.
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http://dx.doi.org/10.1016/j.reprotox.2017.04.012DOI Listing
August 2017

Alterations in male rats following in utero exposure to betamethasone suggests changes in reproductive programming.

Reprod Toxicol 2016 08 28;63:125-34. Epub 2016 May 28.

Department of Morphology, Institute of Biosciences, Univ Estadual Paulista-UNESP, Distrito de Rubião Junior s/n°, 18618-970 Botucatu, SP, Brazil.

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 (0.1mg/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.
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http://dx.doi.org/10.1016/j.reprotox.2016.05.021DOI Listing
August 2016

Implications of caveolae in testicular and epididymal myoid cells to sperm motility.

Mol Reprod Dev 2016 06 10;83(6):526-40. Epub 2016 May 10.

Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.

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.
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http://dx.doi.org/10.1002/mrd.22649DOI Listing
June 2016

Role of Specificity Protein-1 and Activating Protein-2 Transcription Factors in the Regulation of the Gap Junction Protein Beta-2 Gene in the Epididymis of the Rat.

Biol Reprod 2016 06 6;94(6):120. Epub 2016 Apr 6.

Laboratory for Reproductive Toxicology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada

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.
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http://dx.doi.org/10.1095/biolreprod.115.133702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702783PMC
June 2016

Roles of connexins in testis development and spermatogenesis.

Semin Cell Dev Biol 2016 Feb 11;50:22-30. Epub 2016 Jan 11.

INRS-Institut Armand-Frappier, University of Québec, 531 boul. des Prairies, Laval, Québec H7V 1B7, Canada.

The development and differentiation of cells involved in spermatogenesis requires highly regulated and coordinated interactions between cells. Intercellular communication, particularly via connexin43 (Cx43) gap junctions, plays a critical role in the development of germ cells during fetal development and during spermatogenesis in the adult. Loss of Cx43 in the fetus results in a decreased number of germ cells, while the loss of Cx43 in the adult Sertoli cells results in complete inhibition of spermatogenesis. Connexins 26, 32, 33, 36, 45, 46 and 50 have also been localized to specific compartments of the testis in various mammals. Loss of Cx46 is associated with an increase in germ cell apoptosis and loss of the integrity of the blood-testis barrier, while loss of other connexins appears to have more subtle effects within the seminiferous tubule. Outside the seminiferous tubule, the interstitial Leydig cells express connexins 36 and 45 along with Cx43; deletion of the latter connexin did not reveal it to be crucial for steroidogenesis or for the development and differentiation of Leydig cells. In contrast, loss of Cx43 from Sertoli cells results in Leydig cell hyperplasia, suggesting important cross-talk between Sertoli and Leydig cells. In the epididymis connexins 26, 30.3, Cx31.1, 32, and 43 have been identified and differentiation of the epithelium is associated with dramatic changes in their expression. Decreased expression of Cx43 results in decreased sperm motility, a function acquired by spermatozoa during epididymal transit. Clearly, intercellular gap junctional communication within the testis and epididymis represents a critical aspect of male reproductive function and fertility. The implications of this mode of intercellular communication for male fertility remains a poorly understood but important facet of male reproduction.
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http://dx.doi.org/10.1016/j.semcdb.2015.12.019DOI Listing
February 2016

Isolated Rat Epididymal Basal Cells Share Common Properties with Adult Stem Cells.

Biol Reprod 2015 Nov 23;93(5):115. Epub 2015 Sep 23.

Laboratory for Reproductive Toxicology, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Quebec, Canada Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

There is little information on the function of epididymal basal cells. These cells secrete prostaglandins, can metabolize radical oxygen species, and have apical projections that are components of the blood-epididymis barrier. The objective of this study was to develop a reproducible protocol to isolate rat epididymal basal cells and to characterize their function by gene expression profiling. Integrin-alpha6 was used to isolate a highly purified population of basal cells. Microarray analysis indicated that expression levels of 552 genes were enriched in basal cells relative to other cell types. Among these genes, 45 were expressed at levels of 5-fold or greater. These highly expressed genes coded for proteins implicated in cell adhesion, cytoskeletal function, ion transport, cellular signaling, and epidermal function, and included proteases and antiproteases, signal transduction, and transcription factors. Several highly expressed genes have been reported in adult stem cells, suggesting that basal cells may represent an epididymal stem cell population. A basal cell culture was established that showed that these basal cells can differentiate in vitro from keratin (KRT) 5-positive cells to cells that express KRT8 and connexin 26, a marker of columnar cells. These data provide novel information on epididymal basal cell gene expression and suggest that these cells can act as adult stem cells.
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http://dx.doi.org/10.1095/biolreprod.115.133967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712003PMC
November 2015

Claudin-10 is required for relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Dev Biol 2015 May 3;401(2):236-48. Epub 2015 Mar 3.

Departments of Human Genetics, McGill University, Canada; Departments of Paediatrics, McGill University, Canada; The Research Institute of the McGill University Health Centre, Montréal, Québec, Canada. Electronic address:

Species-specific symmetry-breaking events at the left-right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left-right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen's node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen's node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen's node prevented expression of Nodal, Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left-right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen's node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.
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http://dx.doi.org/10.1016/j.ydbio.2015.02.019DOI Listing
May 2015

Tricellulin and its role in the epididymal epithelium of the rat.

Biol Reprod 2015 Mar 7;92(3):66. Epub 2015 Jan 7.

INRS-Institut Armand-Frappier, Université du Québec, Laval, Quebec, Canada

Tricellulin is a tight-junction protein present at tricellular tight junctions. It has been suggested that basal cells are implicated in the blood-epididymis barrier. Basal cells express claudins, a component of tight junctions; however, there is no information regarding the potential architecture or regulation of basal cell-principal cell interactions. The present objectives were to determine the expression and localization of tricellulin in rat epididymis in relation to occludin, basal cell-principal cell interactions, and other junctional proteins. Tricellulin levels were similar in all segments of the adult epididymis, and the protein was localized to the apical region of the epithelium. Postnatal development showed that tricellulin levels increased with age and localization changed from cytoplasmic to membrane-bound as a function of age. Colocalization with occludin indicated that both proteins are in the region of the tight junction. In the initial segment, the proteins did not colocalize compared to the epididymis where they were both colocalized. Tricellulin did not colocalize with cytokeratin 5, a marker of basal cells, in any region of the epididymis, including the corpus and cauda epididymidis, where apical projections of basal cells were apparent. Tricellulin knockdown studies using small interfering RNA in rat caput epididymal principal cells resulted in decreased transepithelial resistance and was correlated with decreased levels of Cldn3, Cldn1, and occludin. Tight-junction protein1, also known as ZO-1, and cadherin1 levels were unchanged. This is the first report of tricellulin in the epididymis and on the interaction between tricellulin and other tight-junction proteins.
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http://dx.doi.org/10.1095/biolreprod.114.120824DOI Listing
March 2015

Regulation of the pannexin-1 promoter in the rat epididymis.

Biol Reprod 2014 Dec 5;91(6):143. Epub 2014 Nov 5.

Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada

Pannexins (PANXs) are channel-forming proteins implicated in cellular communication through the secretion of biomolecules, such as ATP and glutamate. PANX1 and PANX3 are expressed in the male rat reproductive tract and their levels are regulated by androgens in the epididymis. There is currently no information on the regulation of the Panx1 promoter. The objective of the present study was to characterize the Panx1 promoter in order to understand its regulation in the epididymis. RNA ligase-mediated rapid amplification of cDNA ends identified three transcriptional start sites, at positions -443, -429, and -393. In silico analysis revealed that transcription was initiated downstream of binding sites for CREB and ETV4 transcription factors, in a CpG island context. To determine the importance of this region in gene transactivation, a 2-kb fragment of the promoter was cloned into a vector containing a luciferase reporter gene. Deletion constructs indicated that the highest transactivation levels were achieved with shorter constructs (-973 to -346 and -550 to -346). Electrophoretic mobility shift assay and supershifts indicated that both transcription factors were able to bind to the promoter region. Chromatin immunoprecipitation using rat caput epididymis cells confirmed the binding of ETV4 and CREB on the Panx1 promoter. Site mutation of either the ETV4 or CREB binding site decreased the transactivation of the reporter gene. Previous studies indicated that orchidectomy increased epididymal PANX1 levels. Likewise, we observed an increase in both ETV4 and CREB in orchidectomized rats. These results indicate that ETV4 and cAMP response elements play a role in the transcriptional regulation of Panx1 in the epididymis.
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http://dx.doi.org/10.1095/biolreprod.114.122168DOI Listing
December 2014

The blood-epididymis barrier and inflammation.

Spermatogenesis 2014 May-Aug;4(2):e979619. Epub 2014 Dec 31.

INRS-Institut Armand Frappier; University of Quebec ; Laval, QC, Canada.

The blood-epididymis barrier (BEB) is a critical structure for male fertility. It enables the development of a specific luminal environment that allows spermatozoa to acquire both the ability to swim and fertilize an ovum. The presence of tight junctions and specific cellular transporters can regulate the composition of the epididymal lumen to favor proper sperm maturation. The BEB is also at the interface between the immune system and sperm. Not only does the BEB protect maturing spermatozoa from the immune system, it is also influenced by cytokines released during inflammation, which can result in the loss of barrier function. Such a loss is associated with an immune response, decreased sperm functions, and appears to be a contributing factor to post-testicular male infertility. Alterations in the BEB may be responsible for the formation of inflammatory conditions such as sperm granulomas. The present review summarizes current knowledge on the morphological, physiological and pathological components associated with the BEB, the role of immune function on the regulation of the BEB, and how disturbance of these factors can result in inflammatory lesions of the epididymis.
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http://dx.doi.org/10.4161/21565562.2014.979619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4581054PMC
December 2014

Exposure of fathead minnows to municipal wastewater effluent affects intracellular signaling pathways in the liver.

Comp Biochem Physiol C Toxicol Pharmacol 2014 Aug 16;164:1-10. Epub 2014 Apr 16.

INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, Québec H7V 1B7,Canada. Electronic address:

Municipal wastewater effluent can impact its receiving environment. In the St. Lawrence River, male fish living downstream from Montreal exhibit increased hepatic vitellogenin, intersex, delayed spermatogenesis and altered immune function. Few studies have examined genome-wide effects associated with municipal effluent exposure in fish to decipher the mechanisms of toxicity. The present objective was to identify hepatic cellular signaling pathways in fathead minnows following exposure to municipal wastewater effluent. Immature minnows were exposed for 21 days to either 0% (Control) or 20% municipal effluent, the highest concentration in the St. Lawrence River. Hepatic RNA was extracted and used to hybridize a fathead minnow oligonucleotide microarray containing approximately 15k gene sequences. A total of 1300 genes were differentially expressed, of which 309 genes had more than 2-fold change in expression level between control and MWWE-exposed fish. Of those, 118 were up-regulated and 191 were down-regulated. Altered genes grouped according to function, indicated effects on various signaling pathways, apoptosis, immune responses, and cellular metabolism. Pathway analysis software predicted at least 5 signaling pathways that were altered by treatment: cell adhesion, inflammation, various kinases, estrogen receptor signaling and WNT signaling. Various components of the canonical Wnt pathway were dramatically down-regulated, while several other genes involved in the non-canonical Wnt pathway, such as Wnt4, LRP6, and PPP2R5E, which are known to inhibit the canonical Wnt pathway, were increased. These results indicate that municipal wastewater effluent from Montreal can target and inhibit various signaling including those implicated in hepatic Wnt signaling pathway in fathead minnows.
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http://dx.doi.org/10.1016/j.cbpc.2014.04.002DOI Listing
August 2014

The blood-epididymis barrier and human male fertility.

Adv Exp Med Biol 2012 ;763:218-36

INRS-Institut Armand-Frappier, University of Quebec, Quebec, Canada.

Spermatozoa undergo a posttesticular maturation in the epididymis to acquire motility and the capacity to fertilize. Sperm maturation depends in part upon the creation of a specific microenvironment within the epididymal lumen. This environment is conditioned by proteins secreted by the epithelium and by exchange of molecules between the lumen and the blood circulation. These exchanges are selectively regulated by the blood-epididymis barrier. The blood-epididymis barrier is comprised of apical tight junctions between adjacent principal cells. Adherens junctions, which are necessary for cell adhesion, can also be found at the junctional complex present between adjacent principal cells. Progress has been made on the understanding of cellular interactions in the epididymis as well as the regulation of the luminal microenvironment and its importance for sperm maturation in rodents and humans. Clearly, changes in the function of cellular junctions in the human epididymis are associated with male infertility.
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http://dx.doi.org/10.1007/978-1-4614-4711-5_11DOI Listing
April 2013

Epidermal growth factor regulates connexin 43 in the human epididymis: role of gap junctions in azoospermia.

Hum Reprod 2012 Aug 17;27(8):2285-96. Epub 2012 May 17.

INRS-Institut Armand Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, QC, Canada H7V 1B7.

Background: Gap junctions (GJs) allow for direct communication between adjacent cells. They are composed of connexons consisting of transmembrane proteins, connexins (Cxs). The objectives of this study were to determine if GJ proteins GJA1 (Cx43), GJB1 (Cx32) and GJB2 (Cx26) are present in the epididymis of men with a normal epididymis, to assess whether or not Cx expression and localization are altered in azoospermic patients, and to determine if epidermal growth factor (EGF) regulates GJA1 expression.

Methods: Epididymides were obtained from men with localized testis cancer with active spermatogenesis and histologically normal epididymal tubule (group 1), men with non-obstructive azoospermia secondary to Sertoli-cell only syndrome (group 2) and from azoospermic men with normal spermatogenesis and epididymal obstruction (group 3). Epididymides were subdivided into three segments: caput, corpus and cauda. Quantitative real-time RT-PCR was performed to assess GJA1, GJB1, GJB2 and EGF receptor (EGFR) mRNA levels in epididymides from patients from each group (all n=3, except n=1 for caput blockage). A human caput epididymal cell line was then used to determine the role of EGFR signaling on the regulation of human epididymal GJA1.

Results: Real-time RT-PCR analysis revealed that GJA1, GJB1, GJB2 and EGFR were expressed along the human epididymis. In the cauda epididymidis of group 2 and 3 men, we observed a significant decrease in GJA1 (P=0.0456 and P=0.0465, respectively) and GJB1 (P=0.0450 and P=0.0497, respectively) mRNA levels when compared with group 1 men. We also observed a decrease in EGFR mRNA levels (P=0.0358) in the cauda epididymidis of group 3 men when compared with group 1. Immunocytochemistry revealed that in the epididymis, GJA1 and EGFR were localized between basal and principal cells and between adjacent principal cells. In group 2 and 3 patients, however, we noted a dramatic increase in cytosolic immunostaining for both GJA1 and EGFR in both principal and basal cells. Using a human caput epididymal cell line derived from fertile men, we demonstrated that changes in GJA1 phosphorylation could be regulated by EGF (P=0.015) and the extracellular regulated kinase 1/2 signaling pathway (P=0.03). Furthermore, while the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway did not alter GJA1 phosphorylation, treatment with PI3K/AKT inhibitor LY294002 significantly (P=0.024) inhibited the EGF-stimulated increase in GJA1 total protein levels at 24 h. Immunolocalization indicated that loss of PI3K/AKT signaling was associated with increased cytosolic localization of Cx43 in this cell line.

Conclusions: Together, these data suggest that in azoospermic men decreased expression of EGFR may be responsible for decreasing GJA1 levels and increasing its cytosolic localization via the PI3K/AKT signaling pathway.
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http://dx.doi.org/10.1093/humrep/des164DOI Listing
August 2012

Role of microRNAs in controlling gene expression in different segments of the human epididymis.

PLoS One 2012 12;7(4):e34996. Epub 2012 Apr 12.

Centre de Recherche du CHUQ and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Québec, Canada.

Background: The molecular mechanisms implicated in regionalized gene expression in the human epididymis have not yet been fully elucidated. Interestingly, more than 200 microRNAs (miRNAs) have been identified in the human epididymis and could be involved in the regulation of mRNA stability and post-transcriptional expression in this organ.

Methods: Using a miRNA microarray approach, we investigated the correlation between miRNA signatures and gene expression profiles found in three distinct regions (caput, corpus and cauda) of human epididymides from 3 donors. In silico prediction of transcript miRNA targets was performed using TargetScan and Miranda software's. FHCE1 immortalized epididymal cell lines were cotransfected with mimic microRNAs and plasmid constructs containing the 3'UTR of predicted target genes downstream of the luciferase gene.

Results: We identified 35 miRNAs differentially expressed in the distinct segments of the epididymis (fold change ≥2, P-value ≤ 0.01). Among these miRNAs, miR-890, miR-892a, miR-892b, miR-891a, miR-891b belonging to the same epididymis-enriched cluster located on the X chromosome, are significantly more expressed in the corpus and cauda regions than in the caput. Interestingly, a strong negative correlation (r = -0,89, P-value ≤ 0.001) was found between the pattern of expression of miR-892b and its potential mRNA target Esrrg (Estrogen Related Receptor Gamma) and with miR-145 and Cldn10 mRNA (r = -0,92, P-value ≤ 0.001). We confirmed that miR-145 and miR-892b inhibit the expression of the luciferase reporter via Cldn10 and Esrrg 3' UTRs, respectively.

Conclusion: Our study shows that the expression of miRNAs is segmented along the human epididymis and correlates with the pattern of target gene expression in different regions. Therefore, epididymal miRNAs may be in control of the maintenance of gene expression profile in the epididymis, which dictates segment-specific secretion of proteins and establishes physiological compartments that directly or indirectly affect sperm maturation and fertility.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034996PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325285PMC
December 2012

Connexins and pannexins: Coordinating cellular communication in the testis and epididymis.

Authors:
Daniel G Cyr

Spermatogenesis 2011 Oct 1;1(4):325-338. Epub 2011 Oct 1.

INRS-Institut Armand Frappier; University of Quebec; Laval, QC Canada.

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.
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http://dx.doi.org/10.4161/spmg.1.4.18948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271644PMC
October 2011

Development of biological tools to study claudins in the male reproductive tract.

Methods Mol Biol 2011 ;762:259-73

INRS-Institut Armand-Frappier, Laval, QC, Canada.

It is estimated that between 12 and 15% of couples are infertile. More than half of these are related to problems associated with male reproductive dysfunction. Of those, 40% occur from idiopathic or unexplained causes. While spermatozoa are formed in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical functions are acquired as spermatozoa transit through the epididymis in the specific luminal environment created in part by the tight junctions of the blood-epididymis barrier. To understand the normal and pathological conditions attributable to human and animal epididymal function, we have needed to develop biological tools to characterize the physiological, cellular, and molecular functions of tight junctions and claudins (Cldns) in the epididymis. We have shown that by developing epididymal cell lines we have gained valuable insight into the functions of epididymal Cldns, the regulation of the Cldn1 gene and how these can be mistargeted in infertile men. Here we describe some of the techniques that have been used to address these critical aspects of epididymal Cldns.
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http://dx.doi.org/10.1007/978-1-61779-185-7_18DOI Listing
November 2011

Dioxin-induced changes in epididymal sperm count and spermatogenesis.

Cien Saude Colet 2011 Jun;16(6):2893-905

Department of Obstetrics and Gynecology, McMaster University, ON, Canada.

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 contest [corrected] that epididymal function is the key target for the adverse effects of TCDD.
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http://dx.doi.org/10.1590/s1413-81232011000600027DOI Listing
June 2011

Mice lacking the USP2 deubiquitinating enzyme have severe male subfertility associated with defects in fertilization and sperm motility.

Biol Reprod 2011 Sep 4;85(3):594-604. Epub 2011 May 4.

Polypeptide Laboratory, Department of Medicine, McGill University and McGill University Health Centre, Montreal, Quebec, Canada.

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.
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http://dx.doi.org/10.1095/biolreprod.110.088542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480438PMC
September 2011

Characterization of pannexin1 and pannexin3 and their regulation by androgens in the male reproductive tract of the adult rat.

Mol Reprod Dev 2011 Feb;78(2):124-38

INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.

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.
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http://dx.doi.org/10.1002/mrd.21280DOI Listing
February 2011

Regulation and characterization of the ATP-binding cassette transporter-B1 in the epididymis and epididymal spermatozoa of the rat.

Toxicol Sci 2011 Feb 20;119(2):369-79. Epub 2010 Oct 20.

Institut National de la Recherche Scientifique-Institut Armand-Frappier, University of Quebec, Laval Quebec, Canada.

It has been reported that following administration, alkylphenols, such as octylphenol, reach the testis and epididymis but fail to accumulate in these tissues, suggesting the rapid expulsion of these chemicals by transporters. Specialized transporters that function to restrict compounds that enter target cells have been identified. ABCB1 is a member of the ATP-binding cassette family of proteins capable of transporting a broad range of drugs and xenobiotics out of tissues. The objective of this study was to characterize the expression profile and functional role of ABCB1a and ABCB1b along the different regions (initial segment, caput, corpus [CS], and cauda [CA]) of the adult rat epididymis. ABCB1a and ABCB1b transcripts were detected in all four regions of the epididymis. Immunolocalization revealed minimal ABCB1 staining in epithelial cells or spermatozoa of proximal regions of the epididymis; however, this progressively increased in the CS and CA epididymis. This expression gradient was confirmed by Western blot, suggesting that spermatozoa acquire ABCB1 during epididymal maturation. Multidrug resistance (MDR) assays revealed that rat epididymal cells and epididymal spermatozoa display an MDR phenotype that can be inhibited under control conditions. To assess whether or not the system was inducible by alkylphenols, cells from an immortalized epididymal cell line (RCE) were exposed to different concentrations of nonylphenol. Results revealed a significant induction of both ABCB1a and ABCB1b messenger RNA and ABCB1 protein in RCE cells. Our findings demonstrate a role for ABCB1 in protecting both epididymal principal cells and spermatozoa from xenobiotics.
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http://dx.doi.org/10.1093/toxsci/kfq318DOI Listing
February 2011

Regulation of the connexin 43 promoter in the brook trout testis: role of the thyroid hormones and cAMP.

Gen Comp Endocrinol 2011 Jan 12;170(1):110-8. Epub 2010 Oct 12.

INRS-Institut Armand-Frappier, Université du Québec, Laval, QC, Canada.

Gap junctions are critical for spermatogenesis. They are composed of integral proteins, the connexins. In mammals, a loss of Cx43 expression results in the inhibition of spermatogenesis. We have shown that Cx43 is expressed in the Sertoli cells of rainbow trout and that cAMP and triiodothyronine (T(3)) regulate testicular Cx43 expression in brook trout testis. The objective of this study was to determine if cAMP and T(3) act at the level of the cx43 promoter to regulate its expression. A 607 bp 5' flanking sequence of the cx43 promoter was obtained by Genome Walking. A TATA box was predicted to be located between positions -36 and -30 relative to the transcriptional initiation site. 5'-Rapid amplification of cDNA ends indicated a single transcriptional start site. Single C/EBP (-164 to -156) and tr-beta (-112 to -107) response elements were identified and electrophoretic mobility shift assays indicated the presence of competitive protein binding sites at each region. Immortalized rainbow trout gonadal cell line (RTG-2) which express cx43 and tr-beta transcripts were transfected with a vector containing the Cx43 promoter inserted into a luciferase expression vector. Transactivation of the reporter genes was stimulated by either cAMP or T(3). Sequential deletion and point mutations in either the C/EBP or tr-beta response element indicated that T(3) but not cAMP directly induced luciferase transactivation of the luciferase gene by acting on different sites of the Cx43 promoter. Together, these data indicate that T(3) stimulates cx43 expression via direct regulation of gene transcription.
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http://dx.doi.org/10.1016/j.ygcen.2010.09.013DOI Listing
January 2011

Alterations in the human blood-epididymis barrier in obstructive azoospermia and the development of novel epididymal cell lines from infertile men.

Biol Reprod 2010 Oct 26;83(4):584-96. Epub 2010 May 26.

INRS-Institut Armand Frappier, University of Quebec, Laval, Quebec, Canada.

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.
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http://dx.doi.org/10.1095/biolreprod.110.084459DOI Listing
October 2010
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