Julie Dufresne

Claudin-1 Is Not Restricted to Tight Junctions in the Rat Epididymis1

The blood-epididymal barrier creates a unique microenvironment critical for sperm maturation. There is little information on proteins comprising epididymal tight and adhering junctions or on factors regulating their expression. Claudins are a family of transmembrane proteins reported to be exclusively localized to tight junctions. In the present study the expression of claudin-l (Cl-1) was examined with respect to the different cell types of the epididymis and its various regions as well as its expression during postnatal development and regulation by testicular factors, using both immunocytochemistry and Northern blot analysis. RT-PCR of adult epididymal and testicular RNA (positive control) indicated that Cl-1 messenger RNA (mRNA) transcripts were present in all regions of the epididymis. In the adult, Cl-1 was localized immunocytochemically along the entire length of the lateral plasma membranes between adjacent principal cells, including apical areas containing tight junctions, as well as at the inter...

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

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. Mol. Reprod. Dev. 78:124–138, 2011.

Cellular Interactions and the Blood-Epididymal Barrier

The interactions among cells and between cells and the extracellular matrix represents a dynamic process involving cellular recognition, interaction of structural proteins and complex intracellular signalling pathways which we are only now beginning to understand. The complexity of reproductive organs is suggestive of a highly regulated communication system involving the nervous and endocrine systems, as well as finely tuned communication between the cells involved in gamete formation. The luminal environment of the epididymis is highly specialized with specific proteins, ions, pH, required for sperm maturation. Tight junctions between epididymal epithelial cells regulate this luminal environment and distinguish it from blood. Thus the regulation of epididymal tight junctions and the function and regulation of cadherins, cell adhesion molecules involved in adhering junction formation, are essential to understanding epididymal physiology. Gap junctions, also part of the epididymal junctional complex, establish communication between epididymal cells and therefore allow the coordination of luminal changes which are necessary for sperm maturation and storage.

Assessing the Role of Claudins in Maintaining the Integrity of Epididymal Tight Junctions Using Novel Human Epididymal Cell Lines

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.

Activation of an SP Binding Site Is Crucial for the Expression of Claudin 1 in Rat Epididymal Principal Cells

Abstract 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.

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

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.

The Expression of Multiple Connexins Throughout Spermatogenesis in the Rainbow Trout Testis Suggests a Role for Complex Intercellular Communication

Abstract 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.

Regulation of the Pannexin-1 Promoter in the Rat Epididymis

ABSTRACT 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.

Development of Biological Tools to Study Claudins in the Male Reproductive Tract

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.

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

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.