Glycohistochemical, Immunohistochemical and Ultrastructural Studies of the Bovine Epididymis

In the present work, efferent ductules and epididymal duct from male foetuses as well as from sexually mature bulls were investigated using conventional light and electron microscopical techniques as well as glycohistochemical and immunohistochemical staining techniques. The prenatal development of the bovine epididymis was studied in foetuses ranging from 10 cm CRL (75 pcd) to 90 cm CRL (285 pcd). In foetuses with 10 cm CRL (75 pcd) the main event was the establishment of the urogenital junction between the extratesticular rete testis and mesonephric duct via the growing efferent ductules. At the foetal age of 110 pcd (24 cm CRL), efferent ductules underwent a strong coiling. At the same time the mesonephric duct began to lengthen and coil, forming three distinct regions, namely caput, corpus and cauda epididymidis. The coiling was much more distinct in caput and cauda than in corpus epididymidis. At 130 pcd (30 cm CRL) and upwards efferent ductules were organized in lobules which are then arranged in groups separated from each other by connective tissue septa. A similar organization involved the highly convoluted epididymal duct, particularly in the head and tail regions. In addition to the macroscopical modifications in the morphology of extratesticular excurrent duct system, histological differentiation involved both the tubular epithelium and the peritubular mesenchymal cells. The epithelium of efferent ductules was differentiated into ciliated and nonciliated columnar epithelium. The simple epithelium of the epididymal duct increased in height and developed stereocilia on its apical surface. Distribution of WGA-, PNA- and GSA-I-binding sites on luminal surface of the epithelium of efferent ductules, but not of epididymal duct may indicate earlier differentiation of the former. WGA-binding to the peritubular and interstitial mesenchymal cells, but not to the epididymal epithelium indicated that the mesenchymal structures differentiate before epithelial ones. S-100, FGF-1, FGF-2, ACE, laminin and GT were immunolocalized in the epithelium both of efferent ductules and epididymal duct as early as at 75 pcd (10 cm CRL). Also ?-SMA was immunolocalized in the peritubular mesenchymal cells at 75 pcd (efferent ductules) and at 95 pcd (epididymal duct, CRL 18 cm). The epithelium of the adult bovine efferent ductules is simple columnar including ciliated and nonciliated cells as well as some scattered intraepithelial leucocytes. On the basis of their cytological characteristics, nonciliated cells could be categorized into three sub-types. The epididymal duct of the adult bull is lined with pseudostratified columnar epithelium. It consists mainly of tall, slender, stereocilia-bearing columnar cells and small basal cells. On the basis of several morphometric parameters like epithelial height, luminal diameter and width of peritubular muscle coat the epididymal duct could be subdivided into six segments. Ultrastructural studies revealed a well developed Golgi apparatus, numerous profiles of sparsely granulated endoplasmic reticulum and mitochondria as well as rER in the cytoplasm of principal cells particularly in those of the first three segments. Apical surfaces of principal cells particularly those of the proximal segments were equipped with long stereocilia and their apical cell membrane and cytoplasm displayed a well developed endocytotic apparatus. The narrow basal extensions of principal cells were crowded with numerous pleomorphic mitochondria, lysosomes, heteromorphic electron dense granules and residual bodies. Basal cells were insinuated between the narrow basal extensions of principal cells and the basal lamina. They possessed kidney-shaped, mostly deeply-invaginated nuclei and were characterized by a paucity of organelles. Apical mitochondria-rich cells were frequently found in segments II and III and rarely in segments IV and V. Their hyaloplasm was lighter than that of the neighbouring principal cells and their apical surfaces were provided with short microvilli. Apart from a reasonable number of mitochondria, small Golgi apparatus and sporadic strands of rER, they displayed a paucity of organelles. Intraepithelial macrophages were occasionally encountered in the basal third of the epithelium. They possessed many mitochondria, well developed Golgi apparatus and rER as well as small heterochromatic nuclei. Various profiles of lysosomes and dark residual bodies were found in their cytoplasm. Intraepithelial lymphocytes were characterized by their heterochromatic, round and mostly indented nuclei and narrow peripheral cytoplasmic rim. They were often encountered in immediate proximity to subepithelial capillaries. Fluoresceinisothiocyanate (FITC)-labelled lectins (GSA-I, PNA, ECA, WGA, Con A, LCA, PSA, DBA, HPA, SBA, VVA, LTA and UEA-I) were also used for the study of the regional distribution of saccharide groups in adult bovine epididymal tissues. WGA, Con A, LCA, PSA, DBA and HPA bound distinctly to stereocilia of principal cells in the different segments. However, DBA- and HPA-binding sites were confined to stereocilia in caput region. WGA, LCA, PSA, DBA and HPA possessed distinct binding sites in Golgi zone of principal cells, mostly of the caput epididymidis. Basal cells reacted distinctly with WGA, Con A, LCA, PSA and HPA. Intraepithelial leucocytes displayed moderate binding sites for PNA, WGA, LCA and PSA. The basal membrane reacted moderately only with WGA. Epididymal connective tissue showed weak to moderate binding only with ECA and WGA. GSA-I bound distinctly to vascular endothelium and could be applied as a good marker for bovine endothelium. Sperm cell mass bound WGA and PNA distinctly. No binding sites could be found for VVA, LTA or UEA-I. Immunohistochemical studies used the Avidin-Biotin-peroxidase Complex (ABC) method for localization of S-100, FGF-1, FGF-2, ACE, GT, VEGF, ?-SMA, laminin, connexin 43, CD4, CD8 and CD68 in the epididymis. The epithelium of the efferent ductules showed intense immunoreaction for S-100, FGF-1 and FGF-2 and a moderate immunostaining for ACE and GT. Principal cells of the first three epididymal segments exhibited a distinct immunostaining for S-100. They also showed a distinct immunoreactivity for FGF-1 throughout the different segments. Principal cells in the first, second and sixth segment displayed intense immunostaining for ACE. Immunostaining for GT in Golgi zone of the principal cells was intense (segments II and III), distinct (segments IV and V) and moderate (segments I and VI). Basal cells showed moderate (FGF-1) or intense (FGF-2) immunostaining in different epididymal segments. Intense immunostaining for ACE, laminin and ?-SMA was found respectively in the endothelium, endothelial basal lamina and smooth muscle cells of blood vessels. The basal lamina of the epithelium and the peritubular smooth muscle cells displayed a moderate immunoreactivity for laminin. The peritubular smooth muscle cells manifested an intense immunostaining for ?-SMA. CD4+ T cells and CD68+ macrophages were found within the epithelium and in the interstitium. Mast cells were conventionally stained with Alcian blue and Toluidin blue. They also displayed a distinct immunostaining for VEGF and FGF-2. In conclusion, my study supports the previously proposed 6-segment scheme of bovine epididymis. Moreover, lectin histochemistry and immunohistochemistry were not only helpful tools in emphasising this scheme but also in correlating specific functional activities to certain regions. Lectins- and GT-binding sites as well as ultrastructural characteristics point to high synthetic and secretory activities of principal cells in the first three segments, as indicated by the well developed Golgi apparatus. Ultrastructurally, principal cells of the proximal three epididymal segments displayed a well developed endocytotic apparatus. This was reinforced by intense immunostaining for ACE in this region, which reflects extensive absorptive activities in this region. Existence of mast cells in the epididymal interstitium and T-lympho-cytes and macrophages in the interstitium and within the epithelium may reflect their harmonized co-operation in the induction of immune tolerance in the bovine epididymis

Ultrastrukturelle und immunhistochemische Untersuchungen am Nebenhoden des Katers (Felis silvestris "familiaris")

In der vorliegenden Arbeit wurden 45 Nebenhoden von klinisch gesunden Katern im Alter zwischen fünf Monaten und zehn Jahren im Hinblick auf Morphologie, Ultrastruktur und Histochemie untersucht. Es wurden zehn verschiedene immunhistologische Reaktionen auf verschiedene Proteine bzw. Hormonrezeptoren durchgeführt. Der Nebenhoden des Katers entspricht in seinem anatomischen Aufbau dem anderer Tierarten. Durch das Auftreten verschiedener Zellarten, unterschiedlicher Epithelhöhe und der Länge des Zellbesatzes sowie Form und Lage des Nukleus kann der Epididymidis des Katers in fünf verschieden Segmente unterteilt werden. Die Ductuli efferentes bilden mit Segment 1 und 2 den Nebenhodenkopf, Segment 3 und 4 entsprechen dem Corpus epididymidis und der Nebenhodenschwanz besteht aus Segment 5. Die Ductuli efferentes besitzen zilienbesetzte und zilienlose Zellen, die in verschiedenen Färbungen und immunhistologischen Reaktionen auch unterscheiden. Das Epithel des Ductus epididymidis des Katers besteht aus Hauptzellen, Basalzellen und Lymphozyten, sowie Apikalzellen in Segment 2 und 5. Neben den genannten Zellarten bzw. ihrer Kerne oder des Zytoplasmas wurden bei den immunhistologischen Reaktionen Spermien, luminale Epithelzellen, Muskulatur, Gefäße und die Basallamina beurteilt. VEGF (“Vascular endothelial growth factor“) ist immunhistochemisch nur schwach nachweisbar, dafür zeigt sich der VEGF-Rezeptor deutlich positiv, vor allem in den Zellkernen der Hauptzellen von Nebenhodenkopf und –schwanz. GHR (“Growth hormone receptor“) reagiert in Kernen der Hauptzellen und Apikalzellen des gesamten Nebenhodens, sowie deren Zytoplasma. Außerdem ist es in den Gefäßen und der Muskulatur nachweisbar. Vimentin kommt im Zytoplasma der Ductuli efferentes sowie in Muskulatur, Interstitium, Gefäßen und Basallamina des Ductus epididymidis vor. Cytokeratin färbt das Zytoplasma der Ductuli efferentes und der Hauptzellen in Segment 1 und 5 an. Laminin kommt vorwiegend in der Muskulatur, den Gefäßen und der Basallamina, in geringer Menge auch im Zytoplasma der Hauptzellen vor. a-SMA (“Smooth muscle actin“) zeigt sich ausschließlich in den glatten Muskelzellen der Ductuli efferentes, des Nebenhodenganges und der Gefäße, ohne regionale Unterschiede. Androgen-Rezeptor ist in den Kernen aller Hauptzellen, vorwiegend aber von Segment 1 bis 4 sowie im Interstitium nachweisbar. Östrogen-Rezeptor a reagiert dagegen nur in den Kerne der Ductuli efferentes und färbt in Segment 4 und 5 den Zellbesatz und die Spermien an. Progesteron-Rezeptor reagiert im gesamten Nebenhodengang positiv im Interstitium, den Gefäßen und den luminalen Spermien. Außerdem färbt es den Zellbesatz von Segment 2 bis 5 an

Studies on the regional difference of electrogenic chloride secretion in the cultured rat epididymal epithelium.

by Ka-bik Lai.Thesis (M.Phil.)--Chinese University of Hong Kong,Includes bibliographical references (leaves 59-67).Chapter Chapter I --- IntroductionChapter I.1 --- Anatomy and functions of the epididym --- p.isChapter I.1.1 --- Gross anatomy and functions --- p.1Chapter I.1.2 --- Histology of the epithelium and functions of different cell types --- p.3Chapter I.2 --- Regional differenceChapter I.2.1 --- Epithelium --- p.4Chapter I.2.2 --- Electrolyte transport --- p.6Chapter I.3 --- Short circuit current studies on C1- secretion in the cultured rat epididymal epithelium --- p.7Chapter I.4 --- Physiological effects of vasopressin in the epididymis and the male reproductive tract --- p.10Chapter I.5 --- Objectives of the study --- p.14Chapter Chapter II --- Materials and methodsChapter II. 1 --- MaterialsChapter II.1.1 --- Chemicals for cell culture --- p.16Chapter II. 1.2 --- Chemicals and drugs for short circuit current study --- p.16Chapter II. 1.3 --- Materials for preparing pervious supports --- p.16Chapter II.2 --- Preparation of solution for short circuit current study --- p.17Chapter II.3 --- Preparation of pervious supports for the cultured epithelium --- p.17Chapter II.4 --- Culture of rat epididymal epithelial cellsChapter II.4.1 --- "Dissection of the efferent duct, the initial segment and the cauda epididymidis" --- p.17Chapter II.4.2 --- Culture procedures for the epithelium --- p.19Chapter II.4.3 --- Confluent epithelial monolayer --- p.22Chapter II.5 --- Short circuit current measurementChapter II.5.1 --- Mounting of cultured epithelium on Ussing chambers --- p.22Chapter II.5.2 --- Experimental setup for zero voltage clamp --- p.25Chapter Chapter III --- ResultsChapter III.1 --- Regional differencesChapter III.1.1 --- Basal bioelectrical properties --- p.29Chapter III. 1.2 --- Effects of C1- replacement and C1- channel blocker --- p.29Chapter III.1.3 --- Effects of ion transportor inhibitors --- p.33Chapter III. 1.4 --- Effect of adenylate cyclase stimulator --- p.33Chapter III.1.5 --- Effect of exogenous PGE2 --- p.33Chapter III.1.6 --- Effect of Ca2+ ionophore --- p.36Chapter III.2 --- Effect of [arg8]-vasopressinChapter III.2.1 --- Effect of [arg8] -vasopressin on short circuit current --- p.36Chapter III.2.2 --- Effects of C1- replacement and C1- channel blocker --- p.40Chapter III.2.3 --- Effect of repeated stimulation with [arg8]- vasopressin --- p.40Chapter III.2.4 --- Effects of receptor antagonists --- p.40Chapter III.2.5 --- Effects of thapsigargin and trifluoperazine --- p.44Chapter III.2.6 --- Effects of indomethacin --- p.44Chapter III.2.7 --- Effects of forskolin --- p.47Chapter Chapter IV --- Discussion --- p.50Chapter Chapter V --- References --- p.5

Purinergic signalling in the genito-urinary tract.

The main objective of this thesis was to examine the role of purinergic signalling in the contraction of the smooth muscle of the genito-urinary tract of laboratory animals and compare it to that of man. It also examined purinergic signalling in the maturation of sperm within the epididymis. The main methodology involved organ bath studies on the functional physiology of smooth muscle contraction, in conjunction with immunohistochemical examination of smooth muscle P2X receptor expression. In Chapter 3, a comparative study of the smooth muscle cells of the testicular capsule or tunica albuginea of the testis from man, mouse, rat and rabbit was made. The smooth muscle cell arrangement was demonstrated by electron microscopy, and the role of purinergic co-transmission in the contraction of this smooth muscle was investigated. Chapter 4 examined purinergic signalling in the contraction of the human vas deferens smooth muscle. P2X receptors are involved in cell-to-cell signalling. Chapter 5 was a comparative study of the expression of P2X receptors on sperm contained within the head and tail of the epididymides of mice, rats, hamsters and man. This study demonstrated changing expression with maturity. Alterations in the relative purinergic and cholinergic components of detrusor contraction have been demonstrated in the over active bladder. Chapter 6 details the partial bladder outlet obstruction model that was developed in the rat. This model demonstrated an up-regulation of the cholinergic component of detrusor contraction with no significant change in the purinergic component, which implied the rat detrusor adapts to outflow obstruction in a different manner to the human detrusor. In Chapter 7, a general discussion of the role of purinergic signalling in the genito-urinary tract is given. The extent of how well the hypothesis was tested is considered in this chapter and future directions are suggested

Neurohumoral and local controls of the electrogenic chloride secretion in rat and human epididymis with reference to the signal transduction mechanisms.

by Anskar Yu-hung Leung.Thesis (Ph.D.)--Chinese University of Hong Kong, 1992.Includes bibliographical references (leaves 147-163).Chapter Section I --- Literature reviewChapter Chapter I.1. --- The epididymis - its structures and functions --- p.1Chapter Chapter I.2. --- Cellular mechanisms of transepithelial electrolyte transport in epididymis and other exocrine tissues --- p.5Chapter Chapter I.3 --- Signal transduction mechanism of chloride secretion in epididymis and other exocrine tissue --- p.11Chapter Chapter I.4 --- Significance of chloride secretion by the epididymal epithelium and the objectives of the study --- p.17Chapter Section II --- General methodsChapter Chapter II.1. --- Tissue culture from the rat cauda epididymis --- p.20Chapter Chapter II.2. --- The short-circuit current technique --- p.30Chapter Chapter II.3. --- The immunofluorescence technique --- p.39Chapter Chapter II.4. --- Intracellular adenosine 3':5' cyclic monophosphate (cAMP) measurement --- p.43Chapter Chapter II.5 --- Intracellular Ca2+ measurement using the microfluorimetric technique --- p.49Chapter Section III --- ResultsChapter Chapter III.1. --- Studies on the neural and humoral controls of eletrogenic chloride secretion in rat epididymis --- p.55Chapter Chapter III.2. --- Local control of electrogenic chloride secretion in rat epididymis 226}0ؤThe role of the calcitonin gene-related peptide --- p.80Chapter Chapter III.3. --- Characterization of intracellular Ca2+ store using ATP as the calcium mobilizing agonist --- p.94Chapter Chapter III.4. --- Ca2+ handling mechanisms in single cultured rat epididymal cells --- p.106Chapter Chapter III.5. --- Studies on the effector process in the stimulus-secretion coupling- characterization of apical Cl- conductance in cultured rat cauda epididmal cells --- p.114Chapter Chapter III.6. --- Effects of secretory agonists on transepithelial Cl- transport and intracellular Ca2+ concentration in cultured human epididymal epithelium --- p.132Chapter Section IV --- General discussion --- p.142Chapter Section V --- References --- p.147Appendix --- p.16

Some effects of vasectomy on the reproductive system of the ram

The purpose of this study was to examine, the response of the male reproductive system of rams to the altered physiological status existing after vasectomy. Vasectomised rams were examined at different periods up to three years and nine months after the operation, with regard to the structure and functional state of their testes, epididymides, vasa deferentia, vesicular glands, prostate gland and bulbo-urethral glands. The ejaculate was studied for the presence, morphology and metabolic activity of spermatozoa, and the seminal plasma for its fructose concentration. The levels of androgens in the peripheral blood were determined by a radioimmunoassay. Intact rams were studied by similar methods in order to establish the normal status of reproductive function and the seasonal variations occurring in this species. The changes occurring in the testis after vasectomy were restricted to the seminiferous tubules, where spermatogenic activity was found to be both qualitatively and quantitatively affected. The interstitial (Leydig) cells were found to be functionally unchanged, as demonstrated by the similarity of androgen profiles in intact and vasectomised rams during the breeding and the non-breeding seasons. The epididymal changes were most marked in the region of the cauda epididymidis, where enlargement, multiple spermatocoeles and sometimes rupture of the serous covering were observed in vasectomised animals. Although evidence of increased sperm removal by phagocytosis was found, the epididymis was unable to cope successfully with excurrent duct blockage even when the testis was producing smaller numbers of spermatozoa than normal. Evidence was also obtained indicating interference with sperm maturation in the epididymis of some vasectomised rams. The ejaculates of vasectomised rams contained immotile spermatozoa fox well over one year after the operation. Although these spermatozoa were found to be structurally intact for up to three to six months, they were neither fertile nor metabolically active after two to four weeks in most cases. The storage site from which these spermatozoa are voided at ejaculation was located as being the glandular acini surrounding the lumen of the ampulla. The secretory activity of the glandular epithelium in the ampulla and vesicular gland was altered in some vasectomised animals, and this was reflected in the histological appearance of the cells and in the fructose concentration of the ejaculate. These animals had either increased or decreased secretory activity when compared with intact rams, although their androgen profiles were not significantly different from those of intact rams

Regulation of the pannexin-1 promoter in the rat epididymis

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

Transcriptional and epigenetic regulation of steroid 5 [alpha]-reductase type 2 and androgen action in epididymal principal cells

Androgens, in particular dihydrotestosterone (DHT), are crucial for maintaining the structure and functions (sperm maturation and storage) of the epididymis. DHT is synthesized from testosterone by steroid 5alpha-reductase (Srd5aR) type 1 and type 2. Srd5aR1 and srd5aR2 have different tissue-specific distribution patterns and distinct genetic and biochemical properties. Srd5aR2 mRNA is more abundant than that of srd5aR1 in several sex accessory tissues, most dramatically in the epididymis, where it is predominantly expressed in the caput region.The two main objectives of this thesis are to elucidate the transcriptional and epigenetic regulation of srd5aR2 in the rat epididymis and to identify androgen-dependent genes in epididymal principal cells.In the first objective, the 5'upstream region of srd5aR2 was cloned and analyzed. The gene's transcriptional start site was localized, regulatory elements and the core promoter were mapped, and the transcription factors SP1, and to a lesser extent SP3, were found to interact with srd5aR2 in the rat caput epididymidis and mouse proximal caput epididymidis (PC-1) cell line. The epigenetic regulation of srd5aR2 by DNA methylation was subsequently examined and contrasted to that of srd5aRl in tissues where the isozymes are differentially expressed. DNA methylation levels fluctuated along the 5'upstream region of both genes. Tissue-specific variations in adenine and cytosine methylation were also identified for srd5aR1 and srd5aR2, respectively. These studies constitute the first analysis of the 5'upstream region of srd5aR2, at both the transcriptional and epigenetic levels.In the second objective, the direct effects of androgens on principal cell gene expression were examined in PC-1 cells. The majority of androgen-regulated genes displayed an early or late transient increase in expression levels following androgen withdrawal. When DHT was added back to the media, a differential ability of rescue was seen among androgen-regulated genes depending on time of supplementation. This rescue ability was severely compromised after 4 days of androgen deprivation. These results provide novel insights into the response of principal cells to androgens.Together, the two objectives of this thesis impart greater understanding into the mechanisms of androgen action in the epididymis

Isolement des cellules basales épididymaires et caractérisation de nouvelles fonctions.

L’épididyme joue un rôle important dans l’acquisition de la fertilité, en permettant aux spermatozoïdes d’acquérir mobilité et pouvoir fécondant lors de leur progression dans la lumière. Cette maturation ne peut se faire que grâce à un environnement spécifique qui garantit la protection des spermatozoïdes et est maintenu par la barrière hémato-épididymaire. Cette barrière est formée de jonctions serrées entre les cellules de l’épithélium, et isole le compartiment luminal du compartiment basal. Les cellules basales de l’épididyme sont localisées à la base de l’épithélium et possèdent des projections apicales minces régulées par des facteurs testiculaires, qui seraient capables de traverser la barrière hémato-épididymaire et d’atteindre la lumière du tubule, notamment au niveau des jonctions tripartites. En plus de la participation à la barrière hémato-épididymaire, il a été suggéré un rôle de détoxification, une fonction immunitaire et un rôle de régulation des fonctions d’autres cellules épithéliales. L’un des principaux obstacles à l’étude des cellules basales de l’épididyme est l’absence de protocole pour les isoler.L’objectif principal de ce projet était de caractériser la participation des cellules basales à la barrière hémato-épididymaire via la jonction tricellulaire, et d’étudier les voies de signalisation pouvant être impliquées dans cette participation. Trois sous-objectifs étaient nécessaires : (1) Etudier l’expression et la localisation de la tricelluline dans l’épididyme et déterminer si les cellules basales participent à la barrière hémato-épididymaire par le biais de cette protéine. (2) Mettre au point une méthode pour isoler les cellules basales. (3) Etablir le profil d’expression génique des cellules basales afin de pouvoir étudier les gènes et voies de signalisation spécifiques à ces cellules. Les résultats ont montré que la tricelluline était exprimée dans tous les segments de l’épididyme à des niveaux similaires, et que les niveaux de tricelluline augmentaient avec l’âge ; le marquage passe de cytoplasmique aux marges latérales de la membrane plasmique puis au niveau apical. La colocalisation de la tricelluline avec l’occludine a permis de vérifier que la tricelluline se situait bien au niveau des jonctions serrées. La colocalisation de la tricelluline et d’un marqueur des cellules basales, la cytokératine 5 (ou KRT5), a démontré que la cellule basale ne participait pas à la jonction tricellulaire. Enfin, des expériences de knock-down par ARN interférent ont permis de montrer que la résistance transépithéliale décroît lorsqu’on inhibe l’expression de la tricelluline. On a également observé une diminution de l’expression d’autres protéines de jonction telles l’occludine, la claudine-1 et la claudine-3. D’autres protéines de jonction, comme ZO-1 et E-cadhérine, ne sont pas altérées. Nos résultats ont donc permis de démontrer le rôle de la tricelluline dans l’épididyme, et de montrer que les cellules basales n’étaient pas impliquées dans la jonction tricellulaire. Isoler les cellules basales était nécessaire pour étudier leurs fonctions. Les différents protocoles retrouvés dans la littérature n’ont pas permis d’obtenir une population pure de cellules basales. Nous avons donc mis au point une méthode d’isolement par séparation magnétique basée sur l’intégrineα6. L’efficacité de cette séparation a été vérifiée par cytométrie en flux, immunofluorescence et PCR, prouvant un enrichissement de cellules basales d’environ 90%. De la microscopie électronique a permis de caractériser morphologiquement les cellules basales. Nous avons donc mis au point un protocole efficace et reproductible pour isoler les cellules basales, ce qui a permis d’effectuer ensuite leur profil d’expression génique. A partir de l’ARN des différentes fractions obtenues par séparation magnétique, nous avons fait des expériences de microréseaux. Ces données ont permis de déterminer que 552 gènes, dont 45 avec un ratio de 5 fois ou plus, étaient enrichis dans les cellules basales par rapport aux autres cellules de l’épididyme. Ces gènes hautement exprimés ont été montrés comme codant pour des protéines impliquées entre autres dans l’adhésion cellulaire, le fonctionnement du cytosquelette, le transport d’ions, la signalisation cellulaire. Plusieurs gènes hautement exprimés ont été retrouvés dans des cellules progénitrices adultes, suggérant que les cellules basales pourraient représenter une population de cellules souches épididymaires. Ces fonctions de cellules souches dans l’épididyme ont été démontrées in vitro en cultivant les cellules basales pendant 14 jours, ce qui a permis de montrer une prolifération et une réorganisation des cellules basales ainsi que des modifications dans l’expression de certains marqueurs. Les données obtenues dans ce travail de thèse permettent donc de montrer que (1) les cellules basales épididymaires ne sont pas impliquées dans la jonction serrée tricellulaire. (2) La séparation magnétique par l’intégrine-α6 est une méthode reproductible et efficace pour isoler les cellules basales. (3) Le premier profil d’expression génique des cellules basales épididymaires de rat ainsi que le développement d’une méthode de culture de ces cellules suggèrent fortement que ces cellules pourraient agir comme des cellules progénitrices multipotentes de l’épithélium épididymaire. The epididymis plays a critical role in fertilization by enabling spermatozoa to acquire motility and fertilization ability as they transit through the lumen. This maturation is accomplished by a specific luminal microenvironment in the epididymis, which protect spermatozoa from immune attack and which is maintained by the blood-epididymis barrier. This barrier is formed by tight junctions between epithelial cells, and partitions the epithelium into luminal and basal compartments. Basal cells are epididymal epithelial cells localized at the base of the epithelium. They typically display thin apical projections, regulated by testicular factors, which may extend through the blood-epididymis barrier at tricellular junctions. While some epithelial cells have been well studied, basal cells are relatively unknown. In addition to participation in the blood-epididymis barrier, several other roles have been suggested, including detoxification, immune function, and regulation of other epithelial cell functions, either directly via apical projection or indirectly, via hormone synthesis, such as prostaglandins. One of principal difficulties limiting the study of basal cells is the absence of a valid isolation protocol. The objective of this project was to characterize the participation of basal cells in the blood-epididymis barrier through tricellular tight junctions, and to study the pathways implicated in this participation. This objective was divided into three parts : (1) To study the expression and localization of tricellulin in the epididymis, and determine if basal cells participate in the blood-epididymis barrier through this protein. (2) To establish a reliable and reproducible method to isolate epididymal basal cells. (3) To establish the gene expression profile of epididymal basal cells and predict genes and pathways implicated in these cells. Localization of tricellulin was studied in the adult rat epididymis as well as at various stages of postnatal development, by immunofluorescence. Protein localization changed during postnatal development from cytoplasmic to membrane-bound as a function of age. Tricellulin protein expression in the adult and during postnatal development was assessed by Western-blot. In addition, co-localization studies between tricellulin and occludin were conducted in order to verify the localization of tricellulin at tight junctions. Participation of basal cells in tricellular junctions was confirmed by co-localization between tricellulin and cytokeratin 5, a marker of basal cells. Finally, knockdown of tricellulin was achieved using small interfering RNA (siRNA) in our rat caput epididymal principal cell line. These experiments demonstrated a decrease of transepithelial resistance, which was correlated with decreased levels of claudin-1, claudin-3 and occludin. Zonula occludens 1 and cadherin1 levels were unchanged. Our results therefore demonstrate the role of tricellulin in epididymis, and show that basal cells are not necessarily involved in tricellular tight junction.In order to elucidate basal cell functions, it was necessary to develop a reproducible protocol for the isolation of highly purified basal cells. Various isolation protocols reported previously in the literature (density gradients or magnetic separation) have not yielded a sufficiently pure population of basal cells. We have developed a new method, based on magnetic separation of basal cells using integrin-α6 as a basal cell marker. The efficiency of this method was verified by flow cytometry, immunofluorescence and RT-PCR, all of which indicated a purity of the isolated basal cell fraction as close to 90%. Electron microscopy was also performed, in order to further characterize epididymal basal cell morphology. We are therefore satisfied that we have achieved an efficient and reproducible protocol to isolate epididymal basal cells, which has permitted us to elaborate basal cell morphology and further characterize their properties. Using mRNA extracted from the different fractions obtained by magnetic separation (positive or basal cell, and negative or non-basal cell fractions), we performed microarray experiments. These data demonstrated that 552 genes were differentially expressed. Of these, 45 exhibited a ratio of 5 or more fold-change and were enriched in basal cells. These highly expressed genes coded for proteins implicated in cell adhesion, the cytoskeleton, ion transport, and cell signaling. Several genes were also implicated in progenitor functions, suggesting that basal cells may act as stem cells in the epididymis. In other tissues such as prostate or trachea, basal cells can be the origin of other cell types. We demonstrated that basal cells shared some of the features of multipotent stem cells by culturing isolated epididymal basal cells for 14 days. We observed a proliferation and a reorganization of basal cells, as well as modifications in cytokeratins expression. All the data generated for this thesis show : (1) Epididymal basal cells are not implicated in tricellular junctions. (2) The development of a reproducible method to isolate a highly purified fraction of basal cells. (3) The first gene expression profile of rat epididymal basal cells. (4) The characterization of epididymal basal cells, which suggests that they may possess a progenitor function for epididymal epithelial cells