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The transepithelial movement of water into the male reproductive tract is an essential process for normal male fertility. Protein water channels, referred to as aquaporins (AQPs), are involved in increasing the osmotic permeability of membranes. This study has examined the expression of AQP1, AQP2, and AQP7 in epithelial cells in adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis, initial segment, caput, corpus and cauda epididymidis, and vas deferens were investigated by immunohistochemistry and Western blotting procedures to show the localization and distribution of the AQPs. AQP1 was noted in rete testis, in efferent ducts, and in vessels in the intertubular space, suggesting that AQP1 participated in the absorption of the large amount of testicular fluid occurring characteristically in the efferent ducts. AQP2 expression was found in the rete testis, efferent ducts and epididymis, whereas AQP7 was expressed in the epithelium of the proximal regions of the epididymis and in the vas deferens. This is the first time that AQP2 and AQP7 have been observed in these regions of mammalian excurrent ducts, but their functional role in the dog male reproductive tract remains unknown. Investigations of AQP biology could be relevant for clinical studies of the male reproductive tract and to technologies for assisted procreation.

The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis. Fil: Losinno, Antonella Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Sorrivas, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina Fil: Ezquer, Eduardo Marcelo. Universidad del Desarrollo; Chile Fil: Ezquer, Fernando. Universidad del Desarrollo; Chile Fil: Lopez, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Morales, Alfonsina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

Microtubule-associated protein 1B (MAP 1B) is a neuronal cytoskeleton marker with predominant expression in the developing nervous system. The present study provides evidence for the expression of this cytoskeleton protein in non-neuronal and neuronal cells along rat and human efferent ductules and epididymis (initial segment, caput, and cauda). Reverse transcription/polymerase chain reaction and Western blot analysis were used to confirm the presence of MAP 1B (mRNA and protein) in rat tissues. Immunohistochemical studies revealed MAP-1B-positive staining in columnar ciliated cells present in efferent ductules and in narrow cells located in the initial segment, in both rat and human. MAP-1B-positive basal cells, located underneath the columnar cells, were only identified in the initial segment and caput epididymidis of the rat. Qualitative analysis of tissues from 40-day-old and 120-day-old rats indicated that the number of MAP-1B-positive ciliated, narrow, and basal cells per tubule increased with sexual maturation. These immunoreactive cells did not stain for dopamine beta-hydroxylase or acetylcholinesterase, indicating that they were not adrenergic or cholinergic in nature. Immunohistochemical studies also revealed the presence of MAP-1B-positive staining in interstitial nerve fibers in caput and cauda epididymidis from both rat and human. Thus, the expression of MAP 1B is not confined to a specific cell type in rat and human efferent ductules and epididymis. The functional significance of this cytoskeleton protein in tissues from the male reproductive tract requires further investigation.

The presence, location and degree of immunoexpression of various microfilament (MF) and intermediate filament (IF) systems (actin, cytokeratins, desmin, vimentin) were studied in the excurrent ducts of the testis in sexually mature and active galliform (Japanese quail, domestic fowl, turkey) and anseriform (duck) birds. These proteins were variably expressed between the epithelia and periductal tissue (periductal smooth muscle cell layer and interductal connective tissue) types and between species. Variable heterogeneous co-expression of filament systems was also found in the various duct epithelia and periductal tissue types: co-expression of filament systems was the rule rather than the exception. In the duck, neither vimentin nor cytokeratin was present in any of the tissues, whereas actin and desmin (absent in the rete testis) were co-expressed in the efferent ducts and epididymal duct unit (comprising the ductus conjugens, ductus epididymidis and ductus deferens). Actin, desmin and vimentin were generally co-expressed in the rete testis, efferent ducts and epididymal duct unit of the quail, domestic fowl and turkey, with vimentin being more strongly immunoreactive than actin and desmin in the epididymal duct unit, but more weakly immunoexpressed in the efferent ducts. Cytokeratin was present and co-expressed with actin, desmin and vimentin in the rete testis, efferent ducts and epididymal duct unit of the domestic fowl and turkey, but not in the quail and duck. The periductal smooth muscle cell layer and interductal tissue co-expressed actin, desmin and vimentin variably in all birds. Luminal spermatozoa of both the turkey and duck were immunonegative for all protein systems, whereas those of the quail and domestic fowl co-expressed actin, desmin and vimentin moderately or strongly. The tissues of the reproductive tract of male birds thus contain cytoskeletal protein systems that are variably but mostly co-expressed and whose contractile ability appears necessary and sufficient for transportation through the various excurrent ducts of the voluminous testicular fluid and its high sperm content, characteristic features of male avian reproduction.

The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.

As luminal fluid moves from the seminiferous tubule and enters the rete testis, its protein concentration declines from approximately 6 mg/ml to 1 mg/ml. It was therefore suggested that protein is either 1) utilized by the spermatozoa, 2) transported across the epithelium of the terminal segment of the seminiferous tubule, the tubuli recti or rete testis, or 3) absorbed and degraded by the epithelium. Horseradish peroxidase (HRP), a protein marker, was microperfused into single seminiferous tubules or perfused directly into the rete. After fixation, the HRP was localized histochemically and the tissue observed under the light- and electron microscope. HRP was taken up via pinocytotic vesicles into the cytoplasm of the Sertoli cells and germ cells but did not permeate extracellularly beyond the tight junctions. Similar results were obtained in the cells lining the terminal segment and the tubuli recti. The rete epithelium showed uptake of HRP into coated and noncoated vesicles, while some cells additionally revealed diffuse cytoplasmic distribution of HRP. The terminal segment, tubuli recti, and rete testis may be important routes by which proteins may leave the testicular fluid either to be degraded or to enter the blood.

The bovine testis has a central mediastinum consisting of longitudinally oriented rete channels and spacious lymph vessels, embedded in the mediastinal stroma. The latter represents a contractile-elastic unit and is composed of myofibroblasts, collagen bundles and accumulations of elastin, connecting the myofibroblasts. The dimension of the mediastinum varies in cross sections at different levels between 3.5 and 31.8 mm2. In one cross section approximately 30 rete channels and approximately 30 openings of straight testicular tubules are encountered. Nearly 25% of the area is occupied by thin-walled, valveless lymph vessels. Arterial convolutes, interpolated between straight centripetal and straight centrifugal branches of the testicular artery flank the rete on all sides. It is concluded that the pulsation within these convolutes together with the contractile-elastic stroma promotes lymph and rete content in a caudo-cranial direction. Chordae retis as described by Roosen-Runge and Holstein (1978) for the human testis are a common feature in the bovine mediastinum testis. The rete channels are lined by a simple cuboidal or columnar epithelium. Short intraepithelial crypts are present and function as epithelial reserve for dilatation and expansion of the rate. The inventory of organelles is rather inconspicuous in the rete epithelium. The apical border bears short microvilli and gives a strong reaction for alkaline phosphatase. The basal cytoplasm contains many small to medium-sized electron-dense bodies and is site of a strong acid phosphatase reaction. The rete epithelium as a whole reacts strongly with leucine aminopeptidase, the marker enzyme of the testicular excurrent duct system. Many free mononuclear cells, mostly macrophages, are observed in the basal half of the rete epithelium.

The distribution of lymph vessels in the human testis was investigated using ink injection methods, and light and electron microscopy. Lymph capillaries occur in the septula testis but are absent in the intertubular tissue. They consist of endothelial cells provided with an incomplete basal lamina and anchoring filaments of the adjacent connective tissue. Frequently, the endothelial cells are separated by gaps measuring up to 2 micron. The lymph capillaries of the septula testis are connected to lymph vessels in the rete testis and tunica albuginea. These vessels have occasional smooth muscle cells and valves. At the posterior margin of the testis, the network of lymph vessels merges into collecting ducts, which together with vessels derived from the rete testis are drained by the lymphatic system in the spermatic cord.

Fine structure, postnatal development and reaction to efferent duct ligation of the loose connective tissue of the rat rete testis were studied by light and electron microscopy. The loose connective tissue of adult rats consists of elongate fibroblasts in a homogenous ground substance, together with some Leydig cells, lymphocytes, macrophages and mast cells. During postnatal development this tissue increases in amount, while the interstitial areolar tissue decreases. The "looseness" of the tissue becomes more evident between days 22 and 27, and may reflect an increase in hydration. Efferent duct ligation for 15 min to five days has no effect on the histological appearance of the tissue.

Convincingly demonstrated by immunocytological methods in females of several mammalian species, relaxin has not yet been localized in the male. Immunocytologically, a related antigen was identified in adult normal boar testes using and anti- [NIH P-relaxin/HSA] antiserum free of anti HSA Abs. A strong reaction was observed in interstitial cells, a weaker but very clear one in Sertoli cells. NIH P-relaxin and HC1-acetone extracts of either corpora lutea from pregnant sows or boar testes inhibited the immunofluorescence of the reactive structures in the boar testes as well as in ovaries of pregnant sows. Ethanol-acetone precipitates from boar rete testis or caudal epididymal fluids inhibited the reaction of interstitial and Sertoli cells, but this inhibition in the sow was limited only to degenerative ovarian structures, probably due to an insufficient level of inhibiting antigen in these two seminal fluids, in contrast with the very high concentration of relaxin in luteal cells of pregnant sows. Specific immunofluorescence was observed neither in ectopic testes of adult monocryptorchid boars (contrary to scrotal testes in these same animals) nor in testes of prepuberal pigs. The specificity and meaning of these results are discussed.