Search

Sertoli cells (SCs) are supporting cells in the mammalian testis that proliferate throughout fetal and postnatal development but exit the cell cycle and differentiate at puberty. In our previous study, we isolated a population of highly proliferative Sertoli-like cells (SLCs) from the region of the adult mouse testis containing the rete testis and adjacent seminiferous tubules. Here RNA-seq of the adult SLC culture as well as qPCR analysis and immunofluorescence of the adult and immature (6 dpp) SLC cultures were performed that allowed us to identify SLC-specific genes, including Pax8, Cdh1, and Krt8. Using these, we found that SLCs are mostly localized in the rete testis epithelium; however, some contribution of transitional zones of seminiferous tubules could not be excluded. The main feature of SLCs indicating their relationship to SCs is DMRT1 expression. More than 40% of both adult and immature SLCs expressed DMRT1 at different levels in culture. Only rare DMRT1+ cells were detected in the adult rete testis, whereas more than 40% of cells were positively stained for DMRT1 in the immature rete testis. One more SC protein, AMH, was found in some rete cells of the immature testis. It was also demonstrated that SLCs expressed such SC genes as Nr5a1, Dhh, Gdnf, and Kitl and interacted with germ cells in 3D co-culture with immature testicular cells. All these similarities between SLCs and rete cells on one the hand and SCs on the other, suggest that rete cells could share a common origin with SCs.

Background: The primary cilium is a microtubule-based organelle that extends transiently from the apical cell surface to act as a sensory antenna. Initially viewed as a cellular appendage of obscure significance, the primary cilium is now acknowledged as a key coordinator of signaling pathways during development and in tissue homeostasis., Objectives: The aim of this review was to present the structure and function of this overlooked organelle,with an emphasis on its epididymal context and contribution to male infertility issues., Materials and Methods: A systematic review has been performed in order to include main references relevant to the aforementioned topic., Results: Increasing evidence demonstrates that primary cilia dysfunctions are associated with impaired male reproductive system development and male infertility issues., Discussion: While a large amount of data exists regarding the role of primary cilia in most organs and tissues, few studies investigated the contribution of these organelles to male reproductive tract development and homeostasis., Conclusion: Functional studies of primary cilia constitute an emergent and exciting new area in reproductive biology research., (© 2019 American Society of Andrology and European Academy of Andrology.)

Sertoli cells (SCs) play a crucial role in testis differentiation, development and function, determining the magnitude of sperm production in sexually mature animals. For over 40 years, it has been considered that these key testis somatic cells stop dividing during early pre-pubertal phase, between around 10 to 20 days after birth respectively in mice and rats, being after that under physiological conditions a stable and terminally differentiated population. However, evidences from the literature are challenging this dogma. In the present study, using several important functional markers (Ki-67, BrdU, p27, GATA-4, Androgen Receptor), we investigated the SC differentiation status in 36 days old and adult Wistar rats, focusing mainly in the transition region (TR) between the seminiferous tubules (ST) and the rete testis. Our results showed that SCs in TR remain undifferentiated for a longer period and, although at a lesser degree, even in adult rats proliferating SCs were observed in this region. Therefore, these findings suggest that, different from the other ST regions investigated, SCs residing in the TR exhibit a distinct functional phenotype. These undifferentiated SCs may compose a subpopulation of SC progenitors that reside in a specific microenvironment capable of growing the ST length if needed from this particular testis region. Moreover, our findings demonstrate an important aspect of testis function in mammals and opens new venues for other experimental approaches to the investigation of SC physiology, spermatogenesis progression and testis growth. Besides that, the TR may represent an important site for pathophysiological investigations and cellular interactions in the testis.

Efferent ductules are responsible for the transportation of spermatozoa from the testis to the epididymis and their epithelium is responsible for the reabsorption of over 90% of the luminal fluid. The purpose of this research was to characterize the gross morphology and histology of efferent ductules in the male Golden Syrian hamster. The efferent ductules emerge from rete testis with a unique polarity at the apex or cephalic pole of the testis. The number of efferent ductules varied from 3 to 10 with an average of 6.0 and blind ending ducts were observed in approximately 56% of the males. The ductules merged into a single common duct prior to entering the caput epididymidis. The proximal efferent ductule lumen was wider than the distal (conus and common ducts), consistent with reabsorption of most of the luminal fluid, as was morphology of the ductal epithelium. Non-ciliated cells in the proximal region had prominent endocytic apparatuses, showing both coated pits and apical tubules in the apical cytoplasm. Large basolateral, intercellular spaces were also present in the epithelium of the proximal region. Distal non-ciliated cells had an abundance of large endosomes and lysosomal granules. Localisation of sodium/hydrogen exchanger-3 (NHE3; SLC9A3) and aquaporins 1 and 9 (AQP1, AQP9) along the microvillus border was also consistent with ion transport and fluid reabsorption by this epithelium. In comparison, the caput epididymidis epithelium expressed only AQP9 immunostaining. Another unusual feature of the hamster efferent ductules was the presence of glycogen aggregates in the basal cytoplasm of small groups of epithelial cells, but only in the proximal ducts near the rete testis. Androgen (AR), estrogen (ESR1 and ESR2) and vitamin D receptors (VDR) were also abundant in epithelial nuclei of proximal and distal efferent ductules. In comparison, caput epididymidis showed very little immunostaining for ESR1., (© 2014 American Society of Andrology and European Academy of Andrology.)

The anterior testicular ducts of squamates transport sperm from the seminiferous tubules to the ductus deferens. These ducts consist of the rete testis, ductuli efferentes, and ductus epididymis. Many histological and a few ultrastructural studies of the squamate reproductive tract exist, but none concern the Hydrophiidae, the sea snakes and sea kraits. In this study, we describe the anterior testicular ducts of six species of hydrophiid snakes as well as representatives from the Elapidae, Homolapsidae, Leptotyphlopidae, and Uropeltidae. In addition, we examine the ultrastructure of these ducts in the yellow-bellied Sea Snake, Pelamis platurus, only the third such study on snakes. The anterior testicular ducts are similar in histology in all species examined. The rete testis is simple squamous or cuboidal epithelium and transports sperm from the seminiferous tubules to the ductuli efferentes in the extratesticular epididymal sheath. The ductuli efferentes are branched, convoluted tubules composed of simple cuboidal, ciliated epithelium, and many species possess periodic acid-Schiff+ granules in the cytoplasm. The ductus epididymis at the light microscopy level appears composed of pseudostratified columnar epithelium. At the ultrastructural level, the rete testis and ductuli efferentes of P. platurus possess numerous small coated vesicles and lack secretory vacuoles. Apocrine blebs in the ductuli efferentes, however, indicate secretory activity, possibly by a constitutive pathway. Ultrastructure reveals three types of cells in the ductus epididymis of P. platurus: columnar principal cells, squamous basal cells, and mitochondria-rich apical cells. This is the first report of apical cells in a snake. In addition, occasional principal cells possess a single cilium, which has not been reported in reptiles previously but is known in some birds. Finally, the ductus epididymis of P. platurus differs from other snakes that have been studied in possession of apical, biphasic secretory vacuoles. All of the proximal ducts are characterized by widening of adjacent plasma membranes into wide intercellular spaces, especially between the principal cells of the ductus epididymis. Our results contribute to a larger, collaborative study of the evolution of the squamate reproductive tract and to the potential for utilizing cellular characters in future phylogenetic inferences., (Copyright © 2011 Wiley Periodicals, Inc.)

The volumetric proportion of the various ducts of the epididymis of the emu and ostrich and the immunohistochemistry of actin microfilaments, as well as cytokeratin, desmin and vimentin intermediate filaments, were studied in the various ducts of the epididymis of the emu and ostrich. The volumetric proportions of various ducts, which are remarkably different from those of members of the Galloanserae monophyly, are as follows: the rete testis, 5.2 +/- 1.4% for the emu and 2.4 +/- 1.8% for the ostrich; efferent ducts, 14.2 +/- 2.3% (emu) and 11.8 +/- 1.8% (ostrich); epididymal duct unit, 25.8 +/- 5.8% (emu) and 26.1 +/- 4.1% (ostrich) and connective tissue and its content, 54.7 +/- 5.8% (emu) and 60.0 +/- 4.9% (ostrich). Unlike in mammals and members of the Galloanserae monophyly, only vimentin was immunohistochemically demonstrated in the rete testis epithelium of the emu, and none of the cytoskeletal protein elements in the ostrich rete testis. The epithelium of the efferent ducts of the emu co-expressed actin, cytokeratin and desmin in the non-ciliated type I cells, and vimentin in the ciliated cell component. The ostrich demonstrated only cytokeratin in this epithelium. The ratite epididymal duct unit is different from that of mammals in lacking actin (only weaky expression in the ostrich), desmin and cytokeratin, and a moderate/strong immunoexpression of vimentin in the basal cells and basal parts of the NC type III cell in the epididymal duct unit. Immunoexpression of the microfilaments and intermediate filaments varied between the two ratite birds, as has been demonstrated previously in birds of the Galloanserae monophyly, and in mammals.

An immunohistochemical and lectin histochemical study of the efferent ducts was performed in the alpaca. Two types of epithelium, consisting of principal and ciliated cells, were detected on the basis of the different cytokeratins expression and lectin binding pattern. AE1/AE3 and 13 cytokeratin antibodies intensely immunostained the entire cytoplasm of type I PCs, whereas AE1/AE3, but not anti cytokeratin 13, immunoreacted in type II principal cells along the apical, lateral and basal plasma-membrane. The histochemical characterization of the epithelial cells was carried out using a battery of different lectins: Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosilation pre-treatments were also employed. In type I principal cells, the staining of the Golgi zone was interpreted giving evidence for the synthesis and secretion of O- and N-linked oligosaccharides. In particular, alpha-Man/alpha-Glc, GlcNAc, beta-Gal-(1-4)-GlcNAc, Neu5Acalpha2,3Gal and Neu5Acalpha2,6Gal/GalNAc residues were included in both O- and N-linked glycans, whereas alpha-Fuc, beta-GalNAc and alpha-Gal were only found in O-linked oligosaccharides; alpha-GalNac and beta-Gal-(1-3)-D-GalNAc were found subterminal to sialic acid moieties and they were included in O- and N-glycans. In type II principal cells, the lectin staining was observed in the apical cytoplasmic granules and in vacuoles that were interpreted as components of an elaborate endocytotic apparatus specialized for the uptake of particulate material and fluid from the lumen. These results suggest the existence of two structurally different epithelial segments along the ductuli efferentes of the alpaca, with a high degree of compartmentalization of the secretory and absorptive activities.

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 dynamic structures of the human ductuli efferentes, which connect between the rete testis and the ductulus epididymidis, are described in the present paper. Maturation of sperm takes place in the tubule and after this process the group of sperm is transferred to the ductus. All ductuli do not show the same histological contents. Each ductulus shows variety of different configuration depending on the functional phase (the state of sperm maturation) of the tubule. The basic pattern of all the tubuli is the same, however, they differ in the thickness, cytoplasmic components of epithelial cells and luminal contents. Larger tubule has big lumen whereas small is almost without a lumen. During the process, spherical and luminal macrophages act as nurse macrophage by using G1 granules obtained from the epithelial cell, and irregular shaped macrophage in the small tubule acts as scavenger macrophage. It has been shown that the epithelial cell of the large tubule show intense excretory activity and the secretory substance is different from other granules and lysosomes by periodic acid methenamine silver staining.

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 feline urogenital junction is situated between the extratesticular rete and the spacious initial segments of the efferent ductules. The rete epithelium is cuboidal to low columnar. The rete cells forming the junction rest on a wavy basal lamina, display deep mutual invaginations, possess central nuclei with several infoldings and form a distinct border with the columnar epithelial cells of the initial segments of the ductuli efferentes. The epithelium of the initial segments is composed of ciliated cells and non-ciliated principal cells. The latter are the dominating type and characterized by an apical brush-border and a supranuclear endocytotic apparatus. The stroma of the extratesticular rete contains an abundance of collagen whereas contractile cells are here generally absent. In contrast, the initial segments of the efferent ductules are surrounded by elastic fibres and a layer of contractile cells. All nerves for the feline urogenital junction come from the nervus spermaticus superior. In the epididymal head, small nerve bundles deviate into the septa between the ductules. Single fibres establish a dense network within the muscular coat of the ductuli. At the transition to the extratesticular rete, this network ends abruptly. Nerve fibres in the confines of the rete are associated with blood vessels or proceed to the testicular interior, but establish no relationships with the rete epithelium or the myofibroblasts of the mediastinum. The nervous network in the walls of the efferent ductules and their initial segments is not only composed of sympathetic but also parasympathetic, non-myelinated fibres. Particularly noteworthy is the abundance of calcitonin gene-related peptide (CGRP)- and substance P (SP)-containing axons around the initial segments. Both neuroproteins are consistent markers for sensory neurones. Taken together, it can be assumed that the entry of seminal fluid and spermatozoa into the efferent ductules is controlled by a regulatory nervous chain provided with afferent and efferent components.

Purpose: To investigate sperm quality aspirated from the tubuli recti compared to that obtained from microsurgical epididymal sperm aspiration (MESA)., Methods: Sixteen patients with congenital bilateral absence of the vas deferens (CBAVD) underwent MESA. Six MESA procedures were difficult, and therefore sperm were retrieved from the tubuli recti ductules. Intraoperative sperm parameters, recovery after freeze-thaw, and ICSI outcomes were analyzed and compared between tubuli recti and MESA sperm., Results: Mean initial sperm concentration was similar in both groups (18 vs. 16 million sperm/mL). Initial sperm motility was significantly higher in the tubuli recti group (35%) than the MESA group (25%). However, post thaw motility was higher with MESA compared to tubuli recti sperm (8.7 vs. 1.5%). ICSI fertilization rates after sperm freeze-thaw were 66% for tubuli recti sperm and 71% for MESA sperm., Conclusions: Tubuli recti sperm may provide an attractive alternative to testis sperm extraction. Poor sperm recovery after freeze-thaw should be expected.

The epithelium of the rete testis of the ostrich is simple cuboidal to columnar in nature, unlike the more flattened epithelium encountered in other non-passerine birds. A solitary cilium projects into the ductal lumen. A unique lateral cell membrane modification, similar, in some respects, to a hemi-desmosome, occurs frequently along the length of this membrane. The lateral cell membrane is complexly folded, and the cell contains an abundance of intermediate filaments, particularly in the vicinity of the nucleus and the internal surface of the basal cell membrane. The Golgi complex and endoplasmic reticulum are moderately developed. Another unique feature of the cell is the presence of a solitary, large, heterogeneous lipid body/droplet situated in the immediate supranuclear region of the cell. Overall, the cell structure conveys the impression of a metabolically active cell, which has the capability of transporting material from the duct lumen to the basal part of the cell, and of protein synthesis, apparently much in excess of what is necessary for cellular maintenance and sustenance.

The structure of the efferent ducts of animals is known to vary from one species to another, it even varies between segments of the ducts in the same species. In the grasscutter or greater cane rat ( Thryonomys swinderianus), there are three segments of the efferent duct, based on their content of non-ciliated or principal cell types. Type I non-ciliated cell is present exclusively in the long proximal part of the duct, and exhibits a well-developed subapical endocytic apparatus as well as numerous oval or pleomorphic dense bodies. The type II non-ciliated cell predominates in the middle part of the duct, displays a poorly developed subapical endocytic apparatus but contains large, numerous vacuoles and dense bodies, all of which fill about two-thirds of the cell height. The type III non-ciliated cell, found in the epithelium of the terminal part of the duct, is poorly endowed with a subapical endocytic apparatus and contains no conspicuous endocytic vesicles or vacuoles. Only a few, small, dense bodies are present, if at all. The efferent duct of the cane rat is thus similar to that of man, the bull, goat and dog, in containing three varieties or types of non-ciliated cells. This report is the first to describe multiple non-ciliated cells in the epithelium of the efferent ducts of a rodent and, indeed, of a mammal smaller than the dog.

Spermatogonial stem cells form the foundation of spermatogenesis, and their transplantation provides a unique opportunity to study spermatogenesis and may offer an alternative approach for animal transgenesis. This study was designed to extend the technique of spermatogonial transplantation to an economically important, large-animal model. Isolated immature pig testes were used to develop the intratesticular injection technique. Best results of intratubular germ cell transfer were obtained when a catheter was inserted into the rete testis under ultrasound guidance. The presence of infused dye or labeled cells was confirmed in the seminiferous tubules from 70 of 89 injected isolated testes. Infusion of 3-6 ml of dye solution or cell suspension could fill the rete and up to 50% of seminiferous tubules. The technique was subsequently applied in vivo. Donor cells included testis cells from 1- or 10-wk-old boars (from the recipients' contralateral testis or unrelated donors) and those from mice carrying a marker gene. Porcine testis cells were labeled with a fluorescent marker before transplantation. Testes were examined for the presence and localization of labeled donor cells immediately after transplantation or every week for 4 wk. Labeled porcine donor cells were found in numerous seminiferous tubules from 10 of 11 testes receiving pig cells. These results indicate that germ cell transplantation is feasible in immature pigs, and that porcine transplanted cells are retained in the recipient testis for at least 1 mo. This study represents a first step toward successful spermatogonial transplantation in a farm animal species.

Problem: Previous studies demonstrated that F4/80 antigen (murine macrophage-specific antigen)-positive cells in testes of normal adult mice accumulate particularly in the interstitium adjacent to the tubuli recti and rete testis (i.e., the central region). However, it remains unknown whether this accumulation is a congenital or acquired phenomenon., Method of Study: The distribution of F4/80-positive cells on frozen sections of testes obtained from various aged mice was immunohistochemically examined to determine when the positive cells specifically accumulate in the central region., Results: F4/80-positive cells were homogeneously distributed throughout the testicular interstitium with no specific accumulation until 2 weeks of age. However, at 3 weeks of age, the density of positive cells in the central region became slightly, but significantly, higher than that in the interstitium between the seminiferous tubules. Between 3 and 4 weeks of age, the cell density in the central region increased rapidly, the density at 4 weeks of age reaching the level of the mature testes of 8-week-old mice., Conclusion: These results demonstrate that the specific accumulation of F4/80-positive cells in the central region is an acquired phenomenon, which starts and ends before puberty.

The protein composition of epididymal fluid and sperm extracts of rats treated with the nitroimidazole compound ornidazole was investigated by two-dimensional gel electrophoresis. Epididymal luminal fluid from the corpus and cauda regions of male animals rendered infertile by ornidazole treatment contained a prominent protein (contraception-associated protein 1, CAP1) with a molecular mass of approximately 25 kDa and an isoelectric point (pI) of 5.8; it was not found in fluids, but was present in sperm, from fertile vehicle-fed rats. Infrared matrix-assisted laser desorption/ionization mass spectrometry indicated that the molecular mass of CAP1 was 20420+/-120 daltons. Analysis of 17 amino acids demonstrated 49% homology to a diuretic hormone from an insect (Acheta domesticus). Densitometric quantitation of CAP1 on silver-stained gels indicated its presence in greater amounts in cauda than in corpus fluid from treated animals, whereas fluid from the rete testis lacked CAP1. In vitro incubations of tissue from the caput, corpus, and cauda epididymidal regions with [35S]methionine gave no hint that CAP1 was a secretion product of the epididymal epithelium. The absence of CAP1 from luminal fluid obtained from the sperm-depleted corpus epididymidis of efferent duct-ligated ornidazole-fed rats suggested a spermatozoal origin. CAP1 was present in spermatozoa from the caput epididymidis but not from the rete testis in control animals. Less CAP1 was present in detergent extracts of cauda sperm from ornidazole-treated rats than in sperm from control animals, suggesting a contraceptive-related displacement of protein from sperm to fluid. The association of ornidazole- and alpha-chlorohydrin-induced infertility with the presence of CAP1 in epididymal fluid, probably originating from spermatozoa, suggests a critical role for this protein in fertilization.

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis.

The present study was designed to investigate relations of gamma-Glutamyl transpeptidase (gamma-GTP) activities to morphological differentiation and maturation of rat testicular excurrent duct system including rete testis, efferent ductuli and epididymal ducts. Animals used were Wistar rats aged from 3 days to 12 weeks. Histochemical demonstration of gamma-GTP activities was carried out by the method of Rutenburg et al. (1969). Morphological differentiation of the epithelium and structure of the rete testis occurred between 2 and 3 weeks and completed by the age of 6 weeks, which was accompanied by induction and deletion of gamma-GTP activity. Morphological differentiation and maturation of the epithelium of the efferent ductuli and the epididymal ducts became evident by the age of 4 weeks and gamma-GTP was active in both epithelial cells throughout the period examined. Since the maturation of rete testis epithelium was found to proceed as observed as Sertoli cells, the origin and functions of the rete testis epithelium and Sertoli cells are considered to be identical. The simultaneous development of the efferent ductuli and the epididymal ducts suggests that both tissues originate from mesonephric ducts and have similar functions, although mature epithelial cells of both tissues are completely different.

The morphology of the extratesticular rete and ductuli efferentes was reexamined in serial cross sections collected from the entire mass of the efferent ductules and in longitudinal sections collected from the partially unraveled efferent ductules. The extratesticular rete forms a 3-4-mm-long sac-like dilatation, which, within the head of the epididymis, has a wide lumen (up to 4 mm) and gives off along its length numerous evaginations, which, in turn, make connections with the ductuli efferentes. The latter is a mass of 16-18 ductules lined by three types of nonciliated cells: type II cells are characterized by dense, periodic acid-Schiff (PAS)-positive granules; type III cells are characterized by empty-appearing, PAS-negative vacuoles; and type I cells lack both granules and vacuoles. The distribution of the three types of nonciliated cells varies along the length. Whereas only type I cells are present in the beginning portion of the efferent ductule, type II cells predominate in the middle portion and type III cells in the distal portion (near the epididymis). The transition from one cell type to the other type is gradual; thus there are short segments along the length that share characteristics first for type I and type II cells and then for type II and type III cells. These results demonstrate that different nonciliated cell types are not randomly distributed in the epithelium of the ductuli efferentes but, instead, gradually differentiate from type I to type II to type III cells along the length of each efferent ductule. Factors controlling this differentiation remain to be studied.

The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.

In 11 testes of different developmental stages (from 10-week-old embryos to adult) the cytokeratin and vimentin expression patterns of rete testis and epididymis were investigated immunohistochemically in formaldehyde-fixed paraffin-embedded material. In addition, immunofluorescence microscopy including double immunofluorescence was performed on frozen sections of 3 of these 11 cases. Rete testis and epididymis cells displayed a heterogeneous co-expression of cytokeratin and vimentin. In double immunohistochemistry, differences in distribution of keratin and vimentin intermediate filaments with predominance of cytokeratins in the apical cytoplasmic regions and of vimentin filaments in the basal portions of the cells were found. Cytokeratin expression preceded the appearance of vimentin: cytokeratin was already detectable in 10-week-old embryos, while weak vimentin immunoreactivity was first seen in 12-week-old embryos and became conspicuous in testes around the perinatal period. In testes of children up to 2 years of age the cytoplasmic distribution of cytokeratin and vimentin was more homogeneous. Predominance of the basal cell portions for vimentin and the apical regions for cytokeratin staining were less pronounced than in adult testes. In the proximal and distal parts of the epididymis a different intermediate filament expression pattern was found with a clear predominance of cytokeratin near the rete.

The efferent ductules of the boar were investigated by means of corrosion casts, light microscopy, scanning and transmission electron microscopy. They arise from an extratesticular rete and constitute the major, caudolateral part of the ascending limb of the caput epididymidis. Ductules may be subdivided into three segments: a slightly undulating testicular segment, a highly coiled intermediate segment and a moderately coiled epididymal segment. A decrease in diameter is particularly marked from the intermediate to the epididymal segment. The epithelial transitions from the extratesticular rete to the efferent ductules and from these to the epididymal duct are clearly demarcated. The epithelium of the efferent ducts consists of principal and ciliated cells. Mononuclear leukocytes are found in the basal half. Ultrastructural evidence supports a strong absorptive activity of principal cells. Apical protrusions are not considered to be a proof of apocrine secretion but rather seem to be artifacts. The nature of membrane-bound granules of variable density remains speculative.

Goat spermatozoal hyaluronidase and acrosin show significantly increased activities during transition from caput to cauda epididymis. The activity of alkaline phosphatase decreases during spermatozoal transport through epididymis.

The rete testis of the goat can be divided into three parts, septal, mediastinal and extratesticular. The septal rete is short, relatively straight and connects the terminal part of the seminiferous tubules with the mediastinal rete. The mediastinal rete is a labyrinth of intercommunicating channels that occupies about two-thirds of the central axis of the testis. The extratesticular rete is located outside the testis at its extremitas capitata and forms sac-like dilations up to 2 mm in diameter. The rete testis, regardless of its location, is lined by simple cuboidal to simple squamous epithelium that invariably contains a few intra-epithelial lymphocytes and macrophages. The epithelial cells possess few microvilli and a centrally located flagellum at the luminal border. With the exception of a few small vesicles in the apical cytoplasm, morphological features associated with absorption such as coated vesicles, canaliculi, vacuoles and multivesicular bodies are absent. However, basolateral interdigitations between adjacent cells, another feature of absorptive epithelium, are frequently noticed. Most cytoplasmic organelles except mitochondria and free ribosomes are poorly developed, suggesting that caprine rete epithelial cells are not associated with protein/glycoprotein secretion. There is no evidence of sperm phagocytosis by the rete epithelium, but luminal macrophages containing sperm fragments are occasionally encountered. The structural-functional relationships of the rete epithelial cells are discussed.

The gross and microscopic features of the normal adult, prepubertal, and aging testis are described. Qualitative and quantitative parameters of spermatogenic activity are presented in practical terms. Emphasis is placed on these findings, which are important to the pathologist in interpretation of diagnostic biopsy specimens other than neoplasms.

The fine structure of cells capable of phagocytosing spermatozoa in the testes and epididymes of monkeys and men was studied by means of transmission and scanning electron microscopy. Both resident cells, such as Sertoli cells and epithelia of the rete testis, and free cells, such as macrophages (spermatophages) were found to phagocytose spermatozoa or fragments of spermatozoa in the seminiferous tubules and excurrent ducts. The spermatophages develop from precursor cells which invade the seminiferous tubules. After phagocytosis of spermatozoa they increase in size and sometimes possess more than one nucleus. Large spherically shaped spermatophages are found in the epididymis. In the rete spaces of Macaca fascicularis spermatophages are able to penetrate the rete epithelia and reside in the subepithelial connective tissue layers. Spermatophagy is frequently seen in the gonads of older men and in certain cases of oligozoospermia. In Macaca an increased number of spermatophages occurs at the end of the spermatogenetic season.

The testicular seminal pathway in the camel is divided into two portions: an intratesticular and an extratesticular portion. The intratesticular seminal pathway consists of three portions: a 'tapering terminal segment' of the seminiferous tubules opening into the receptacle, the tubuli recti with three parts and the rete testis. The extratesticular portion consists of the rete testis, as an extension of the intratesticular portion and an elongated sac from which 6-7 ductuli efferentes originated. The tapering terminal segments are lined by modified Sertoli cells. Both, the tubuli recti and rete testis, are lined by simple cuboidal to columnar cells. The epithelium of the ductuli efferentes which, at its origin from the sac is simply cuboidal, gradually changes to an epithelium that consists of alternating groups of high-columnar ciliated and low-columnar non-ciliated cells. The strongly PAS-positive basement membrane of the ductuli efferentes is surrounded by a thin circular layer of smooth muscle fibres. The pseudo-stratified columnar epithelium of the epididymis is supported by an intensely PAS-reactive basement membrane. Morphological changes in the epithelium of the head and body regions of the epididymis due to seasonal variations are well marked in the camels of all age groups.

Cultures of rete testis epithelial cell-enriched preparations from testes of adult rams have been investigated, and some of their properties have been determined. In monolayers, the cells form mosaic-like borders, and retain many ultrastructural features characteristic of rete epithelial cells in situ, including an indented nucleus with prominent heterochromatin clumps, short rod-shaped or round mitochondria that are easily distinguished from the elongated mitochondria of Sertoli cells, the presence of desmosomes, and few if any lipid droplets or vacuoles. Unlike Sertoli cell-enriched aggregates in culture, rete testis epithelial cell preparations do not form cytoplasmic extensions, and no associated germ cells are present. Rete cells in culture express cytokeratin and vimentin in the cytoskeleton, whereas Sertoli cells prepared from testes of adult rams contain vimentin but not cytokeratin. Both rete cells and Sertoli cells stain positively for laminin but not for fibronectin, Collagen Type I, or Collagen Type III. The rete cells synthesize and secrete several proteins into the culture medium, evident in gel electrophoresis patterns of radiolabeled proteins. This pattern is similar, but not identical, to that secreted by Sertoli cell-enriched preparations. Rete cells in culture in the presence of serum continue to undergo mitotic division, but Sertoli cells do not. A variety of criteria were employed to estimate the relative numbers of Sertoli cells present in the rete testis epithelial cell-enriched preparations from testes of adult rams, including morphological and ultrastructural differences between the two cell types, and the presence of desmosomal proteins and cytokeratin in rete cells but not in Sertoli cells. The relative number of fibroblast-like cells was determined by measuring the expression of fibronectin and Collagen Type I, and an immunocytochemical probe for the detection of Factor VIII was used to estimate the degree of contamination by vascular endothelial cells. Using these markers, we determined that the rete testis epithelial cell-enriched preparations were about 93% pure. Primary cultures under defined conditions contained relatively few Sertoli cells (0.4%), but were contaminated to a larger extent by fibroblast-like cells (approximately 4%) and by endothelial cells (about 3%). The possible functions of rete testis epithelial cells are discussed herein.

Transformation of the rete ovarii into a rete testis and of the epoophoron into an epididymis after experimental sex reversal in Gallus domesticus is described. After castration of female chicks, the medulla of the left ovary and/or the right gonadal rudiment develop into a testoid or an ovotestis. From general view, this is observed as sex reversal. In case of formation of a testoid, the epoophoron develops into an epididymis and the rete ovarii develops into a rete testis which consists of intra- and extratesticular and intracapsular parts. Thus, a luminated duct system is developed which allows the transport of semen. In case of formation of an ovotestis, the discontinuously side-by-side located parts of the rete ovarii and of the epoophoron are maintained.

The epididymal region of the Japanese quail was studied histologically. The organ consists of the extratesticular portion of the rete testis, the ductuli efferentes proximales and distales, the ducti conjugentes and ductus epididymidis. Distinct tubuli recti link the seminiferous tubules with the rete testis. The non-ciliated cells of the ductuli efferentes proximales and distales show, between them, certain internal structural differences which were highlighted. In 40% of the birds, the ductus deferens showed dark-grey pigments, regarded as melanin. The epididymal region was generally similar in structure to that of the domestic fowl, turkey and duck.

A systematic search for endocrine cells in the excurrent duct system of the testis was carried out by means of histochemical and immunohistochemical techniques. A panel of antibodies against amine and polypeptide hormones was used. 80 specimens comprising representative areas of rete testis, ductuli efferentes, ductus epididymis and 30 examples of ductus deferens were investigated. Cells immunoreactive for serotonin were detected in four out of 110 specimens. They were invariably in normal-appearing ductuli efferentes. A salient feature was their rarity and focal distribution. We failed to detect any endocrine cells in other segments of the excurrent duct system and notably not among epididymal epithelium. It seems of interest that serotonin cells are specifically distributed throughout remnants of excretory mesonephric tubules in both males and females.

Rabbit polyclonal antibodies, directed specifically against clusterin purified from ram rete testis fluid, were employed in an investigation of the biosynthesis of clusterin by cultures of rete testis epithelial cells and by Sertoli cells prepared from testes of adult rams. Cells in serum-free medium were incubated in the presence of either [35S]methionine, [3H]leucine, or [3H]glucosamine, and radiolabeled proteins secreted were immunoprecipitated. The pellet was subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and the gels were then fluorographed. In other experiments, protein bands were transferred to nitrocellulose and visualized immunochemically. Under non-reducing conditions, a single band was detected, having a molecular weight of 80,000. Under reducing conditions, doublet bands were detected, having approximate molecular weights of 40,000 (major band) and 37,000 (minor band). These properties were indistinguishable from those obtained with authentic samples of pure clusterin subjected to gel electrophoresis and Western immunoblot procedures. Amounts of clusterin synthesized by rete testis cells in culture, quantitatively determined with a sandwich enzyme-linked immunosorbent assay procedure, were approximately 4 micrograms/micrograms cell DNA/48 h. Immunocytochemical localization investigations, using monoclonal antibodies against clusterin, revealed the presence of clusterin in the perinuclear of juxtanuclear regions, in both rete testis epithelial cells and Sertoli cells in culture. The possible functions of clusterin produced by rete testis epithelial cells and by Sertoli cells are discussed.

Cells considered to be lymphocytes are reported in the epithelial lining of the excurrent ducts of the testis of normal and vasoligated domestic fowl. They resemble those already reported in the rat and monkey epididymal epithelium, the human intestinal mucosa, and in the bursa of Fabricius. The cytoplasm is usually less dense than that of adjacent epithelial cells, and contains only a few organelles. The nucleus is highly heterochromatic and with no definite nucleolus. Cytoplasmic processes are found to extend from the cell in between epithelial cells. The possible role of these cells in the reproductive tract of the cockerel is discussed.

Ram testicular spermatozoa, collected continuously from the cannulated testis, were frozen in rete testis fluid in straws using the cryoprotective agents egg yolk and glycerol. The effect of cryopreservation on the viability of the spermatozoa was assessed by studying their metabolism, morphology, ultrastructure, and radioiodination patterns. Freeze-thawing significantly depressed the respiration rate and glycolytic activity of testicular spermatozoa. Morphologically, there was little evidence of cryodamage in frozen-thawed testicular spermatozoa. Except for some slightly corrugated acrosomes and a more loosely attached plasma membrane over the sperm head, frozen-thawed testicular spermatozoa were indistinguishable from nonfrozen control spermatozoa. Surface radioiodination of frozen-thawed testicular spermatozoa was highly selective and resulted in a labeling pattern similar to that of the nonfrozen controls. In contrast, the radiolabeling pattern of frozen-thawed electroejaculated spermatozoa was characterized by high background radioactivity and low selectivity. These results confirm previous suggestions that testicular spermatozoa have a greater low-temperature tolerance than do ejaculated spermatozoa and indicate that cryopreservation of immature testicular spermatozoa in rete testis fluid with added egg yolk and glycerol may be a useful approach to extend the availability of these cells.

The epididymal region of the guinea-fowl was studied in sexually mature birds. The structure of the epididymal region was generally similar to that already described for the domestic fowl, turkey and Japanese quail. Well formed, intratesticular tubuli recti was seen connecting the seminiferous tubules with the rete testis. The latter consists of both intracapsular and extracapsular portions. Six main cell types were recognised in the region: the rete testis was lined by squamous cells, the proximal efferent ductules by ciliated and non-ciliated Type I cells, the distal efferent ductule by ciliated and non-ciliated Type II cells, and the connecting ductules, ductus epididymidis and ductus deferens were lined by non-ciliated Type III and basal cells. The cell classification adopted in this study is discussed.

The tubules connecting the seminiferous tubules proper to the rete testis in the fowl were studied with the aid of light and electron microscopy. The material examined ultrastructurally was fixed by vascular perfusion through the thoracic aorta. The seminiferous tubules were joined to the rete testis in three different ways; they were either linked by a terminal segment and a tubulus rectus, by a terminal segment only, or opened directly into the rete cavities. The terminal segment of the seminiferous tubules was lined with columnar cells (modified Sertoli cells). These cells were characterized by having an indented nucleus with a prominent nucleolus, many mitochondria, a sizable Golgi apparatus, electron dense bodies and many cytoplasmic protrusions into the lumen. Intraepithelial lymphocytes as well as macrophages in the lumina of the terminal segment, the tubuli recti and the rete testis were also observed. Myoid cells were found in the boundary tissue of the terminal segment.