Efferent Tubules Research Articles

UNILATERAL SPERMATIC CYST AND GRANULOMA IN THE BOVINE EPIDIDYMIS

In the bull, efferent tubules have gray-green apparence and can be visually defferentiaed from the rest of the tissue in the epididymal. Often, some sperm are trapped and die, relieving mycolic acid to produce a granulomatous reaction. Spermiostasis and sperm accumulation a consequence of the distention of abnormal efferent or epididymal tubules, with secondary formation of sperm granulomas in the region of the epididymal head. This condition is common in male goats and perhaps in sheep, less common in bulls. This study aimed to describe the macroscopic, histopathological and ultrasound aspects of sperm cyst and granuloma in a Sindhi breeder in Belém, Pará.

OP-3 - Immunohistochemical analyses of steroid hormone receptors in the Wolffian remnants of female mice

The developing mesonephros contains the mesonephric duct, mesonephric tubules, and rete cells. In males, they differentiate into the epididymal duct, efferent tubules, and rete testis, respectively. These structures in females cease development and persist as the epoöphoron and rete ovarii. The morphology and function of the female reproductive organs are closely related to steroid hormones. Therefore, we examined the expression of steroid hormone receptors in the epoöphoron and rete ovarii to clarify whether these remnants can react to the steroid hormones in adult female mice. Serial paraffin sections of ovaries collected from adult C57BL/6 J mice were prepared. Androgen receptor (AR), estrogen receptor (ESR1), progesterone receptor (PR), Ad4BP as a marker for the rete ovarii, and Pax2 as a marker for the epoöphoron were detected by immunohistochemistry. Additionally, the rete ovarii and epoöphoron were reconstructed in Amira software from image data of the serial sections. The rete ovarii was positive for Ad4BP at the hilum of ovary, and Pax2 was detected in the epoöphoron within the adipose tissue around the ovary. Pax2 was also found in a part of epithelial cells of the rete ovarii. The Pax2 positive epithelial cells of the epoöphoron and rete ovarii were also positive for AR and ESR1. The PR expression was confirmed in neither the rete ovarii nor epoöphoron. Three-dimensional reconstruction confirmed a convoluting duct, which divided 3-4 branches extending toward the rete ovarii. The Pax2-positive epithelium of the rete ovarii also protruded to the epoöphoron. However, no contact between the rete ovarii and epoöphoron was found in almost all cases. The epithelium of mesonephric tubules, which have connected to the rete ovarii, is suggested to be partially stranded on the rete ovarii during the regression in the female mouse. These epithelial cells left in the postnatal rete ovarii probably retain the sensitivity for androgen and estrogen.

OP-12 - Three-dimensional and immunohistochemical analysis of sex steroid receptors in the developing murine mesonephros

Sex steroids are essential for the development of the reproductive tracts, and their function is mediated by activating their receptors. The expression patterns of the receptors have been investigated in previous studies, but the results differ. One of the reasons is a lack of a method to distinguish each region of the reproductive tracts. To distinguish the cranial and caudal Wolffian duct (WD), which contribute to the common efferent duct and epididymal duct, respectively, it is necessary to check the connection with the mesonephric tubules (MTs) that differentiate into the efferent ductules. In this study, we examined the expression patterns of sex steroid receptors in detail to combine the three-dimensional analysis. Serial paraffin sections of the developing murine mesonephros were prepared, and immunohistochemistry for sex steroid receptors, such as estrogen receptor (ESR1), androgen receptor (AR), and progesterone receptor (PR), was performed. Additionally, the sections in which Collagen Type IV was detected were used to reconstruct the MTs and WD. ESR1 was found in the mesenchymal cells around the WD, and the MTs and cranial WD also became positive from the middle period of the development, but the caudal WD was negative for ESR1. AR was first found at the distal end (gonadal side) of the MTs and weakly in the mesenchymal cells. The epithelial expression became strong in the whole length of the MTs and cranial portion of the WD in the middle period of the development. Finally, the caudal part of the WD also showed strong positive just before birth. PR was weakly positive in the MTs and cranial WD, but the distal end of the MTs where the rete cells were adherent was negative for PR. Androgen secreted from the gonad probably acts on the distal end of the MTs to maintain and develop the efferent tubules. ESR1 and PR may also contribute to the development of the efferent duct. Additionally, estrogen treatment in the neonatal male mice induces epididymal inflammation after puberty, but its mechanisms are still unclear. This study found the ESR1 expression in not epithelial cells but mesenchymal cells around the caudal WD. These results suggest that neonatal estrogen treatment probably affects the mesenchymal cells around the epididymal duct, resulting in the inability to suppress the autoimmune response to spermatozoa after puberty.

Three-dimensional analysis of mesonephric tubules remodeling into efferent tubules in the male mouse embryo.

Spermatozoa are transported to the epididymal duct through efferent tubules. Although the origin of the efferent tubules is thought to be mesonephric tubules (MTs), their detailed developmental process, for example, where the rete testis and efferent tubules are connected, is unclear. We investigated the structural changes of the MTs in the male mouse embryo using a three-dimensional reconstruction method. Three to six MTs were connected to the Wolffian duct, and some of them branched, resulting in five to nine tips. Rete cells contacted the three to six tips. The MTs showed a folded shape when the gonadal fate was determined. After the testis development started, they became short and straight but emerged as long and twisting by birth. Before birth, the efferent duct was composed of MTs and a cranial portion of the folded Wolffian duct. The contact between the rete testis and efferent tubules is possibly established at the tip of each MT. The MTs regress after gonadal fate is determined but is remodeled to the twisting efferent tubules by birth. The efferent tubules are composed of the MTs but also a cranial portion of the folded Wolffian duct in the mouse.

A possible highway system for the rapid delivery of sperm from the testis to the penis in the naked mole‐rat, Heterocephalus glaber

Gametogenesis is suppressed in most members of the eusocial naked mole-rat (NMR) colony, while the queen selects mainly one breeding male during her life span. Recently, it was reported that the NMR testicular organization seems to produce spermatozoa on demand after suppression of spermatogenesis during most of gestation. A Sertoli cell "pump" is then used to flush the spermatozoa into short tubuli recti and simplified rete testis to reach the excurrent duct system. We hypothesize that the components of this duct system are adapted for rapid delivery of spermatozoa to the penis and for numerous copulations with the queen. Therefore, the aim was to study the ultrastructure of the male NMR reproductive duct system using light microscopy and transmission electron microscopy. The NMR rete testis gives rise to six to eight efferent tubules joining the caput epididymis. The caput epididymis resembles that of other rodents but with less distinction in terms of histological zoning. The remainder of the epididymis is considerably reduced in length compared to other rodents. In contrast, the vas deferens epithelium is highly specialized in that a vast range of vesicles, often closely associated with the spermatozoa, were visible. The large ampulla is a factory for merocrine and apocrine secretions, producing even more diverse vesicles. The transitional epithelial cells of the bladder appear to secrete abundant mucous and the penis as well as its baculum is relatively small. We speculate that these modifications strongly suggest that the excurrent duct system has been simplified and adjusted to compensate for the absence of long maturation and storage of spermatozoa. We propose that these adaptations to the NMR reproductive tract are associated with a state of degenerative orthogenesis that was selected for due to the absence of sperm competition and apparently rapid delivery of spermatozoa from the testis.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia)

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

Intra-Seminiferous Tubular Injection of Vascular Endothelial Growth Factor C Sustained-Release Ultrafine Particles: A Novel Method for Improving the Regeneration of Spermatogenesis After Chemotherapy.

Busulfan and other chemotherapeutic drugs used in the treatment of cancer may result in temporary or even permanent damage to spermatogenesis. During spermatogenesis, the rapidly dividing spermatogonia are highly susceptible to chemotherapy. Consequently, there is significant interest in developing an approach that could provide stimulation and regenerate spermatogenesis after chemotherapy. In a previous study, we suggested the potential application for vascular endothelial growth factor C (VEGFC) because of its key role in stimulating the proliferation of spermatogonia. However, methods to facilitate the recovery of spermatogenesis in such patients using VEGFC, or other regulatory factors, are sorely lacking because of the rapid degradation of these proteins and restrictions created by the blood-testis-barrier. To this end, we loaded VEGFC into polyanion dextran sulfate incorporated in a polycation chitosan shell to produce VEGFC sustained-release ultrafine particles (UFPs, CS-DS-VEGFC). We tested such particles in an azoospermic mouse model, created using busulfan. For each mouse, CS-DS-VEGFC was injected into the seminiferous tubules of one testis, while unloaded UFPs (CS-DS), or the VEGFC protein alone, was injected into the opposite testis as a control. All mice were sacrificed and evaluated 5 weeks later. Spermatogenesis in the tubules that were injected with CS-DS-VEGFC was clearly better than those injected with controls, and contained more spermatogonia and spermatocytes, along with Ki67 and PCNA positive-cells per tubule. In addition, the phosphorylation levels of AKT and MAPK in these tubules were also higher than in controls, indicating that CS-DS-VEGFC could induce the sustained activation of these pathways. In conclusion, CS-DS-VEGFC, combined with the efferent tubule injection technique, is a feasible approach with which to improve the regeneration of spermatogenesis in busulfan-induced azoospermic mice.

Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120–240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epthetial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer.

Lesion of the maxillary gland of spirochete-infected brine shrimp: high-voltage electron microscopy of focal epithelial-cell injury

High-voltage electron microscopy of serial sections (0.25 μm thick) was used to find and study lesions of the efferent tubule of the maxillary gland in a spirochete-infected brine shrimp (Artemia franciscana). The efferent-tubule lesion consists of a cluster of spirochetes and small vesicles situated in an indentation of the base of the epithelium and often involves discontinuity of the basal lamina. Of 12 lesions examined, 2 revealed obvious signs of focal epithelial-cell injury. In both cases a large vacuole was located in the epithelium adjacent to the basal cluster of spirochetes and appeared to be a damaged, swollen epithelial-cell process. In the larger of the two lesions the vacuole contained spirochetes, vesicles, altered mitochondria, and abnormal membranous configurations. It is postulated that host-cell injury was initiated by spirochetes entering a cellular process by direct penetration of the plasmalemma (i.e., by diacytosis). Observations of spirochetes inside a damaged process and intracytoplasmic spirochetes devoid of a host-derived membrane support the hypothesis that diacytosis occurred. It is also suggested that each lesion of the efferent tubule originated from one or more spirochetes that migrated from the hemolymph into the basal lamina and underwent replication to form a microcolony beneath the basal surface of the epithelium.

High-voltage electron microscopy of maxillary gland of spirochete-infected brine shrimp: lesion of efferent tubule

Natural populations of the brine shrimp, Artemia, may possess spirochete- infected animals in low numbers. The ultrastructure of Artemia's spirochete has been described by conventional transmission electron microscopy. In infected shrimp, spirochetal cells were abundant in the blood and also occurred intra- and extracellularly in the three organs examined, i.e. the maxillary gland (segmental excretory organ), the integument, and certain muscles The efferent-tubule region of the maxillary gland possessed a distinctive lesion comprised of a group of spirochetes, together with numerous small vesicles, situated in a cave-like indentation of the base of the tubule epithelium. in some instances the basal lamina at a lesion site was clearly discontinuous. High-voltage electron microscopy has now been used to study lesions of the efferent tubule, with the aim of understanding better their three-dimensional structure.Tissue from one maxillary gland of an infected, adult, female brine shrimp was used for HVEM study.

The seminal vesicle of the African catfish, Clarias gariepinus. A histological, histochemical, enzyme-histochemical, ultrastructural and physiological study.

The seminal vesicle of the African catfish, Clarias gariepinus, consists of 36-44 fingerlike lobes built up of tubules in which a fluid is secreted containing acid polysaccharides, acid-, neutral- and basic proteins, and phospholipids. In this fluid sperm cells are stored. The seminal vesicle fluid immobilizes the sperm cells. After ejaculation, it prolongs the period of sperm activity. The seminal vesicle fluid is secreted by the epithelium lining the tubules. The tubules in the proximal part of the lobes are predominantly lined by a simple cylindrical and those of the distal part by a simple squamous epithelium. These epithelial cells contain enzymes involved in energy-liberating processes, the enzyme activities being proportional to the height of the cells. Interstitial cells between the tubules have enzyme-histochemical and ultrastructural features indicative of steroid biosynthesis. Similar characteristics are found in testicular interstitial cells. The most rostral seminal vesicle lobes and the most caudal testicular efferent tubules form a network of tubules that opens at the point where the paired parts of the sperm ducts fuse with each other. The tubules of most seminal vesicle lobes, however, form a complex system that fuses with the unpaired part of the sperm duct.

The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study

The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct. At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall. The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis. The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.

Utilization of the chick chorioallantoic membrane for in vitro growth of the embryonic murine kidney.

Previous experimental investigations of embryonic kidney development with in vitro organ-culture techniques have been limited by the short duration of graft viability and lack of detailed anatomic development. In a modified application of the chick chorioallantoic-membrane (CAM) grafting technique, we have transplanted 11-day-old undifferentiated embryonic murine kidney rudiments and studied their differentiation and early morphogenesis. Surviving grafts, maintained for 5-10 days on the CAM, demonstrated well-developed convoluted secretory tubules and highly branched collecting ducts. Definitive glomeruli were also identified in these grafts by the presence of efferent tubules, visceral and parietal epithelium, capillary tuft, and Bowman's space. In vitro branching of the ureteric bud preceded formation of definitive tubules. Sections of the CAM grafts appeared histologically comparable to the 14-15-day in vivo embryonic metanephros. Grafts of 10-day-old rudiments showed minimal kidney development; fewer grafts survived, fewer tubules were formed, and fewer glomeruli developed. Our results indicate that the CAM is an efficient site for in vitro murine kidney development with both vascularization of the explant and extensive maturation, including the appearance of primitive nephron units.

Host-Parasite Interrelationships Between the Freshwater Pulmonate Biomphalaria Pfeifferi and the Trematode Schistosoma Mansoni

The influence of infection with S. mansoni on reproduction, growth, food consumption and survival in B. pfeifferi was studied experimentally (Ch. II). The prepatent period in infected snails was shown to last 22-24 days under the conditions prevailing in the relevant experiment. The number of eggs laid by infected snails as compared with controls was significantly reduced from 7-10 days postinfection onwards. After day 13 postinfection it was completely suppressed in the majority of the infected snails. The increase in shell diameter in infected specimens was larger than that of the controls between 7 and 18 days, that in weight between 11 and 21 days postinfection. Gigantism did not occur. There was no difference in mortality between infected and control snails prior to the 11th week after the beginning of the experiment. After that, the mortality among infected specimens showed a sharp rise. Uninfected and infected snails consumed similar amounts of food (dried lettuce) in the first two weeks of the experiment. After that, infected snails consumed less food than controls. The digestive gland of B. pfeifferi was studied with histological, histochemical and ultrastructural methods (Ch. III). The highly folded epithelium of the ducts connecting the digestive gland with the pyloric part of the stomach, consists of cilia and/or microvilli bearing columnar cells with interspersed mucous cells. In the epithelium of the digestive gland two main cell types are distinguished: the digestive cell and the secretory cell. In addition some mucous cells occur. It is concluded that the main functions of the digestive cell are absorption and endocytosis of predigested food material, followed by intracellular digestion. Indigestible residues are accumulated in a large vacuole, which eventually is excreted. The secretory cell produces and secretes proteinaceous substances, presumably digestive enzymes. It does not store calcium. The so-called excretory cells, which are in fact degenerating secretory cells, have vacuoles containing yellow globules. These yellow globules together with cell debris are finally released into the lumen of the digestive gland. The observations indicate that the digestive gland is not important as a storage organ for reserve material. The lobules of the digestive gland are covered by a thin connective tissue sheath, consisting of ground substance, collagen-like fibrils, smooth muscle cells and cells with the appearance of pigment cells. The connective tissue between the lobules of the digestive gland and the acini of the ovotestis, and between these organs and the mantle, consists mainly of vesicular connective tissue cells. These cells are supposed to play an important metabolic role as storage cells for glycogen. The tegument of the daughter sporocyst of S. mansoni (Ch. IV), consisting of an outer syncytial anucleate layer which is joined by cytoplasmic connections to nucleated cell bodies (tegumental cells), has the same basic architecture as that described for various stages of trematodes and cestodes. Well-developed mitochondria, GER cisternae and tubules, free ribosomes and polysomes, and lipid droplets occur throughout the tegument. The ultrastructural features suggest that the outer layer is involved in uptake of nutrients by absorption and endocytosis. The poorly developed musculature is loosely arranged in two layers beneath the outer tegumental layer. The nucleated portions of the muscle fibres are situated between the tegumental cells. The protonephridial system consists of flame cells with efferent tubules. The bundle of cilia, implanted in the flame cell, beats in the cilia chamber or barrel. The proximal part of the barrel consists of alternating rib-like extensions of the flame cell and of the first tubule cell, respectively. Ultrafiltration probably takes place in the ribbed part of the barrel. The development of cercariae from germinal cells is briefly described. The pathological effects of infection with S. mansoni on the digestive gland epithelium of B. pfeifferi (Ch. V) are limited. From about 12 weeks after infection slight changes occur, but they are difficult to distinguish from changes due to ageing of the cells. The effect of starvation on the digestive gland epithelium is much more marked than the adverse influence of infection. Compression of digestive gland lumina by daughter sporocysts was not observed. Changes in the connective tissue of the digestive gland due to the presence of daughter sporocysts were: development of muscle cells into cells with the appearance of pigment cells and/or degeneration of these muscle cells, and an increase in the number of amoebocytes, which become involved in the elimination of cell debris and in the formation of a loose layer over the parasites.

Intercoil connections of the kidney of the brine shrimp, Artemia salina.

Maxillary glands (kidneys) of adult brine shrimp were examined by light and electron microscopy. Within a single maxillary gland there are numerous places where adjacent coils of the efferent tubule are linked by structures designated “intercoil connections”. At the site of such a connection the basal laminae of the two coils are continuous, and one or more cytoplasmic processes originating from a cell of one coil form a bridge to the adjacent coil. The connecting processes are branched and interdigitate complexly with complementary processes from cells of the neighboring coil. Speculations are made about the functional significance of intercoil connections in the maxillary gland of Artemia.

Quinone and hydrocarbon production in the defensive glands of Eleodes longicollis and Tribolium castaneum (Coleoptera, Tenebrionidae)

The defensive glands of tenebrionid beetles produce a mixture of benzoquinones and hydrocarbons. These reactive toxicants are manufactured within a complex morphological system, consisting of secretory cells, secretory vesicles within these cells, and cuticular organelles and tubules which carry the products to the gland reservoir. Histochemical and thin-layer chromatographic techniques were employed to follow the process of toxicant production within these cells. Chromatographic evidence indicated the presence of both p-diphenols and their glucosides in extracts of the secretory cells, which suggested that quinones are generated from phenol glucosides by hydrolysis and subsequent oxidation. Histochemical tests demonstrated concentrations of phenolic materials in the cytoplasm of one cell, type 2a, and on the basis of their sensitivity to emulsin, it was concluded that these are phenol glucosides. Glucosidase activity, as shown by a modified bromonaphthol-glucoside technique, was associated with the margins of the vesicle in the same cell type. The cuticular organelle, within this vesicle, yielded positive reactions for a phenolase both with dopa and with N-phenyl- p-phenylene diamine as substrates. Along the efferent tubule which runs from this organelle to the gland reservoir, a haemoprotein peroxidase was detected by the benzidine peroxidase test. Each of these steps in quinone production was associated with a particular morphological compartment, and it is apparent that the cytoplasm of the secretory cell is never exposed to the toxic product of the secretory unit. Lipid reserves, which are possible precursors of the hydrocarbon components in the secretory end-products, were found in all secretory cells. However, the densest deposits were concentrated in the type 1 units, and in these units, a paraoxon-resistant carboxylic esterase was demonstrated with indoxyl substrate, along the margins of and within the secretory vesicle. Deposits of unsaturated lipids were detected in the cuticular organelle of this vesicle.

Further Effects of Androgenic Substance on Sexual Development in the Female White Rat

SummarySixty-seven intersexed rats, produced by injecting the pregnant rat with testosterone, have been examined. Testosterone given to the pregnant rat may in some instances arrest the fusion of the caudal portions of the mullerian ducts and the unfused portions are patent and open into the urethra. In addition, a number of animals have been produced in which wolffian duct derivatives, efferent tubules, epididymis, vas deferens and seminal vesicles are present in association with a fallopian tube, uterus and the upper portion of the vagina and with masculinization of the female gonad.