Morphological examination of epididymal epithelium in the mule (E. hinnus) in comparison with parental species (E. asinus and E. caballus).

Following previous studies about the ultrastructure of male genital tract in parental species, a comparative study of epididymis of one of the possible hybrids, the mule, has been undertaken. Apart from small differences, general features of epididymal epithelium in the mule are similar to those of parental species. However, extension of our studies from the donkey to the horse to the hybrid permits a deeper insight into the morphology of this tract of excurrent duct. In the meantime, it is possible to evidence some features, sometimes shared with other species if taken separately, which in the whole characterize the epididymis in Equidae: the presence in principal cells of intranuclear inclusions and peculiar small granules in the basal cytoplasmic edge; the organization of groups of cells, likely to be principal ones, in such a way as to constitute intraepithelial crypts; a cumbersome presence of lipofuscinic matter all along the epithelium. Another interesting observation in the presence in the mule epididymis of well recognizable macrophages. All these data are discussed in comparison with parental species and with other species described in literature. Beyond any other consideration, it can be outlined that the complex morphology of the epithelium lining ductus epididymis in the mule is unaffected by the absence of spermatozoa, which are normally the target of the manifold functions of the epithelium itself.     

Immunolocalization of androgen receptor in the epididymis of rats with dihydrotestosterone deficiency

Dihydrotestosterone (DHT), 5alpha-reduced metabolite of testosterone, is the most potent androgen in the epididymis. The conversion of T into DHT is carried out by 5alpha-reductase. The activity of 5alpha-reductase type 2, preferentially expressed in the epididymis can be inhibited by a finasteride (a steroid-based specific inhibitor of 5alpha-reductase type 2) which results in DHT deficiency. The aim of the study was to examine the morphology of epididymis and the immunolocalization of an androgen receptor (AR) in the initial segment, caput and cauda epididymis of rats treated with finasteride for 56 days. There were no morphological changes in the morphology of epididymal epithelium in the experimental rats. Immunostainable AR was localized in nuclei of epithelial cells, smooth muscle cells and mainly in the cytoplasm of interstitial cells in the epididymis of control rats. In the epididymis of experimental rats, AR immunostaining was noticed mainly in the cytoplasm of epithelial cells and interstitial cells. The single cells of the initial segment epithelium, basal cells and smooth muscle cells of cauda epididymis showed nuclear AR staining. In conclusion, finasteride affected the expression of the AR in the rat epididymis without changing the morphology of epididymal epithelium. Altered AR expression reflected the hormonal status within the epididymis.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Epithelial crypts: A complex and enigmatic olfactory organ in African and South American lungfish (Lepidosireniformes,Dipnoi)

African lungfish (Protopterus ) seem unique among osteognathostomes in possessing a potential vomeronasal organ homolog in form of accessory epithelial crypts within their nasal cavity. Many details regarding structural and functional properties of these crypts are still unexplored. In this study, we reinvestigate the issue and also present the first data on epithelial crypts in the South American lungfish Lepidosiren paradoxa . The nasal cavities of L. paradoxa and Protopterus annectens were studied using histology, scanning electron microscopy, and alcian blue and PAS staining. In both species, the epithelial crypts consist of a pseudostratified sensory epithelium and a monolayer of elongated glandular cells, in accordance with previously published data on Protopterus . In addition, we found a new second and anatomically distinct type of mucous cell within the duct leading into the crypt. These glandular duct cells are PAS positive, whereas the elongated glandular cells are stainable with alcian blue, suggesting distinct functions of their respective secretions. Furthermore, the two lungfish species show differently structured crypt sensory epithelia and external crypt morphology, with conspicuous bilaterally symmetrical stripes of ciliated cells in L. paradoxa . Taken together, our data suggest that stimulus transport into the crypts involves both ciliary movement and odorant binding mucus.     

A stem cell niche dominance theorem

Background  

Multilevelness is a defining characteristic of complex systems. For example, in the intestinal tissue the epithelial lining is organized into crypts that are maintained by a niche of stem cells. The behavior of the system 'as a whole' is considered to emerge from the functioning and interactions of its parts. What we are seeking here is a conceptual framework to demonstrate how the "fate" of intestinal crypts is an emergent property that inherently arises from the complex yet robust underlying biology of stem cells.     

Specific distribution of an epididymal 50 kDa protein revealed by an anti-Mos protein monoclonal antibody.

An anti-Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c-mos gene is reportedly expressed. The 4A6-reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell-deficient C57BL/6J-Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c-Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c-Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis.     

Growth and characterisation of primary bovine colon epithelial cells in vitro

Epithelial crypts from the bovine colon were obtained by using a combined mechanical and enzymatic isolation method, followed by differential D-sorbitol gradient centrifugation. By using this isolation technique, a pure fraction of epithelial crypts with minimal mesenchymal contamination was obtained. The crypts were seeded in collagen-coated plastic flasks. The attached epithelial cells proliferated and formed a confluent monolayer after 6 days in culture. Under low-serum culture conditions (1% fetal calf serum), the cells had a population doubling time of 21-22 hours. During the culture period, the colonocytes were characterised morphologically and enzymatically. The morphology of the cultured cells was confirmed by scanning electron microscopy and transmission electron microscopy. The presence of microvilli, tight junctions and desmosomes demonstrated the ability of the cultured cells to restore an epithelial-like cell monolayer. The epithelial origin of the cells was demonstrated by labelling the cells with antibodies against epithelial-specific cytokeratins 7 and 13. The functional integrity of the cells was evaluated by measuring various marker enzymes (gamma-glutamyltranspeptidase, acid phosphatase, alkaline phosphatase, NADH-dehydrogenase) and membrane-associated Na+-K+-ATPase activity. Membrane integrity was determined by measuring the leakage of lactate dehydrogenase into the culture medium. This new culture system for bovine colon epithelial cells could be used as an in vitro model of the colon epithelium in physiological and toxicological studies.     

Seasonal changes in epididymis and the effects of FSH and testosterone in the spiny-tailed lizard,Uromastix hardwicki

Seasonal changes in epididymal weight and histology were studied in relation to testicular function in the adult spiny-tailed lizard, Uromastix hardwicki, over a period of 1 year. The eipdidymal weights, tubular diameter, and epithelial height increased in March, reaching a peak in April. This peak coincided with sperm maturation, elevated plasma testosterone levels, and release of sperm into the epididymis. The epididymal weights decreased in May following a sudden regression of the testis early in the month. The epididymal weights decreased further during June and remained low until February. The diameter of the duct and the height of the epithelial cells also decreased in May and the epididymal epithelium maintained a low histological profile from June to February. The fall testicular recrudescence was not accompanied by a change either in the weight or the histological structure of the epididymis. Administration of oFSH (0.1 mg) daily for 7 days during the sexually quiescent period induced a significant increase in the weight of the epididymis and epithelial height of the duct. Administration of testosterone alone, (2.0 mg) daily for the same period and under identical conditions, did not induce a change in the weight of epididymis or its histology. A possible permissive role of gonadotrophin in the hormonal regulation of the lizard epididymis has been suggested.     

Postnatal study on the histochemistry of epididymis in buffalo (Bubalus bubalis).

The histochemical localisation and development of carbohydrates, acid mucopolysaccharides, lipids and desoxyribonucleic acid has been determined in the head, body and tail segments of buffalo epididymis at different stages of postnatal development from 3-76 weeks of age. The cytoplasm contains numerous Schiff-positive, diastase-resistant granules which are abundant in the apical region of the epithelial cells at 3-52 weeks. They decrease in number at 72-76 weeks and instead, a diffuse Schiff-positive material is observed in the cytoplasm of the cells, particularly in regions III-VI of the epididymis. In all the stages of development, the basement membrane and the luminal border of the epithelial cells have strong PAS-positive reaction. The intertubular connective tissue is mildly PAS-reactive, with moderate activity on the endothelial lining and smooth muscles of blood vessels. The capsule shows an intense Schiff-positive material in the fibers while the stereocilia are mildly reactive. PAS activity is less in region I and greater in regions V-VI as compared with the other regions of the epididymis. A moderate quantity of lipids is present in the epithelial cells and smooth muscles of the tubules. Sudanophilia is more pronounced in the tail region as compared with the head and body regions of the epididymis. Acid mucopolysaccharides are present, minutely, in the epithelial cell cytoplasm, with moderate activity on the stereocilia of the tubules. The Feulgen reaction is deep in region I and light to moderate in the other regions of the epididymis.     

Phagocytosis of sperm cytoplasmic droplets by a specialized region in the epididymis of the brushtailed possum, Trichosurus vulpecula

During sperm maturation in the brushtailed possum, Trichosurus vulpecula, cytoplasmic droplets are shed from maturing spermatozoa in the distal regions of the head of the epididymis. Examination of luminal contents from various regions of the epididymis showed that the proportion of detached droplets in the luminal contents was reduced from about 45% in the proximal corpus epididymidis to less than 10% in the distal corpus and cauda epididymides. In contrast, the proportion of droplet-free spermatozoa increased from about 45% to more than 90% in the luminal contents. Disappearance of detached cytoplasmic droplets from the lumen was found to be associated with a region of specialized principal cells lining Regions 6 and 7 of the epididymis which selectively sequester and phagocytose free droplets from the luminal milieu. The luminal surfaces of these cells are characterized by a complex system of interdigitating processes which appear as waves of microfolds . These processes contrast with the stereocilia which cover the luminal surfaces of principal cells in adjacent, nonphagocytic regions of the duct. Cytoplasmic droplets are phagocytosed with their limiting membrane intact and gradually become condensed as they are transported deeper into the cell. Membrane lamellae are gradually compacted, transformed into concentrically arranged membrane stacks and then condensed into small electron-dense vesicles, which are probably degraded by the epithelial cells. The presence of a specific recognition factor on cytoplasmic droplets is suggested by the observation that phagocytic principal cells are able to selectively remove detached cytoplasmic droplets from the lumen in the presence of sperm-associated droplets and spermatozoa.     

Peroxisomes and ether lipid biosynthesis in rat testis and epididymis

Plasmalogens are a main component of the spermatozoon membrane, playing a crucial role in their maturation. The initial steps in plasmalogen biosynthesis are catalyzed by two peroxisomal enzymes, dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase. The localization of both enzymes in the membrane of peroxisomes implies that plasmalogen-producing cells should contain this organelle. To unravel the putative source of spermatozoan plasmalogens we investigated which cell types in the testis and epididymis are endowed with peroxisomes. To this extent, testicular and epididymal tissue was analyzed at the protein and RNA levels by means of light and electron microscopical immunocytochemistry as well as by Western and Northern blotting. Proteins and mRNAs of peroxisomal enzymes, especially those of dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, were detected in the testis and epididymis. In the testis, peroxisomes were localized exclusively in Leydig cells and not in cells of the seminiferous tubules, implying that the latter do not contribute to the biosynthesis of plasmalogens of the sperm membrane. In contrast, peroxisomes could be clearly visualized in the epithelial cells of the epididymis. The results suggest that peroxisomes in epithelial cells of the rat epididymis play a pivotal role in the biosynthesis of plasmalogens destined for delivery to the sperm plasma membrane.     

Glutathione peroxidase-3 produced by the kidney binds to a population of basement membranes in the gastrointestinal tract and in other tissues

Glutathione peroxidase-3 (Gpx3), the extracellular glutathione peroxidase synthesized largely in the kidney, binds to basement membranes of renal cortical epithelial cells. The present study assessed extrarenal expression of Gpx3 using RT-PCR and presence of Gpx3 protein using immunocytochemistry. Gpx3 expression was higher in kidney and epididymis than in other tissues. Gpx3 bound to basement membranes of epithelial cells in the gastrointestinal tract, the efferent ducts connecting the seminiferous tubules with the epididymis, the bronchi, and type II pneumocytes. It was not detected on the basement membrane of type I pneumocytes. Gpx3 was also present in the lumen of the epididymis. Transplantation of Gpx3(+/+) kidneys into Gpx3(-/-) mice led to Gpx3 binding to the same basement membranes to which it bound in Gpx3(+/+) mice but not to its presence in the epididymal lumen. These results show that Gpx3 from the blood binds to basement membranes of specific epithelial cells and indicate that the cells modify their basement membranes to cause the binding. They further indicate that at least two Gpx3 compartments exist in the organism. In one compartment, kidney supplies Gpx3 through the blood to specific basement membranes in a number of tissues. In the other compartment, the epididymis provides Gpx3 to its own lumen. Tissues other than kidney and epididymis express Gpx3 at lower levels and may supply Gpx3 to other compartments.     

Fermentable dietary fiber potentiates the localization of immune cells in the rat large intestinal crypts

Intestinal crypts are composed of a well-defined hierarchy of epithelial cells, and proliferating epithelial cells reside close to the bottom of the crypts-even in the large intestine. We investigated whether CD8(+)and CD4(+)intraepithelial lymphocytes (IELs) and CD161(+) natural killer (NK) cells localized in proliferating or differentiated epithelial region of cecum and colon. Both proliferating epithelial layer cells and the immune cells along the longitudinal crypt axis of the large intestine were measured histochemically. Dietary intervention revealed that the physiological localization of the immune cells in the longitudinal crypt axis depended on the immune cell type. CD8(+) IELs were preferentially located among differentiated epithelial cells. In contrast, CD161(+) NK cells were located adjacent to the epithelial cells at the bottom of crypt. Cecal crypts contained significantly larger numbers of CD8(+) IELs than did colonic crypts. However, there was only a minor population of CD4(+) IEL in the cecal and colonic epithelia. Some dietary fibers increased the densities of CD8(+) IELs and CD161(+) NK cells in the cecum, with the magnitude of response varying among the types of fiber. There was a significant relationship between SCFA and the localization of immune cells, especially CD8(+) IEL and CD161(+) NK cells, which are considered to be involved in the maintenance of epithelial homeostasis.     

Isolation of hamster intestinal epithelial cells using hypoosmotic media and PVP

Vibration of hamster small intestinal segments in hypotonic media containing PVP is a rapid method for obtaining quantitative yields of viable intestinal epithelial cells. This preparation of epithelial cells offers a unique system for the study of epithelial cell function in vitro. The method for cell separation combines hypoosmotic swelling of cells, which separates them at the desmosomes, with mechanical agitation which releases the cells from the lamina propria. No chemical agents known to affect cell proteins and cell surfaces are employed in this procedure. Only a short time is elapsed between in vivo and in vitro conditions, i.e., a preparation time of approximately 75 minutes. Although the technique yields a pure population of epithelial cells, the cells are of different morphologies, are removed from different areas of the crypts and villi, and therefore presumably have different functions. Examination of the intestinal tissue remaining after several vibration intervals by light and scanning electron microscopy indicates that the sequences of release of cells is removal of: (1) cells from the villus based, (2) cells from the lower one-half to two-thirds of the villi, (3) cells from the villus tips (and some crypts), and (4) cells from the crypts. When pools of a + b cells are compared to pools of c + d cells, it is found that villus cells can be characterized by: (1) processes, such as monosaccharide absorption, associated with the brush border, and (2) synthesis of components (e. g., glycoproteins) of the brush border. Surprisingly, disaccharide hydrolytic activity is found in cells which transport monosaccharides poorly. The subpopulations of cells synthesize proteins equally.     

Impact of the Processes of Total Testicular Regression and Recrudescence on the Epididymal Physiology of the Bat Myotis nigricans (Chiroptera: Vespertilionidae)

Myotis nigricans is a species of vespertilionid bat, whose males show two periods of total testicular regression within the same annual reproductive cycle in the northwest São Paulo State, Brazil. Studies have demonstrated that its epididymis has an elongation of the caudal portion, which stores spermatozoa during the period of testicular regression in July, but that they had no sperm during the regression in November. Thus, the aim of this study was to analyze the impact of the total testicular regression in the epididymal morphophysiology and patterns of its hormonal regulation. The results demonstrate a continuous activity of the epididymis from the Active to the Regressing periods; a morphofunctional regression of the epididymis in the Regressed period; and a slow recrudescence process. Thus, we concluded that the processes of total testicular regression and posterior recrudescence suffered by M. nigricans also impact the physiology of the epididymis, but with a delay in epididymal response. Epididymal physiology is regulated by testosterone and estrogen, through the production and secretion of testosterone by the testes, its conduction to the epididymis (mainly through luminal fluid), conversion of testosterone to dihydrotestosterone by the 5α-reductase enzyme (mainly in epithelial cells) and to estrogen by aromatase; and through the activation/deactivation of the androgen receptor and estrogen receptor α in epithelial cells, which regulate the epithelial cell morphophysiology, prevents cell death and regulates their protein expression and secretion, which ensures the maturation and storage of the spermatozoa.     

Regeneration of nasopharyngeal epithelium in Macaca fascicularis.

Regeneration of the nasopharyngeal epithelium in Macaca fascicularis occurs as a result of migration of epithelial cells from the margins of the lesions as well as from the neighbouring glandular ducts and epithelial crypts. The study further reveals that the basal cells are the progenitors of both goblet and ciliated cells. The regenerating epithelium at first consists of mucus-containing cells which are finally converted into normal globlet and ciliated cells. The formation of centrioles and concurrent reduction in the amount of 'mucus' droplets, and rearrangement of centrioles towards the luminal surface of the cells along with simultaneous development of cilia in some of these mucus-containing cells are stages in the differentiation of ciliated cells. However, some cells which do not possess secretory droplets may also develop into ciliated cells directly.     

Mode of growth of the jejunal crypt cells of the rat: an autoradiographic study using double labelling with 3H- and 14C-thymidine in lower and upper parts of crypts.

The frequency distribution of cells through the mitotic cycle in lower and upper portions of jejunal crypts of the rat was examined by the 3H-14C-thymidine double labelling technique. Isolated crypts were cut perpendicular to the longitudinal axis so that the percentage of cells in the lower portion varied from 16 to 74%. The lower and upper portion of the same crypt were squashed separately on one microscope slide and the number of 3H- and 14C-only labelled cells were scored to determine the flow rate into and out of S for the two portions. The mitotic cycle and its phases of the crypt epithelial cells were also determined. For lower portions of crypts which contained less than 40% of the total cell number in that crypt the flow rate into S was about 1-7 times that of the flow rate out of S indicating that nearly every mitosis in this region produced two proliferative daughter cells. As the proportion of cells in the lower part of the crypt increased the quotient of the flow rate into S divided by the flow rate out of S decreased, and approached the steady state value of 1-0 in lower portions containing 60-74% of the cells. For upper portions of crypts which contained less than 40% of the total crypt cells the flow rate into S was about 0-2 times that of the flow rate out of S, indicating that in this region mitoses predominantly produced non-proliferative daughter cells. The results obtained were in good agreement with the model of crypt cell proliferation proposed by Cairnie, Lamerton & Steel (1965b).