Morphological features of the epididymal epithelium of gerbil, Meriones unguiculatus

Principal cells of the ducts epididymis of the Mongolian gerbil showed ultrastructural characteristics of lining epithelium cells close related to processes of protein secretion, and transcytosis occurring between adjacent principal cells which were mainly verified in the initial segment. Principal cells also presented roles of fluid phase and adsorptive endocytoses, as well as autophagic and heterophagic lysosomal activities mainly observed in the caput epididymis. Columnar (principal) cells of the corpus epididymidis presented great number of variable vesicles and vacuoles distributed in all the cytoplasmic levels occurring a progressive coalescence pattern among them, which help to guarantee formation of cytoplasmic channels for fluid phase transport between the tubular lumen and epididymal interstitium. Clear cells were presented in the initial segment and predominately in the cauda epididymis epithelium of the gerbil and showed marked ultrastructural characteristics of endocytosis activities occurrence, perhaps directly related to the turnover of fluid phase of spermatozoa stored into the lumen of the distal tail. Other epididymal epithelium cells were verified and described such as basal, halo, apical and dark cells, but they did not presented special ultrastructural features.     

Monoclonal antibodies recognize a cell surface marker of epithelial differentiation in the rabbit reproductive tract

Monoclonal antibodies against the cell surface were produced by immunizing mice with endometrial scrapings prepared from 6-day pregnant rabbits. Spleen cells from an immune mouse were fused with myeloma cells and cultured by standard hybridoma technology methods. Hybridoma supernatants were screened for reaction with the apical epithelial surface by immunohistochemistry on frozen sections of uterus from 6-day pregnant rabbits, and positive colonies were cloned by limiting dilution. Ascites fluid was produced in mice from hybridoma clones that gave a consistent pattern of apical epithelial surface staining through 6 sub-clonings. Antibodies in the ascites fluid were tested by immunohistochemistry on frozen sections of uterus, oviduct, lung, liver and kidney from nonpregnant or 6-day pregnant rabbits. At a dilution of 1:5000, the antibodies recognized an antigen that was specific to the apical surface of luminal but not glandular epithelium of the 6-day pregnant uterus and could not be detected in the nonpregnant uterine epithelium. At higher concentrations of antibody (1:100 to 1:1000), crossreaction was seen with antigens in stromal and myometrial cells of pregnant and nonpregnant uterus. At a dilution of 1:5000, the antibody also crossreacted with some components of lung, liver and kidney but without discriminating between the two reproductive states. In the oviduct, staining of the surface epithelium was specific to the pregnant state. We conclude that this monoclonal antibody has a high affinity for a luminal epithelial cell surface antigen in the reproductive tract of the pregnant rabbit and shows multiple organ reactivity with other tissues that is not affected by pregnancy. This antigen will provide a useful cell surface marker of epithelial differentiation in the progestational reproductive tract.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.     

The fine structure and histochemistry of the caecal epithelium of Calicotyle kröyeri (Monogenea: Monopisthocotylea)

The caecal epithelium of Calicotyle kröyeri consists of a single cell type which functions in the uptake and intracellular digestion of host epidermis and associated mucus. Each cell is columnar with a small basal nucleus and prominent nucleolus. Perinuclear cytoplasm contains narrow profiles of GER and mitochondria with numerous cristae. Golgi complexes are small and indistinct. Most of the cell is filled with vacuoles of heterogeneous content, the largest occupying the cell apex. There is in each cell an apical endocytotic complex comprising cell surface lamellae, apical vesicles and numerous tubular invaginations of the plasmalemma. The limiting membrane of all these components is structurally modified and bears a highly organized array of peg-like structures on its luminal surface. The complex is capable of ingesting particulate food material from the gut lumen for transfer, via vesicles, to the vacuoles for digestion. Most of the vacuoles represent the digestive elements of the cell and, histochemically, are reactive for protein, mucus and carboxylic esterases. Indigestible residues and lipid droplets accumulate in the large apical vacuole and are periodically released to the lumen by exocytosis. Small, undifferentiated caecal cells were occasionally observed in the epithelium, but their development has not been recorded.     

The vaginal surface epithelium during postnatal development in gerbils

The postnatal development of the gerbil vagina has been investigated and special attention has been paid to the luminal surface epithelium structures. Different types of microvilli and solitary cilia are discussed.     

Transcytosis in the epididymis studied by local arterial perfusion

Summary The transport of protein across the cells of the epididymal epithelium was studied using horseradish peroxidase. Transient vascular perfusion of the epididymis of the rat and golden hamster was achieved by pulsatile retrograde infusion into the testicular artery. Peroxidase was found in the interstitium and in the epithelium, located in vesicles, vacuoles and multivesicular bodies of principal, clear and apical cells. Similar findings were obtained in mice after systemic injection of the tracer. In the rat, discharge to the lumen was confirmed by the appearance of enzyme activity in luminal fluid from the caput epididymidis after local injection. The extent of transport amounted to no more than what has been considered leakage in physiological experiments, and the time-course of appearance complemented that found by electron microscopy. The level of transcytosis after pulsatile administration of peroxidase in vivo, as judged from the occurrence of tracer in the epithelium, was much less than that obtained during constant immersion in vitro. The protein was present in multivesicular bodies of principal cells and in vesicles of clear cells at short times after presentation in vitro, when it could not have arrived by endocytosis from the lumen. This suggests that routing of basal endocytic vesicles to the lysosomal apparatus occurs.     

Postnatal development of the Mongolian gerbil uterine glands

The development of gerbil uterine glands has been described. Gland formation starts about day 6 postnatally (p.n.). At day 10 p.n. the glandular lumen contains already some fluid, and the glandular cells are in an activated state. The apical shape of the glandular cells depends mainly on the width of the lumen. Secretory vesicles in early developmental stages are electron-lucent. As sexual maturity is reached, secretory granules become electron-dense. This change in secretory granular structure indicates a change in the quality of uterine gland fluid.     

Regular structures on the microvillar surface membrane of ileal epithelial cells in suckling rat intestine

Freeze-fracture deep-etch studies were done to examine regular patterns of surface membrane particles in the suckling rat ileal epithelium by using quick-freezing method. In addition to the presence on the luminal surfaces of well-developed endocytic complexes, latticed particles were consistently observed on almost the entire apical membrane between microvilli and frequently on the microvilli. These particles seemed to be partly integrated in the outer half layer of membranes. After rinsing with phosphate-buffered saline, patterns on the microvilli appeared to be parallel circle lines rather than latticed particles found in fresh preparations. Filipin treatment showed the presence of filipin-sterol complexes on most of the particle-covered membrane areas except small tubules and vesicles. Possibilities were suggested that these latticed particles were transferred to the apical surface along the outer half layer of membranes, and released or secreted into the intestinal lumen.     

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus)

Summary The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8+1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15–20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells serve as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.     

Growth of separated and recombined neonatal rat uterine luminal epithelium and stroma on extracellular matrix: Effects of in vivo tamoxifen exposure

Summary We have developed a system for serum-free culture of separated uterine epithelium and stroma from 11-day-old rats recombined on extracellular matrix extracted from Englebreth-Holm-Swarm tumors. Epithelium grew and, after 2 days in culture, developed into luminal epithelial spheres (LES) surrounding a fluid-filled lumen. Individual LES cells maintained epithelial cell characteristics such as basally located nuclei, apical microvilli (oriented toward the lumen), lateral membranes with interdigitations and desmosomes, secretory Golgi complexes, and abundant mitochondria and rough endoplasmic reticulum. Secretory vesicles were ubiquitous throughout the luminal fluid. Addition of 17β-estradiol to the growth medium increased the number and longevity of the LES. Prior exposure of uteri to tamoxifen via s.c. injection in vivo on postnatal Days 1 to 5 reduced or completely inhibited formation of LES in vitro. These effects occurred regardless of whether the stromal or epithelial component of the recombinant tissue was exposed to tamoxifen. These data suggest a directive property of neonatal stroma in culture resulting in the formation of highly secretory spherical epithelial structures completely enclosing a lumen. LES formation is responsive to both estrogen (positive response) and antiestrogen (negative response).     

Uterine epithelial-sperm interaction, endometrial cycle and sperm storage in the terminal zone of the oviducal gland in the placental smoothhound, Mustelus canis.

The fate of spermatozoa deposited within the female reproductive tract has been described in the smoothhound, Mustelus canis. Evidence of uterine epithelial-sperm interaction is presented, as well as documentation of sperm storage specifically in the terminal zone of the oviducal gland. Sperm fate is correlated with morphology of the endometrial cycle and specificity of storage in the oviducal gland. The endometrium of M. canis undergoes dramatic tissue remodeling associated with gestation. In females harboring fertilized ova or preimplantation yolk-reliant embryos, the uterine epithelium is simple cuboidal with mucous droplets for lubrication. The presence of the embryo elicits a response from the uterus, which becomes modified for nutrient and respiratory exchange into vascular uterine attachment sites that abut the distal aspect of the yolk sac. Areas of the uterus adjacent to the uterine attachment sites are termed paraplacental sites. Uterine attachment sites are simple squamous while the paraplacental epithelium is simple columnar. Paraplacental cells have basal metachromatic vesicles and a dense array of apical cytoplasmic filaments. Immediately postpartum the uterine attachment sites, now termed uterine or placental scars, begin to remodel to a mucous epithelium for the next gestational cycle. Paraplacental cells slough off the apical filamentous portion, and sperm become embedded in the epithelium. Bundled sperm occur throughout gestation in the terminal zone of the oviducal gland. Sperm are not embedded in the terminal zone epithelium as in the uterus. Following sperm release from the uterus, the paraplacental epithelium reverts to a mucous epithelium for the next reproductive cycle. Fertilization is presumed to occur in the anterior oviduct above the oviducal gland. The physiological mechanisms that mediate sperm-uterus attachment, release, and storage in the terminal zone of the oviducal gland are currently under investigation.     

Structural changes to the uterus of the dwarf ornate wobbegong shark (Orectolobus ornatus) during pregnancy

Embryos of the viviparous dwarf ornate wobbegong shark (Orectolobus ornatus) develop without a placenta, unattached to the uterine wall of their mother. Here, we present the first light microscopy study of the uterus of O. ornatus throughout pregnancy. At the beginning of pregnancy, the uterine luminal epithelium and underlying connective tissue become folded to form uterine ridges. By mid to late pregnancy, the luminal surface is extensively folded and long luminal uterine villi are abundant. Compared to the nonpregnant uterus, uterine vasculature is increased during pregnancy. Additionally, as pregnancy progresses the uterine epithelium is attenuated so that there is minimal uterine tissue separating large maternal blood vessels from the fluid that surrounds developing embryos. We conclude that the uterus of O. ornatus undergoes an extensive morphological transformation during pregnancy. These uterine modifications likely support developing embryos via embryonic respiratory gas exchange, waste removal, water balance, and mineral transfer.     

Vesicular transport of cationized ferritin by the epithelium of the rat choroid plexus

We have studied the transport of ferritin that was internalized by coated micropinocytic vesicles at the apical surface of the choroid plexus epithelium in situ. After ventriculocisternal perfusion of native ferritin (NF) or cationized ferritin (CF), three routes followed by the tracers are revealed: (a) to lysosomes, (b) to cisternal compartments, and (c) to the basolateral cell surface. (a) NF is micropinocytosed to a very limited degree and appears in a few lysosomal elements whereas CF is taken up in large amounts and can be followed, via endocytic vacuoles and light multivesicular bodies, to dark multivesicular bodies and dense bodies. (b) Occasionally, CF particles are found in cisterns that may represent GERL or trans-Golgi elements, whereas stacked Golgi cisterns never contain CF. (c) Transepithelial vesicular transport of CF is distinctly revealed. The intercellular spaces of the epithelium, below the apical tight junctions, contain numerous clusters of CF particles, often associated with surface-connected, coated vesicles. Vesicles in the process of exocytosis of CF are also present at the basal epithelial surface, whereas connective tissue elements below the epithelium are unlabeled. Our conclusion is that fluid and solutes removed from the cerebrospinal fluid by endocytosis either become sequestered in the lysosomal apparatus of the choroidal epithelium or are transported to the basolateral surface. However, our results do not indicate any significant recycling via Golgi complexes of internalized apical cell membrane.     

Uterocalin: A mouse acute phase protein expressed in the uterus around birth

Mouse SIP24/24p3 is a 24 kDa lipocalin expressed in the liver and secreted into the bloodstream during the acute phase response (APR). In this report we show that SIP24/24p3 mRNA and protein are expressed in the uterus around parturition at levels higher than are found in the liver during the APR. Because of the unique expression of this lipocalin in the uterus, we have named this protein uterocalin. Contrary to its expression pattern during the APR, there is little or no expression of uterocalin in the liver during or after pregnancy. Also, unlike the APR, and despite its high level of expression in the uterus, uterocalin was not detected in the blood or amniotic fluid. Day 19 and postpartum uterine samples were examined by immunocytochemistry. Uterocalin was found in the luminal epithelium at day 19 and in the glandular epithelium in postpartum samples. Although some uterocalin remained in the luminal epithelium, most of the uterocalin was found deposited on its luminal surface. The uterus undergoes extensive tissue remodeling during pregnancy and suffers stress and tissue damage around parturition. Uterocalin could be part of the local inflammatory response associated with parturition. Mol. Reprod. Dev. 46:507–514, 1997. © 1997 Wiley-Liss, Inc.     

High‐resolution imaging of the actin cytoskeleton and epithelial sodium channel,CFTR, and aquaporin‐9 localization in the vas deferens

Vas deferens is a conduit for sperm and fluid from the epididymis to the urethra. The duct is surrounded by a thick smooth muscle layer. To map the actin cytoskeleton of the duct and its epithelium, we reacted sections of the proximal and distal regions with fluorescent phalloidin. Confocal microscopic imaging showed that the cylinder‐shaped epithelium of the proximal region has a thick apical border of actin filaments that form microvilli. The epithelium of the distal region is covered with tall stereocilia (13–18 µm) that extend from the apical border into the lumen. In both regions, the lateral and basal cell borders showed a thin lining of actin cytoskeleton. The vas deferens epithelium contains various channels to regulate the fluid composition in the lumen. We mapped the localization of the epithelial sodium channel (ENaC), aquaporin‐9 (AQP9), and cystic fibrosis transmembrane conductance regulator (CFTR) in the rat and mouse vas deferens. ENaC and AQP9 immunofluorescence were localized on the luminal surface and stereocilia and also in the basal and smooth muscle layers. CFTR immunofluorescence appeared only on the luminal surface and in smooth muscle layers. The localization of all three channels on the apical surface of the columnar epithelial cells provides clear evidence that these channels are involved concurrently in the regulation of fluid and electrolyte balance in the lumen of the vas deferens. ENaC allows the flow of Na⁺ ions from the lumen into the cytoplasm, and the osmotic gradient generated provides the driving force for the passive flow of water through AQP channels.     

The maintenance of the permeability barrier of bladder facet cells requires a continuous fusion of discoid vesicles with the apical plasma membrane

The luminal surface of the bladder epithelium is continuously exposed to urine that differs from blood in its ionic composition and osmolality. The apical plasma membrane of facet or umbrella cells, facing the urine, is covered with rigid-looking plaques consisting of hexagonal uroplakin particles. Together with tight junctions these plaques form a specialized membrane compartment that represents one of the tightest and most impermeable barriers in the body. Plaques also occur in the membrane of cytoplasmic discoid vesicles. Here it is shown shown that synaptobrevin, SNAP23 and syntaxin are perfectly colocalized with uroplakin III at the apical plasma membrane as well as with membranes of discoid vesicles. Such a distribution suggests that discoid vesicles in facet cells may gain access to the apical plasma membrane probably by combination of homotypic and heterotypic fusion events. Furthermore, we detected uroplakin III-containing membranes of different sizes in the urine of healthy humans and rats. Probably facet cells maintain their permeability barrier by a process of continuous membrane regeneration that includes the cutting off of areas of the apical membrane and its replacement by newly fused discoid vesicles.     

Structure of the uterine luminal epithelium of the brush-tailed possum (Trichosurus vulpecula)

Morphology and morphometry of the luminal surface of the uterus of the brush-tailed possum were studied during the oestrous cycle, in anoestrous animals and after ovariectomy. At oestrus the secretory cells were small and the epithelium heavily ciliated. The relative surface area occupied by secretory cells reached a maximum on Day 13 when plasma progesterone concentrations are maximal. The mean apical surface area of the secretory cells also reached a maximum at this time. Both these measures decreased on Day 18 when involution of the epithelium was taking place. This process was essentially complete by Day 24 and was followed by extensive ciliogenesis. Secretory cells from anoestrous animals appeared to have an apical surface area similar to the minimum recorded during the oestrous cycle and extensive loss of cilia did not occur. Ovariectomy caused loss of ciliated cells and a reduction in the mean apical surface area to a dimension much smaller than that measured in intact animals.     

Ultrastructure of the full-term shark yolk sac placenta. III. The maternal attachment site

During mid- and late gestation, the uterus of sandbar sharks possesses specialized sites for exchange of metabolites between the mother and fetus. Attachment sites are highly vascular, rugose elevations of the maternal uterine lining that interdigitate with the fetal placenta. The maternal epithelium remains intact and there is no erosion. The attachment site consists of a simple, low columnar juxtaluminal epithelium underlain by an extensive vascular network. Juxtaluminal epithelial cells possess branched microvilli, saccular invaginations of the apical surface, and coated pits. They contain numerous coated vesicles, lipid-like inclusions, a prominent rough endoplasmic reticulum, and many free ribosomes. Tight junctions join the luminal aspect of adjacent cells. Lateral cell boundaries are highly folded and interdigitated. Capillaries are closely apposed to the basal cell surfaces. The endothelium is pinocytotically active. Comparison with the uterine epithelium of non-placental sharks, mammalian epitheliochorial placentae, and selected transporting epithelia reveals that the structure of the maternal shark placenta is consistent with its putative multiple functions, viz: (1) nutrient transfer; (2) transport of macromolecules, e.g., immunoglobulins; (3) respiration; and (4) osmotic and ionic regulation.