Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human

Blood supply is essential for the maintenance of epididymal function. Since there is no considerable neovascularization in the epididymis, this tissue could represent a suitable model to study the vascular endothelial growth factor (VEGF) effect for vascular permeability. We studied the expression and function of VEGF and its receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase (designated as kinase insert domain-containing receptor, KDR in the human) in the human epididymis. VEGF and VEGF receptors mRNA were detected in the human epididymal tissue. VEGF protein was localized in peritubular and in ciliated cells of efferent ducts as well as in peritubular and basal cells of the epididymal duct. Vascular endothelial cells did not express VEGF. Flt-1 protein was localized in ciliated cells of efferent ducts and in lymphatic vessels. Vascular endothelial cells were negative for Flt-1 but positive for KDR. In vitro VEGF165 treatment of epididymal tissue induced endothelial fenestrations and opening of interendothelial junctions. Additionally, we observed for the first time that VEGF could induce transendothelial gaps. We conclude that these gaps might be of importance not only for molecular transport but also for cell passage across the vessel wall, which may be significant for tumor metastasis. VEGF may act as a paracrine effector to influence the permeability of lymphatic vessels via Flt-1, and of blood vessels via KDR.     

Papillary cystadenoma of the epididymis. An ultrastructural and immunohistochemical study

Papillary cystadenoma of the epididymis is a rare neoplasm that is sometimes associated with von Hippel-Lindau's syndrome. Electron microscopic study of the present case revealed that neoplastic cells contained abundant glycogen granules and large lipid droplets, but a few organelles. On the apical surface there were numerous microvilli and a few single cilia, but no ciliated cells. Subepithelial basal lamina was noted, but it was occasionally disrupted. Furthermore, microvilli sprang from the circumference of the small tumor-cell nest and became associated with matrix components (microvillus-matrix associations). On immunohistochemical study, neoplastic cells showed epithelial characteristics, but positive reactivity for S-100 protein. These findings resembled those of the epithelial cells of the efferent ductules of the epididymis. In the stroma, prominent vasculature was characteristic and fenestrated-type capillaries were found in the peripheral portion of the tumor. Papillary cystadenoma of the epididymis may originate from non-ciliated epithelial cells of the efferent ductules.     

Preoperative treatment and survival of patients with pheochromocytomas

A 9-year-old male Shetland Sheepdog had a small mass in the left testis. Grossly, the round to oval cyst was present at the upper pole of the testicular parenchyma near the head of the epididymis. Histologically, the cyst was lined by a single layer of nonciliated and ciliated epithelial cells. Immunohistochemically, the epithelial cells of the cyst showed expression of the low- and high-molecular-weight cytokeratins, vimentin, and desmin similar to that of normal efferent ductules in the dog. The testicular cystic dysplasia was thought to originate from the efferent ductules.     

Morphology and function of rooster efferent ductule epithelial cells in culture

Regulation of the excurrent ducts of the testis is not well understood, particularly in avian species. To investigate the role of steroid hormones in the male reproductive tract, we developed a primary cell culture of epithelia isolated from rooster ductuli efferentes (efferent ductules). Efferent ductules of the avian testis comprise 77% of the epididymal region and form a mass of tubules containing a heavily folded epithelium enmeshed in connective tissue. The epididymal region was separated by microdissection and small epithelial plaques isolated by serial digestion with collagenase, elastase and repeated pipetting. Isolated cell plaques were cultured in a bicameral chamber on Millicell-CM inserts coated with two layers of basement membrane matrix, consisting primarily of laminin and Types I and IV collagen. Active ciliary beat was observed before plating and this activity was maintained for 14 days in culture. Cell plaques attached within 24 h and outgrowths formed a confluent monolayer by 5-6 days. The epithelial nature of cultured cells was demonstrated by immunocytochemical staining for cytokeratin. Light and electron microscopy confirmed that morphology and polarity of the original epithelial cells were maintained in culture. Cultured efferent ductal epithelium was cuboidal in shape and maintained many of the cytoplasmic organelles typical of these cells in vivo. The uptake of cationic ferritin indicated the endocytotic activity of these cultured cells was maintained. Estrogen receptor mRNA expression was maintained in cultured cells. These data demonstrate avian efferent ductal epithelium can be isolated and grown in defined culture medium for the purpose of determining the role of hormones and other factors in regulating the function of the epididymal region in the bird.     

Location of a membrane-bound carbonic anhydrase isoenzyme (CA IV) in the human male reproductive tract

We studied the location of a membrane-bound carbonic anhydrase (CA IV) in the human male reproductive tract using a specific antiserum to human CA IV in conjunction with immunoblotting, immunoperoxidase, and immunofluorescence techniques. The microvilli and apical plasma membrane of the epithelial cells and the subepithelial smooth muscle layer of the epididymis, ductus deferens, and ampulla of the ductus deferens showed specific staining for CA IV. The epithelial cells of the prostate and seminal vesicle failed to stain for CA IV, however, whereas the subepithelial smooth muscle layer showed positive staining. No specific staining for CA II was seen in the epithelium of the epididymal duct or the proximal ductus deferens. The presence of CA IV in the epididymis was confirmed by immunoblotting, which revealed 35 KD and 33 KD polypeptides. The results show that the microvilli and the apical plasma membrane of the lining epithelium of the epididymal duct, ductus deferens, and ampulla of the ductus deferens contain the membrane-bound carbonic anhydrase isoenzyme IV. The presence of the enzyme in the epithelium of the epididymis and ductus deferens is probably linked to the acidification of the epididymal fluid that prevents premature sperm activation. Its physiological role in the smooth muscle cells remains to be elucidated.     

An evaluation of the effectiveness of osteopathic treatment on symptoms associated with myalgic encephalomyelitis. A preliminary report

To improve the study of epithelial function in rat ductuli efferentes (efferent ductules) and initial segment epididymis, we developed a primary cell culture system with modification of the Klinefelter method (1992). The cultured efferent ductal epithelium was grown to confluence and the cells maintained many of the organelles characteristic of these cells in vivo, including dense-staining granules, indented nuclei and apical cilia. Ciliary beat was observed for up to 10 days in culture, Cultured initial segment epithelial cells were elongated and characterized by apical branched microvilli. Electron microscopy revealed intact cell junctions, and endocytotic apparatus and lysosomal granules. Ultrastructurally, the initial segment epithelium contained a well developed Golgi apparatus. For both epithelia, cell characteristics were also confirmed by indirect immunofluorescent staining for cytokeratins 8, 18. Endocytotic activity was detected by the uptake of cationic ferritin at the apical surface and within vesicles. Estrogen receptor and clusterin mRNAs were expressed in the cultured epithelia and no difference was found in their expressions when cultured with or without 10(-9)M 17-beta estradiol. Indirect immunofluorescent staining for clusterin further indicated that this protein was present in the cultures. In conclusion, these in vitro methods will be useful for the investigation of epithelial function in the head of the epididymis.     

Mice with mottled mutation--a model for defective copper metabolism in humans

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.     

Autometallographic demonstration of zinc ions in rat sperm cells

An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.     

Effect of chronic experimental poisoning with benzene and ethylene vapors on the circulatory system

To examine the content and the composition of free amino acids in the intraluminal fluid of rat epididymis, the fluids were obtained by light pressure on the dissected tissues. The amount of the total free amino acids in the pressed fluid from the caput epididymis was significantly higher than those of the cauda epididymis and the testis. Glu and Gln were predominant amino acids in the caput, and their amounts occupied more than half of the total ninhydrin reactive compounds. Such a high concentration of Glu and Gln was not observed either in the cauda or in the testis. Castration decreased Glu and increased Gln in amount. Testosterone treatment to castrated animals did not restore Glu and Gln contents in the pressed fluid from the caput epididymis to the level observed in intact rats completely. Therefore, it was assumed that a large amount of Glu in the caput was due to many factors; secretion and metabolism of epithelial cells of the gland which might be regulated by androgen, inflow of rete testis fluid, and sperm metabolism of amino acids in the epididymis. The results obtained from the caput epididymis to which the efferent duct of the testis was ligated also supported this interpretation.     

Phylogenetic relationship within Fusarium sambucinum Fuckel sensu lato, determined from ribosomal RNA sequences

The importance of abnormalities of function and epididymis structure in the etiology of male infertility is still not well understood. We studied 52 individuals distributed in five age groups: fetuses, children, adolescents, adults and the elderly. The region of the body of epididymis was obtained by autopsy and immediately fixed by immersion in a solution of 10% buffered formaldehyde, embedded in paraffin and histologically prepared. The samples were observed under an optic microscope. Test-points were counted in 12 random microscopic fields with the M42 test-system. The following stereological parameters were determined: ductal area, volumetric densities (Vv) of the duct, smooth muscle, connective tissue, epithelial duct and blood vessels. The main results distinguished by those whose averages were statistically significant (p < 0.05), showed that the ductal area is 9.7 times greater in the adolescent/adult/elderly group than the children's group. The Vv of the lumen of the epididymis duct occupies 11.7% of the epididymis body in the fetal period, 5.3% in the child and in individuals after puberty this figures reaches more than 15%. The Vv of smooth muscle occupies 28.3% of the body of the epididymis in the fetus and 35.9% in children, but after puberty this figures stays around 22%. The Vv of the connective tissue occupies 26% in prenatal life, 37% in children, and after puberty these figures range from 21 to 27.5%. Comparing the results of the adult group with that of the elderly group there is an increase in the volumetric density of the connective tissue by 18.1%. In conclusion, the epididymal duct area and the Vv of the ductal lumen, smooth muscle and connective tissue were significant comparing the different groups. However, the quantitative relative differences of the duct's epithelium and the blood vessels were not significant comparing these groups. The study of quantitative aspects of the normal human epididymis can increase our knowledge about male fertility.     

Basic fibroblast growth factor receptors and their prognostic value in human breast cancer

We performed a saturation binding study with 125I-labeled FGF (fibroblast growth factor)-2 in a nonselected series of 250 human primary breast cancers. Two hundred twenty-five breast cancer biopsies possessed bFGFR (basic FGF receptor). The median dissociation constant was 0.35 nM (range, 0.014-1.9), and the median concentration was 1126 fmol/mg protein (range, 49-7328). FGFR-1 was localized, using a specific monoclonal antibody, in cancerous cells and in epithelial cells in normal breast or in benign tumors. In all of the tissues studied, light stromal cell staining was also observed. Thus, the localization of FGFR-1 in carcinoma cells supports the hypothesis that an important part of FGF-2 binding reflects binding to FGFR-1. bFGFR concentrations were positively correlated to estrogen receptor and progesterone receptor levels. Cox univariate analyses showed that the bFGFR (> or = upper quartile) was associated to longer relapse-free survival [P = 0.004; RR (risk ratio), 0.46] and overall survival (P = 0.001; RR, 0.35); age, estrogen receptor levels, progesterone receptor levels, node involvement, tumor diameter, and histoprognostic grading were prognostic, also. In Cox multivariate analyses, only the bFGFR, age, node involvement, and histoprognostic grading were prognostic factors; the bFGFR was associated with longer relapse-free survival (P = 0.03; RR, 0.4) and overall survival (P = 0.009; RR, 0.3). The present study confirms that FGF could be an important regulator of human breast cancer growth and that patients with a high level of bFGFR had a better prognosis.     

Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures

Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.     

Hormone-dependent changes in A and B forms of progesterone receptor

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied. These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.     

Characterization of the testis and epididymis in mouse models of human Tay Sachs and Sandhoff diseases and partial determination of accumulated gangliosides

Beta-hexosaminidase (Hex) is an essential lysosomal enzyme whose activity is higher in the epididymis than in other tissues. The enzyme is also present in sperm and has been postulated to be required for fertilization. To better understand the role of Hex in reproduction, we have examined the testes and epididymides of mouse models of human Tay Sachs and Sandhoff diseases, produced by targeted disruption of the Hexa (alpha-subunit) or Hexb (beta-subunit) genes, respectively, encoding the enzymes Hex A (structure, alphabeta) and Hex B (betabeta). Testis weight, morphology, and sperm counts were unaffected in Hex-deficient mice. In the epididymis of the Hex A-deficient Hexa-/- mice, there was a large increase in the size and number of lysosomes in the initial segment/intermediate zone. In Hexb-/- mice (Hex A and B-deficient), the epididymal defects were much more extensive and the cytoplasm of all cell types throughout the efferent ducts and epididymis was filled with pale, uncondensed, enlarged lysosomes. In contrast to the brain where GM2 ganglioside accumulates, both mutant mice accumulated two non-GM2 gangliosides in the epididymis. The major accumulated species was characterized by electrospray ionization tandem mass spectrometry. The Hexa-/- male mice were fertile; however, litter sizes were reduced. The Hexb-/- males were able to sire normal sized litters up to nine weeks of age and remained healthy until 16-20 weeks of age. The extensive abnormalities in the Hexb-/- mice, in contrast to region-specific effects in the Hexa-/-mice, indicate an important and novel role for the Hex B isozyme in the epididymis and a region-specific role for Hex A in the initial segment/intermediate zone. In contrast to other reports, our results indicate that Hex is not essential for fertilization in young adult male mice. To explain the extensive epididymal abnormalities in the Hexb-/- mice, we propose that substrates for Hex, such as testis-derived glycolipids, cannot be catabolized and accumulate in lysosomes, leading to epididymal dysfunction and abnormalities in the epididymal luminal environment that supports sperm maturation.     

Immunocytochemical localization of progesterone receptor in the reproductive tract of adult female rats

The distribution of the progesterone receptor (PR) was investigated immunocytochemically in female reproductive tracts of rats during the estrous cycle and early pregnancy through use of an anti-PR monoclonal antibody. PR was localized predominantly in the nuclei of epithelial, stromal, and muscle cells in the uterus and vagina during the estrous cycle. In the uterus, the nuclei of epithelial cells were stained intensively at diestrus, while the PR staining of the stromal cells was more intense at proestrus than at any other stage of the cycle. PR expression during the cycle in muscle cells of the myometrium was similar to that in the endometrial stromal cells. In the vagina, however, PR expression during the cycle was approximately the same among epithelial, stromal, and muscle cells, the nuclei of which were stained deeply at proestrus. Ovariectomy at various stages of the cycle altered the PR expression appearing in the uterus and vagina during the cycle. In ovariectomized rats, estrogen increased the PR immunoreaction of various types of cells examined in the uterus and vagina except for the uterine epithelial cells. The reaction of these uterine epithelial cells was decreased by estrogen but was increased by progesterone given after estrogen; however, progesterone given alone reduced the reaction. In the epithelial and stromal cells of the uterus, intensity of the staining was increased after mating, reaching maximum on Day 3 of pregnancy, and then decreased on Day 4 (day of implantation), while in epithelial and stromal cells of the vagina the staining remained weak during early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.