Anatomic distribution of lectin-binding sites in mouse testis and epididymis

Abstract. Testis and epididymis of sexually mature mice were studied histochemically using 25 fluorescein-isothiocyanate-labeled lectins. Several lectin-specific binding patterns were recognized. Thus, HAA, HPA, GSA-I, and UEA-I1 reacted only with spermatozoa. PNA, GSA-11, SBA, VVA, BPA, RCA-I, and RCA-I1 reacted with spermatozoa and spermatocytes. WGA, PEA, LCA, and MPA reacted with spermatogonia, spermatocytes, and spematozoa in increasing order of intensity. ConA, SUC. ConA, LAA, STA, LTA, LPA, PHA-E, PHA-L, IJEA-I, and LBA reacted with all spermatogenic cells with equal intensity. In the epididymis, 12 lectins reacted uniformly with the epithelial cells lining all segments of this organ. One lectin (VVA) did not react with epididymal lining cells. The remaining 12 lectins reacted in a specific manner with portions of the head, body, or tail, thus selectively outlining different portions of the epididymis. RCA-I and RCA-I1 selectively accentuated the so-called halo cells of the epididymis. These findings provide a detailed map of lectin-binding sites in the mouse testis and epididymis and show that certain lectins can be used as specific markers for spermatogenic cells and segments of the epididymis.     

Postnatal development of lectin-binding pattern in the rat testis and epididymis

Seven rhodamine-conjugated lectins were utilized to study the distribution of glycoproteins in the developing rat testis and epididymis. In the testis a clear developmental pattern was found in Leydig cells and the cell boundaries between Sertoli and spermatogenic cells, as well as during acrosome formation. Some of the first degenerating meiotic cells and the apical extensions of the Sertoli cells at the time of spermiation also displayed a characteristic lectin binding. The epididymal differentiation was characterized by an increasing lectin binding of the subapical Golgi zone and apical surface, and intratubular secretion prior to the arrival of sperm. After the accumulation of tubular secretion and sperm some epithelial cells were transformed into narrow (initial segment) and light cells (distal caput, cauda) with a strong affinity for some lectins. These cells appeared to be responsible for the absorption and digestion of tubular material derived from the testis and of surplus secretion and/or sperm structures.     

Differential distribution of dihydrotestosterone and estradiol binding sites in the epididymis of the mouse

Summary The distribution of androgen and estrogen binding sites in the mouse epididymis was assessed by autoradiography with ³H dihydrotestosterone (³H DHT) and ³H estradiol (³H E₂). Nuclear labeling with ³H DHT in principal cells of the epithelium is high in the caput, low in the corpus, and high again in the cauda. ³H E₂ also binds to the nuclei of principal cells. The pattern is distinct from ³H DHT: nuclear labeling is highest in the ductulus efferens and high in the caput, but low or absent in corpus and cauda. Apical cells in caput and clear cells in corpus and cauda are moderately labeled with ³H DHT but heavily labeled with ³H E₂. Connective tissue cells show variable labeling with both hormones, being more pronounced with ³H E₂. Smooth muscle cells are also labeled to varying degrees with both hormones.The different binding patterns of ³H DHT and ³H E₂ and the results of the competition studies with unlabeled compounds demonstrate that in the epididymis besides the specific nuclear receptors for androgen also estrogen receptors are present.Supported by US P.H.S. grant NS 09914 and Deutsche Forschungsgemeinschaft Dr 94/4     

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Summary Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.Supported in part by USPHS Grant HL-32870     

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Differential distribution of dihydrotestosterone and estradiol binding sites in the epididymis of the mouse. An autoradiographic study

The distribution of androgen and estrogen binding sites in the mouse epididymis was assessed by autoradiography with 3H dihydrotestosterone (3H DHT) and 3H estradiol (3H E2). Nuclear labeling with 3H DHT in principal cells of the epithelium is high in the caput, low in the corpus, and high again in the cauda. 3H E2 also binds to the nuclei of principal cells. The pattern is distinct from 3H DHT: nuclear labeling is highest in the ductulus efferens and high in the caput, but low or absent in corpus and cauda. Apical cells in caput and clear cells in corpus and cauda are moderately labeled with 3H DHT but heavily labeled with 3H E2. Connective tissue cells show variable labeling with both hormones, being more pronounced with 3H E2. Smooth muscle cells are also labeled to varying degrees with both hormones. The different binding patterns of 3H DHT and 3H E2 and the results of the competition studies with unlabeled compounds demonstrate that in the epididymis besides the specific nuclear receptors for androgen also estrogen receptors are present.     

Ultrastructural distribution of lectin-binding sites on gastric superficial mucus-secreting epithelial cells

Normal human gastric epithelial cells were examined by electron microscopy using each of five biotinylated lectins [Ulex europaeus agglutinin I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), soybean agglutinin (SBA) andDolichos biflorus agglutinin (DBA)] as a probe. We employed 35 gastric surgical specimens removed from complicated peptic disease. The lectin-binding sites were revealed with streptavidin-colloidal gold complex. All specimens were embedded in Spurr and LR White resins. In superficial foveolar epithelial cells, the lectins used were generally positive in all cell types (mainly UEA-1 and PNA) on the Golgi region and mucus cytoplasmic vacuoles, with many variations among cells in the same case. On the other hand, extracellular mucus was negative for WGA. Labelling with PNA revealed a biphasic pattern (peripheral positivity) on mucous droplets in surface and foveolar cells. Thecis side of the Golgi apparatus was labelled with SBA and PNA and rough endoplasmic reticulum with SBA (only five cases). Lectin-binding variability could be related to heterogeneous composition of gastric mucus. Our results with SBA suggest initiation ofO-glycosylation at the Golgi apparatus; however a role of the rough endoplasmic reticulum cannot be excluded (N-glycosylation). We propose the following sequence of sugar addition to the carbohydrate side-chains of gastric glycoproteins: (1) GaNAc (Golgi apparatuscis-side), (2) GlcNAc (Golgi apparatus intermediate face), (3) GalNac or Gal, -l-fucose (Golgi apparatustrans-side).Supported by a grant from Junta de Andalucía (Consolidación de Grupos de Investigación. Ref. 541A.6.60.609.018311)     

Developmental changes in the distribution of cecal lectin-binding sites of Balb-c mice.

The existence of lectin-binding sites was investigated in the cecum of Balb-c mice at seven developmental stages ranging from 18 days post conception (p.c.) to 8 weeks after birth. Nine horseradish-peroxidase-conjugated lectins (concanavalin A, Triticum vulgaris, Dolichus biflorus, Helix pomatia, Arachis hypogaea, Glycine maximus, Lotus tetragonolobus, Ulex europaeus, Limulus polyphemus) were applied to 5- to 7-microns thin paraffin sections of Bouin-fixed tissue. After DAB staining the sections were evaluated by light microscopy. It was shown that each lectin exhibits a unique developmental pattern. The adult binding patterns were established at the age of 3-4 weeks with only minor changes occurring thereafter. Considerable differences in binding patterns occurred not only between lectins of different groups but also between lectins with the same nominal monosaccharide specificity.     

Development of mouse testis and epididymis following intrauterine exposure to a static magnetic field

In order to test if the in utero exposure to static magnetic fields affects testis and epididymis development in mice, females were exposed to 0.5-0.7 T, generated by a permanent magnet, from day 7 of gestation to the day of birth. No significant differences were found between exposed and sham-exposed animals with respect to body weight gain of dam during the gestational period, litter size, body weight of male pups at the day of birth, and body or testis-epididymis weight gain of pups from birth to day 35. Histopathologic evaluation of testis and epididymis of pups of 1, 5, 15, and 35 days of age showed no detectable alterations due to in utero exposure to static magnetic fields.     

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.This work was supported by Research Initiate Grant funded by Faculty of Science, Mahidol University to W.W.     

Presence of two types of estrogen binding sites in mouse testis

The characteristics of cytosol estrogen binding sites in BALB/c mouse testis were investigated. The cytosol prepared from the whole testis contained two classes of the specific estrogen binding sites by Scatchard and Rosenthal plot analyses. The first binding site (first binder) had high affinity for 17 beta-estradiol (E2; Kd = 4.9 X 10(-9) M) and binding specificity as observed in the typical estrogen receptor. The second binding site (second binder) had lower affinity for E2 (Kd = 4.8 X 10(-8) M) and the binding was inhibited less vividly by diethylstilbestrol (DES) and antiestrogens in comparison with that for the first binder. Postlabeled sucrose density gradient analysis in a low salt medium revealed that the major radioactive peak of the first binder appeared at 7S region, while that of the second binder sedimented at 4S region. The 7S component showed an appreciable binding to the nuclei, while the 4S component did not show a significant binding ability to the nuclei. Much higher concentrations of the first and the second binders were found in Leydig cells preparations. These results demonstrate the presence of two types of the specific estrogen binding sites in the mouse testis especially in Leydig cells.