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

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-II reacted only with spermatozoa. PNA, GSA-II, SBA, VVA, BPA, RCA-I, and RCA-II reacted with spermatozoa and spermatocytes. WGA, PEA, LCA, and MPA reacted with spermatogonia, spermatocytes, and spermatozoa in increasing order of intensity. ConA, Suc. ConA, LAA, STA, LTA, LPA, PHA-E, PHA-L, UEA-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-II 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.     

Lectin histochemistry of human placenta

Abstract. The human placenta was studied histochemically using 23 fluorescein-isothiocyanate-labeled lectins Distinct patterns of staining, as well as some differences between first-trimester and term placenta, were discerned. Eleven lectins (HPA, VVA, BPA, HAA, SBA, PNA, GSA-I, MPA, RCA-I, RCA-II, and UEA-I) did not react with the trophoblast. Two lectins (LCA and PEA) reacted with the trophoblast of first-trimester placenta but not with the trophoblast of third-trimester placenta. The remaining ten lectins (ConA, Suc.ConA, WGA, GSA-II, LAA, STA, DBA, LBA, PHA-E, and PHA-L) reacted with the trophoblast of both first- and third-trimester placenta, and two of these lectins (ConA and Suc.ConA) reacted preferentially with the syncytiotrophoblast. Five lectins (LAA, STA, DBA, GSA-II, and LBA) reacted with nuclei of the cytotrophoblast. The nuclei of some stromal and syncytiotrophoblastic cells were also reactive. Eighteen lectins reacted with the trophoblastic basement membrane, and all reacted with Hofbauer cells and the stroma of the villi. Latin binding was influenced by the mode of fixation and tissue processing. These data show that some lectins can be used to identify components of the placental villi (e.g., basement, membrane) to characterize differences between the first- and third-trimester trophoblast, and to distinguish the cytotrophoblast from the syncytiotrophoblast.     

Pregnancy-related changes in the mouse oviduct and uterus revealed by differential binding of fluoresceinated lectins

The binding of 20 fluorescein isothiocyanate (FITC)-labeled lectins to various portions of the pregnant and non-pregnant murine oviduct and uterus was studied by fluorescence microscopy. Five lectins (from Ricinus communis (RCA-I), Maclura pomifera (MPA), Triticum vulgare (wheat germ-WGA), Bauhinia purpurea (BPA), and Ulex europeus (UEA-I] reacted differentially with the epithelium of pregnant as compared with the non-pregnant uterus. The binding of RCA-I, MPA and WGA delineated pregnancy-related changes in the distal oviduct and colliculus tubaris. WGA recognized also pregnancy related changes in the proximal oviduct. The reactivity of the remaining 15 lectins did not distinguish the pregnant and non-pregnant oviduct and uterus, although some of them served to identify specific components of the mouse genital tract. Thus, Soybean lectin (SBA) reacted almost exclusively with the colliculus tubaris. UEA-I alone reacted exclusively with the epithelium of the non-pregnant uterus. RCA-II reacted preferentially with the epithelium of the oviduct and uterus as compared with its weak reactivity with the stroma. Two lectins (from Pisum sativum and Lens culinaris) reacted selectively with stromal cells of the uterus and oviduct. Present data indicate that the differential binding properties of these FITC-labeled lectins can be exploited to identify certain components of the mouse oviduct and uterus and to indicate changes in the cell surface and/or cytoplasm in these structures during pregnancy.     

Pregnancy-related changes in the human endometrium revealed by lectin histochemistry

The binding of 22 fluorescein isothiocyanate (FITC) conjugated lectins to human proliferative phase and pregnant endometrium was studied histochemically. Only the lectin from Bauhinia purpurea (BPA) reacted exclusively with the epithelial cells. All the others reacted to a certain extent with glandular and/or stromal cells. Lectins from soybean (SBA), and Vicia villosa seeds (VVA) reacted with endometrial glands of pregnancy but not with the glands of the proliferative endometrium. In the proliferative endometrium SBA reacted only with cells of the surface endometrium. Lectin from peanuts (PNA) reacted only with some glands in the proliferative endometrium but was unreactive with others. In pregnant endometrium PNA reacted with all glands. Lectins from lentils (LCA) and red kidney beans (PHA-E and PHA-L) reacted with endometrial glands of the proliferative phase but not with the glands from pregnant endometrium. We thus show that FITC labeled lectins define specific carbohydrate moieties selectively expressed on either proliferative phase or pregnant endometrial glands.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Importance of N-linked Sugar Residues in the Development of Auerbach’s Plexus in the Rat Colon: A Lectin Histochemical Study

The lectin-binding patterns in Auerbach's plexus in the distal portions of the rat colon from 15- to 21-day-old foetuses, newborns, and adults were examined by light and electron microscopy using 16 different lectins (ConA, RCA-1, WGA, PNA, SBA, UEA-1, DBA, LCA, PHA-L, DSA, GS-1, VVA, MPA, BPA, MAA, and PSA). The binding of ConA was shown to increase after day 19 of gestation in parallel with differentiation of Auerbach's plexus, whereas the staining intensity for DSA and RCA-1 increased after day 17 of gestation in accordance with the appearance of the plexus. At the electron microscopical level, DSA binding sites were observed to be localized mainly in the plasma membrane, Golgi apparatus, and nuclear membrane of nerve cells. Positive sites were also observed in the axolemma and in the plasma membrane of nerve cell processes, Schwann cells, and the surrounding smooth muscle cells. PSA, PHA-L, LCA, and WGA showed constant staining during the development after day 15 of gestation. Other lectins, most of which are specific for O-glycosidic mucin-type sugar residues, were essentially negative throughout the developmental stages. Moreover, N-glycanase digestion significantly diminished the positive reactions. N-linked oligosaccharides may thus play important roles in the development and maturation of the Auerbach's plexus, and may be involved in the developmental defect of the plexus, e.g. as occurs in Hirschsprung's disease.     

Importance of N-linked sugar residues in the development of Auerbach's plexus in the rat colon: a lectin histochemical study

The lectin-binding patterns in Auerbach's plexus in the distal portions of the rat colon from 15- to 21-day-old foetuses, newborns, and adults were examined by light and electron microscopy using 16 different lectins (ConA, RCA-1, WGA, PNA, SBA, UEA-1, DBA, LCA, PHA-L, DSA, GS-1, VVA, MPA, BPA, MAA, and PSA). The binding of ConA was shown to increase after day 19 of gestation in parallel with differentiation of Auerbach's plexus, whereas the staining intensity for DSA and RCA-1 increased after day 17 of gestation in accordance with the appearance of the plexus. At the electron microscopical level, DSA binding sites were observed to be localized mainly in the plasma membrane, Golgi apparatus, and nuclear membrane of nerve cells. Positive sites were also observed in the axolemma and in the plasma membrane of nerve cell processes, Schwann cells, and the surrounding smooth muscle cells. PSA, PHA-L, LCA, and WGA showed constant staining during the development after day 15 of gestation. Other lectins, most of which are specific for O-glycosidic mucin-type sugar residues, were essentially negative throughout the developmental stages. Moreover, N-glycanase digestion significantly diminished the positive reactions. N-linked oligosaccharides may thus play important roles in the development and maturation of the Auerbach's plexus, and may be involved in the developmental defect of the plexus, e.g. as occurs in Hirschsprung's disease.     

Expression pattern of glycoconjugates in rat retina as analysed by lectin histochemistry

The present study sought to characterize the expression and distribution of complex glycoconjugates in the rat retina by lectin histochemistry, using a panel of 21 different lectins with different carbohydrate specificities. Paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with various biotinylated lectins, followed by the streptavidin-peroxidase and glucose oxidase-diaminobenzidine-nickel staining procedures. The results showed that the retinal pigment epithelium was stained intensely with LCA, Jacalin, WFA, S-WGA, PWA, DSA, UEA-I, LTA and PHA-E, suggesting that this epithelium contained glycoconjugates with alpha-Man, alpha-Glc, alpha-Gal/GalNAc, beta-GalNAc, alpha-Fuc, NeuAc and other oligosaccharide residues. The outer and inner segments of the photoreceptor layer showed different lectin binding affinities. The outer segments reacted with S-WGA and GS-II, whereas the inner segments reacted with UEA-II, UEA-I, LTA and MAA, suggesting that the inner segments contained glycoconjugates rich in alpha-Fuc and NeuAc(alpha2,3)Gal residues. PNA labelled specifically the cones and could be used as a specific marker for these photoreceptors. RCA-I, WFA, S-WGA, DSA, MAA and PHA-E reacted with both the outer and inner plexiform layers. On the other hand, UEA-I and LTA specifically labelled the outer plexiform layer, while PNA labelled the inner plexiform layer. The retinal microglial cells were labelled specifically by GS-I-B4 and SNA. Interestingly, we also observed that WFA bound specifically to Müller cells and could be used as a novel marker for this retinal glial cell. The capillaries and larger vessels in the retina and choriocapillaris reacted intensely with GS-I-B4, RCA-I, S-WGA, PWA, DSA and PHA-E. No significant differences in lectin binding were observed in the microvessels at these two sites. In summary, the present study demonstrated the expression patterns of glycoconjugates in the rat retina and that certain lectins could be used as histochemical markers for specific structural and cellular components of the rat retina.     

Lectin binding sites as markers of neonatal porcine uterine development.

To identify lectin binding sites and to determine if lectin binding patterns change with age in developing neonatal porcine uterine tissues, gilts (n = 3/day) were hysterectomized on Day 0 (birth), 7, 14, 28, 42, or 56. Lectin binding was visualized in Bouin's-fixed uterine tissues with seven biotinylated lectins (ConA, DBA, PNA, RCA-I, SBA, UEA-I, and WGA) and avidin-peroxidase staining procedures. Lectin specificities were demonstrated by pre-incubating lectins with appropriate inhibitory sugars (0.2 M). Staining intensity was evaluated visually (absent, weak, moderate, or strong) for three endometrial tissues; luminal epithelium, glandular epithelium, and stroma. Staining intensities for DBA, PNA, SBA, and WGA were not affected by neonatal age. Staining with these lectins was greater in uterine epithelium (moderate or strong) than in stroma (weak). In contrast, binding patterns for ConA, UEA-I, and RCA-I were affected by neonatal age. Strong epithelial staining associated with ConA binding was observed on all days, whereas stromal ConA staining decreased in intensity from moderate to weak after Day 14. Epithelial staining with UEA-I increased from moderate to strong after Day 28, whereas stromal UEA-I staining decreased from moderate to weak after day 28. Staining with RCA-I was homogeneous for luminal epithelium and stroma but variegated for glandular epithelium on and after Day 7. These observations indicate that a variety of lectin binding sites are present in developing neonatal porcine endometrial tissues and that developmentally related alterations in the distribution and/or orientation of glycoconjugates containing alpha-D-mannose, beta-D-galactose, beta-D-acetyl-N-galactosamine, and alpha-L-fucose residues occur between birth and Day 56 as these tissues mature.     

Lectin histochemistry of glycolipid storage diseases on frozen and paraffin-embedded tissue sections

Lectin histochemical studies were performed on frozen and paraffin-embedded brain tissue sections from six cases of galactosylceramide lipidosis (i.e., globoid cell leukodystrophy, or Krabbe's disease) in Twitcher mice and one case of canine infantile GM1-gangliosidosis. The globoid cells in Krabbe's disease stained with Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), and Bandeirea simplicifolia agglutinin-I (BS-I) in frozen sections. However, paraffin sections and frozen sections pretreated with chloroform-methanol or xylene, from the same animals, stained with Concanavlia ensiformis agglutinin (ConA), wheat germ agglutinin (WGA), and succinylated-WGA (S-WGA), in addition to staining with RCA-I, PNA, and BS-I. The affected neurons of canine infantile GM1-gangliosidosis stained only with RCA-I in frozen sections. In paraffin sections, however, these cells were negative with RCA-I but positive with BS-I, ConA, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Ulex europaeus agglutinin (UEA-I) in paraffin sections. These results indicate that in paraffin processing of glycolipid storage disease tissue, some lectin receptors are lost and others are unmasked. The retained receptors can be stained with specific lectins and could serve as markers to characterize and differentiate among the various glycolipid storage diseases.     

Characterization of the glycoconjugates of boar testis and epididymis

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.     

Lectins as differentiation markers of human gliomas.

The lectins Concanavalin A (Con A), Ricinus communis agglutinin (RCA-I), Peanut agglutinin (PNA) and Wheat germ agglutinin (WGA) as well as the immunomarkers for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used in a series of 21 glial tumors (4 pylocytic astrocytomas, 5 grade II astrocytomas, 3 anaplastic astrocytomas, 4 glioblastomas and 5 oligodendrogliomas). ConA binds to all tumoral astrocytes in low grade astrocytomas, as well as to well differentiated tumoral astrocytes in anaplastic astrocytomas and glioblastomas. RCA-I has a similar behaviour. PNA, and to a lesser degree WGA, binds selectively to the oligodendroglial plasma membrane in well differentiated oligodendrogliomas. The results suggest that these lectins are markers of differentiation in gliomas rather than of malignancy.     

Glycoconjugates in normal human kidney

Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine‐rich protein containing both Kunitz and whey acidic protein (WAP)‐type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down‐regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen‐dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility. Mol. Reprod. Dev. 79: 832–842, 2012. © 2012 Wiley Periodicals, Inc.     

Expression pattern of Glycoconjugates in Rat Retina as Analysed by Lectin Histochemistry

The present study sought to characterize the expression and distribution of complex glycoconjugates in the rat retina by lectin histochemistry, using a panel of 21 different lectins with different carbohydrate specificities. Paraffin sections of Carnoy-fixed Sprague–Dawley rat eyes were stained with various biotinylated lectins, followed by the streptavidin-peroxidase and glucose oxidase–diaminobenzidine–nickel staining procedures. The results showed that the retinal pigment epithelium was stained intensely with LCA, Jacalin, WFA, S-WGA, PWA, DSA, UEA-I, LTA and PHA-E, suggesting that this epithelium contained glycoconjugates with α-Man, α-Glc, α-Gal/GalNAc, β-GalNAc, α-Fuc, NeuAc and other oligosaccharide residues. The outer and inner segments of the photoreceptor layer showed different lectin binding affinities. The outer segments reacted with S-WGA and GS-II, whereas the inner segments reacted with UEA-II, UEA-I, LTA and MAA, suggesting that the inner segments contained glycoconjugates rich in α-Fuc and NeuAc(α2,3)Gal residues. PNA labelled specifically the cones and could be used as a specific marker for these photoreceptors. RCA-I, WFA, S-WGA, DSA, MAA and PHA-E reacted with both the outer and inner plexiform layers. On the other hand, UEA-I and LTA specifically labelled the outer plexiform layer, while PNA labelled the inner plexiform layer. The retinal microglial cells were labelled specifically by GS-I-B₄ and SNA. Interestingly, we also observed that WFA bound specifically to Müller cells and could be used as a novel marker for this retinal glial cell. The capillaries and larger vessels in the retina and choriocapillaris reacted intensely with GS-I-B₄, RCA-I, S-WGA, PWA, DSA and PHA-E. No significant differences in lectin binding were observed in the microvessels at these two sites. In summary, the present study demonstrated the expression patterns of glycoconjugates in the rat retina and that certain lectins could be used as histochemical markers for specific structural and cellular components of the rat retina.     

Microanatomy of the testes and testicular ducts of the butterfly lizard,Leiolepis ocellata Peters, 1971 (Reptilia: Squamata: Agamidae) during the active reproductive period

The microanatomy of the testes and testicular ducts (rete testis, ductuli efferentes, ductus epididymis and ductus deferens) of Leiolepis ocellata (Agamidae) was investigated using light microscopy including histochemistry. Each testis contains seminiferous tubules and interstitial tissues. The former house spermatogenic cells (spermatogonia A & B, preleptotene, primary and secondary spermatocytes, spermatids (steps 1–8) and spermatozoa) and Sertoli cells, while the latter comprise peritubular and intersitial tissues. The rete testis is an anastomosing duct, having intratesticular and extratesticular portions. The proximal region of ductuli efferentes has wider outer ductal and luminal diameters than those of the distal region. The convoluted ductus epididymis is subdivided into four regions (initial segment, caput, corpus and cauda), based on the ductal diameter, epithelium characteristics and cell components. The ductus deferens has the greatest diameter and is divided into the ductal and ampulla ductus deferens. The ductal portion is subdivided into the proximal and distal regions, based on the epithelium types and ductal diameters. The ampulla ductus deferens is a fibromuscular tube, having numerous mucosal folds projecting into the lumen. Spermiophagy is detectable in the ductus epididymis and ductus deferens. The present results contribute to improved fundamental knowledge on the microanatomy of the reptilian reproductive system.     

Selective partitioning of plasma membrane antigens during mouse spermatogenesis

The selective partitioning of cell membrane components during mouse spermatogenesis has been examined using a heterologous antibody raised against isolated type B spermatogonia. The anti-type B spermatogonia rabbit IgG (ATBS) binds to isolated populations of mouse primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, round spermatids, residual bodies, and mature spermatozoa. Although immunofluorescent labeling is uniformly distributed on the cell surface of early spermatogenic cells, a discrete topographical localization of IgG is observed on testicular, epididymal, and vas deferens spermatozoa. The convex surface of the acrosome, postacrosomal region, and tail are labeled. Antibody does not bind to a broad area corresponding to the concave region of the acrosome. The antibody also binds to mouse somatic cells including Sertoli cells, Leydig cells, thymocytes, and splenocytes, but not to mature spermatozoa of the vole, rat, hamster, guinea pig, rabbit, or human. ATBS, after absorption with mouse splenocytes or thymocytes, does not react with any somatic cells examined by fluorescence except with Sertoli cells. In addition, all reactivity with testicular, epididymal, and was deferens spermatozoa is abolished. However, spermatogenic cells at earlier stages of differentiation, including residual bodies, still react strongly with the absorbed antibody. The number of surface receptor sites per cell for absorbed ATBS ranges from approximately 3 million on primitive type A spermatogonia to 1 million on round spermatids and on residual bodies. Spermatozoa, however, have only 0.003 million binding sites for absorbed ATBS, in contrast to 10 million sites for the unabsorbed antibody. It appears that receptor sites for absorbed ATBS are not masked by components of epididymal secretions. These data imply, therefore, that specific mechanisms operate at the level of the cell membrane during spermiogenesis to insure that some surface components, not required in the mature spermatozoon, are removed selectively by partitioning to that portion of the spermatid membrane destined for the residual body.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Distribution of sugar residues in the bovine testis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates.

In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to alpha-D-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. alpha-D-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal D-Gal-(beta 1----3)-D-GalNAc, D-Gal and D-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited D-Gal, D-GlcNAc or sialic acid and D-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (alpha-D-Man), RCA-I (D-Gal), UEA-I (alpha-L-Fuc) and WGA (D-GlcNAc or sialic acid).     

DIFFERENTIAL LOCALIZATION OF CELL SURFACE AND SECRETORY COMPONENTS IN RAT INTESTINAL EPITHELIUM BY USE OF LECTINS

Sections through various levels of small intestine from adult male rats were examined by fluorescence microscopy after treatment with fluorescein isothiocyanate-labeled lectins from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis, and Triticum vulgare (wheat germ). The latter three lectins reacted with the microvillar portion of the epithelial cells lining the crypts and villi in sections of intestine adjacent to the pylorus. This pattern of reactivity was sharply altered along the first 15 cm of intestine so that in sections distal to this point the luminal surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with the L. tetragonolobus and R. communis lectins, whereas the wheat germ lectin reacted with the surfaces of the cells lining the villi. In sections from the distal end of the small intestine, all three lectins reacted with the surfaces of cells only at the base of the villi and in the crypts. These results show a difference in surface components in cells at various portions on the villi and the dependence of these differences on the region of intestine. The D. biflorus lectin reacted with approximately 25% of the goblet cells at each level of intestine studied whereas the reactivities of the goblet cells with the other three lectins were dependent upon the region of intestine.