Distribution of Lectin Binding in Rat Testis and Epididymis

Summary: Seven lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) conjugated with rhodamine were employed to analyse the staining pattern of glycoproteins with varying sugar residues in the testis and epididymis of adult Wistar rats. Some lectins (UEA I, SBA, DBA) gave rather specific staining of the mature acrosome, while others (PNA, RCA I) showed affinity for the early stages of acrosome formation or had a wide affinity for germinal and non-germinal cells and structures (Con A, WGA). In the epididymis the sperm mass had a homogenous staining reaction with some lectins (PNA, RCA I, Con A, WGA, DBA) which also showed a rather strong reaction on the epithelial surface. It was concluded that this reaction is at least partially due to the secretory products synthetized by principal, apical, narrow and light cells of the epididymal epithelium. Some differences in the staining pattern of these cells were recorded indicating specialization of the cells for the production of distinct glycoproteins. The staining pattern of the interstitial and intertubular compartment of the testis and epididymis was also recorded. Zusammenfassung: Die Verteilung der Lektin-Bindung im Hoden und Nebenhoden der Ratte Sieben Lektine (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) wurden verbunden mit Rhodamin verwendet, um das Darstellungsmuster von Glykoproteinen mit verschiedenen Zuckerresten im Hoden und Nebenhoden erwachsener Wistar-Ratten zu analysieren. Einige Lektine (UEA I, SBA, DBA) gaben eine recht spezifische Darstellung des reifen Akrosoms, wohingegen andere (PNA, RCA I) eine Affinität zu den frühen Stadien der Akrosomformation zeigten oder eine breite Affinität zu Germinal- und Nichtgerminalzellen und -strukturen aufwiesen (Con A, WGA). Im Nebenhoden zeigte die Spermatozoenmasse ein homogenes Darstellungsmuster mit einigen Lektinen (PNA, RCA I, Con A, WGA, DBA), welche auf der epithelialen Oberfläche auch eine recht starke Reaktion aufwiesen. Es wurde daraus geschlossen, daβ diese Reaktion zumindest teilweise auf die sekretorischen Produkte, die in den Haupt-, Apikal-, Schmal- und Hellzellen des Nebenhodens synthetisiert werden, zurückzuführen ist. Einige Unterschiede im Darstellungsmuster dieser Zellen wurden aufgezeichnet, die die Spezialisierung der Zellen für die Produktion bestimmter Glykoproteine anzeigen. Das Darstellungsmuster des interstitiellen und intertubulären Anteils des Hodens und Nebenhodens wurde ebenfalls aufgezeichnet.     

Lectin affinity of the seminiferous epithelium in healthy and cryptorchid post-pubertal boars

The present study describes the sugar content of the seminiferous epithelium, using lectin histochemistry, in healthy boars and in boars with unilateral and bilateral abdominal cryptorchidism. In healthy boars the apical cytoplasm of Sertoli cells exhibited abundant glucosyl (Con A and WGA lectins), galactosyl (HPA, DBA, SBA and PNA lectins), and fucosyl (AAA lectin) residues. Spermatogonia and spermatocytes contained abundant glucosyl (Con A and WGA lectins) and fucosyl (AAA lectin) residues. In spermatids, galactosyl (SBA and PNA lectins) and glucosyl (Con A and WGA lectins) residues increased progressively throughout spermiogenesis, and fucosyl (AAA lectin) residues decreased. As compared with healthy boars, the scrotal testis of unilateral cryptorchid boars showed decreased amounts of fucosyl (AAA lectin) and galactosyl (HPA and DBA lectins) residues on the Sertoli cell apical cytoplasm; spermatocytes exhibited higher content of glucosyl (Con A lectin) residues and spermatids showed altered nature of glucosyl (Con A and WGA lectins) and galactosyl (SBA and PNA lectins) complexes. In abdominal testes of unilateral and bilateral cryptorchid boars, immature Sertoli cells and spermatogonia showed decreased fucosyl (AAA lectin), and increased glucosyl (Con A and WGA lectins) and galactosyl (SBA and PNA lectins) contents. These results suggest that the seminiferous epithelium of healthy boars has polarized activity with the apical compartment implicated in germ cell-Sertoli cell adhesion and interaction, in transport of ions, substrates and fluids, and in acrosomal differentiation. In scrotal testes, unilateral abdominal cryptorchidism could lead to defective germ cell-Sertoli cell adhesion, impaired acrosomal differentiation and increased ionic transport in the apical compartment of the seminiferous epithelium. Unilateral and bilateral cryptorchidism could induce increased ionic transport and membrane permeability in the seminiferous epithelium of abdominal testes.     

Lectin-binding sugar chain in bladder tumors

We studied the lectin binding patterns of 40 initial superficial and 10 subsequent invasive bladder tumors by the avidin-biotin-peroxidase complex (ABC) method using the following biotin-labeled lectins: PNA, DBA, UEA-I, BS-I, ConA and WGA. We observed the relationship between lectin binding and subsequent course of initial superficial tumors, grade and stage (T). DBA or WGA staining tumors and Con A negative tumors revealed no recurrence or superficial recurrence. Low grade tumors were DBA or BS-I positive and high grade tumors were ConA positive. Low staging tumors possessed DBA or WGA positiveness and high staging tumors had ConA positiveness. From these results we considered that negative staining of WGA or DBA, or positive staining of ConA was a change accompanying the malignant potentiality.     

早早孕人子宫蜕膜及绒毛膜凝集素的组化分析

以凝集素作为组化探针研究早早孕期人子宫蜕膜、绒毛膜表面糖复合物的变化。结果表明：６种凝集素在早早孕期与子宫蜕膜及绒毛膜结合显示不同变化。其中刀豆凝集素（ＣｏｎＡ）、花牛凝集素（ＰＮＡ）、双花藕豆凝集素（ＤＢＡ）与子宫蜕膜结合随妊娠无数增加着色逐渐减弱。荆豆凝集素（ＵＥＡ－Ⅰ）与子宫蜕膜未见着色。麦胚凝集素（ＷＧＡ）、蓖麻凝集素（ＲＣＡ－Ⅰ）与子宫内膜在早早孕期着色程度未见有变化。ＰＮＡ、ＣｏｎＡ在与绒毛滋养层细胞结合随妊娠天数增加着色逐渐增强。ＵＥＡ－Ⅰ和ＤＢＡ与滋养层细胞在各期中未见结合。     

Lectin‐binding pattern of glycoconjugates during spontaneous testicular recrudescence in Syrian hamster (Mesocricetus auratus) after exposure to short photoperiod

Lectin histochemistry was used to characterise glycoconjugates and cellular apoptosis in the seminiferous epithelium and interstitium of hamster testis during spontaneous recrudescence. An increase in the LTA lectin affinity was observed in spermatids in the Golgi phase. An increase in labelling of PNA and Con‐A lectin in acrosome of spermatids (acrosome phase) as well as increased labelling with Con‐A in spermatids (cap phase) was observed. Spermatocytes showed decreased affinity with PNA and AAA lectins and an increase in positivity for LTA and GNA lectins. Spermatogonia showed a slight decrease in positivity to WGA and an increase in labelling with Con‐A and a decreased affinity for the AAA lectin. At the end of recrudescence, all these germinal cells showed a similar pattern to the control. The Sertoli cells showed a gradual decrease in labelling with the GNA lectin and the Leydig cells an increase in labelling with Con‐A and GNA. Particularly unusual was the observation of apoptotic spermatocytes and spermatids positive for PNA, GNA, AAA and Con‐A, together with spermatocytes positive to LTA. In conclusion, the normal lectin pattern is recovered during testis recrudescence and germ cell apoptotic activity is low, as is observed by specific lectins for germ cells in apoptosis.     

Effect of Castration on Lectin Staining in Rat Epididymis

Seven rhodamine-conjugated lectins (PNA, RCA I, Con A, WGA, UEA I, SBA, DBA) were used to follow the staining pattern of the rat epididymis at different time points after castration. The affinity of the intratubular sperm mass for the lectins increased rapidly with concurrent augmentation of the staining in the principal cells but a decline of the reaction in the light cells. The light cells showed some differences in their response to castration, which was compatible with secretory/absorptive activity in caput and absorptive activity in cauda. The active phase of sperm mass destruction and epithelial involution was accompanied by local accumulation of macrophages and round cells, which also acquired an increased affinity for most of the lectins. It is concluded that the androgen-deprived epididymis is rapidly programmed for autolytic and phagocytic processes, which include the destruction of macromolecules including glycoproteins of the spermatozoa.     

Lectin-Binding Sites in Testis of Men with Acrosomeless Round-Headed Spermatozoa/Testikuläre Lektinbindungsstellen bei Männern mit akrosomenlosen Rundkopfspermatozoen

We report herein about lectin histochemistry of seminiferous epithelia in two infertile men with exlusely acrosomeless round-headed spermatozoa. FITC-conjugated lectins (ConA, PNA, RCA II, WGA) have been employed on tissue sections of Bouin fixed testicular biopsies. RCA II gave a dot-like fluorescence of the acrosomal region and WGA gave a cap-like acrosomal fluorescence of spermatids. PNA-a marker of acrosomal differentiation-failed to stain spermatids. The binding of ConA to germ cells was not influenced by this syndrome. In conclusion, the syndrome of acrosomeless round-headed spermatozoa is associated with selective perturbations of testicular lectin-binding sites. They might contribute of the inability of sperm cells to adhere to and penetrate into ova.     

Lectins in diagnosis of bladder carcinoma.

With the purpose of studying changes in the expression of glycoconjugate structures in nonmalignant and cancerous lesions of urothelium the lectins ConA, TKA, PNA, DBA, STA, LFA, UEA, MPA, RCA, LCA, GSA1, SBA, GSA2, WGA, PHA and Lot were tested in formalin-fixed, paraffin-embedded tissue sections of (1) cold biopsies from normal urothelium and bladder cancer of different grades (G1-G3) in humans, (2) normal transitional epithelium and N-butyl-N(4-hydroxybutyl)nitrosamine (BBN)-induced bladder cancer in animal experiments (Wistar rat), and (3) human transitional cancer cell line HT 1376. In human urothelium TKA and SBA were positive markers demonstrating positive staining reactions in all tumor grades without binding to normal epithelium. They stained also the human transitional carcinoma cell line HT 1376 (G3). In Wistar rats DBA, ConA, LCA, SBA, GSA2 and WGA had a specific affinity to BBN-induced carcinoma. Findings of positive lectin marker in transitional cell cancer may offer progress in diagnostics and therapy.     

Distribution of glycoconjugates in normal rat ventral prostate and their use as markers of androgen-controlled secretory function in culture

The distribution of glycoconjugates in uncultured and cultured rat ventral prostate was studied by using eight fluorescent lectins. The prostate pieces were cultured in defined medium with or without testosterone for 1-14 days. Each lectin revealed a characteristic binding pattern. Con A, LCA, WGA, and RCA I stained both epithelial and interstitial components. SBA and PNA were specific for the epithelium: SBA stained the region of the Golgi complex; PNA showed the brightest fluorescence in the apical part of the cells representing the region of secretory granules. In culture without testosterone the epithelial cells gradually lost their fluorescence, whereas the stromal fluorescence increased. The basement membrane was disorganized. With testosterone the integrity of the epithelium and stroma was maintained, and the staining pattern of the lectins was in the main similar as in vivo. However, at 14 days a change in the staining pattern of apical cytoplasm with PNA was noted, indicating that in long-term cultures, in addition to testosterone, other hormones and growth factors are necessary to complete especially the last stages of the secretory process in the epithelial cells.     

Lectin-Binding Sites in Normal Human Testis/Lektinbindungsstellen normaler humaner Hoden

Nine fluorescein isothiocyanate (FITC)-labelled lectins have been used to investigate the distribution of glycoconjugates in unfixed frozen and Bouin-fixed sections of normal human testis. Interstitial cells and lamina propria of seminiferous tubuli were stained by PNA, HPA, RCA II, SBA, ConA, and WGA indicating an abundance of the following glycoconjugates: N-GlcNAc, N-GalNAc, Gal, and Man. The germinative cells were stained cytoplasmatically by ConA (Alpha-D-Man/-Glc). Sertoli cells showed the same pattern with ConA. Early spermatids fixed PNA and RCA II in the acrosomal region. Elongated spermatids fixed WGA on their acrosomes and fainty on the flagellae too indicating abundance of N-GlcNAc residues. The findings argue for differentiation-related modifications of lectin-binding sites on germinative cells and the usefulness of Bouin-fixed samples for lectin histochemistry.     

Lectin-binding pattern of bull testis and epididymis

Seven rhodamine-conjugated lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) were used to study the distribution of glycoproteins in the testis and epididymis of immature, juvenile, and adult bulls. A marked change was found in the staining pattern of the lectins in the seminiferous tubules during acrosomal development, and the Sertoli cells seemed to have a cyclic affinity for some of the lectins. The distribution of lectin staining in six regions of the bull epididymis showed some typical differences that were associated with the secretory and absorptive functions of the organ. Region 1 was characterized by strong surface and villous staining and a patchy reaction in the principal cells. Regions 2 and 3 showed a strongly reactive apical Golgi zone and secretory material. In regions 4 and 5, the Golgi zone was subapical but strongly reactive with most lectins, while in region 6 a weakly reactive apical Golgi zone was found. During sexual maturation, an increasing number of basal cells with a strong affinity for some lectins was found at the periphery of the epithelium in regions 2 to 6. These regions also had lectin-stained material along the basal border of the principal cells. These findings suggest that the basal cells may be active in the digestion of absorbed material and that they derive from the principal cells, which may be active in transporting absorbed material to them. The staining pattern of the spermatozoa changed during their transit through the epididymis. The degenerating cells in the testis and epididymal tubules also showed an altered affinity for the lectins.     

Analyses of avidin-biotin complexes with lectins of membrane glycoproteins in the urinary bladder of rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine

The bladder epithelium was examined by staining with avidin-biotin complexes with lectins to determine the early membrane changes during bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BHBN). Concanavalia ensiformis (Con A), Triticum vulgaris (WGA), Ricinus communis (RCA) and Arachis hypogaea (PNA) were used as probes. The cellular distributions of lectin particles were distinguished into membranous and cytoplasmic patterns. Normal bladder cells stained very slightly and showed a spotty membranous pattern. After treatment of rats with BHBN for two or three weeks, staining became stronger and its pattern changed from a membranous to a cytoplasmic type. The staining of bladder cancer cells induced by BHBN varied from area to area and with different lectins. These data indicated that changes in carbohydrates occur in the bladder cell membrane during the early phase of carcinogenesis.     

Lectins expression of parathyroid glands with primary hyperparathyroidism

The binding sites of lectins in parathyroid glands were determined by an immunohistochemical method in normal parathyroid gland, hyperplasia, adenoma and carcinoma, the used lectins were commercially available Glycine max (SBA), Concanavalin enciformis (Con A), Triticum vulgaris (WGA), Richinus communis (RCA), Banderiaea simplicifolia II (BSA II) and Arachis hypogaea (PNA). For normal parathyroid glands (2 cases) and hyperplasia (2 cases), WGA and BSA II were stained in cytoplasma and cell membrane. For carcinoma (1 cases), all lectins but BSA II were positively stained. In particular, SBA revealed more stronger stain than any other hystological types. From the staining patterns of lectins, it was suggested that adenomas (22 cases) be divided into one group similar to carcinoma and the others to normal parathyroid gland and hyperplasia. But there was no difference in clinical data of patients between the two groups.     

Localization of carbohydrate component in human synovial lining cells with fluorescent lectins and enzyme digestion

FITC-conjugated lectins, Con-A, DBA, GS-I, GS-II, PNA, MPA, RCA-I, SBA, UEA-I, WGA were used for demonstration of lectin bindings of human synovial lining cells, obtained from the patients with rheumatoid arthritis (RA), osteoarthritis (OA), aseptic necrosis (AN), and traumatic injury (TI). In the RA samples, GS-I binding to the cytoplasmic sites was predominantly noted and moderate SBA and MPA bindings were observed. However, PNA was not significant. In the OA samples, predominant binding was found in GS-I and SBA lectins, moderate binding in MPA and PNA. In the AN samples, binding was predominant in MPA, moderate in GS-I, SBA and PNA. After neuraminidase treatment the intensity of fluorescence increased significantly with PNA and moderately with SBA in the RA samples. These results suggested that the inflammatory lining cells produce galactose group and the content of neuraminic acids in the synovial membranes of the RA appears to be greater than in those of other diseases.     

Carbohydrate residues in non-malignant prostatic epithelium as revealed by lectins

Summary Non-neoplastic prostatic epithelium from 39 patients obtained at transurethral resection for outflow tract obstruction and 5 normal prostates from men under 35 years of age obtained at postmortem were formalinfixed and paraffin-embedded. The distribution of 8 lectin receptors were studied using a peroxidase anti-peroxidase method and an avidin-biotin method. Con A, WGA, and PNA bound to most epithelial cells. Con A,and WGA also showed major stromal binding. Approximately 5% to 10% of cells bound UEA1, GS1, DBA, and BPA. No major differences in lectin receptor expression were observed between normal and hyperplastic epithelium with either of the immunohistochemical techniques except that hyperplastic cells stained more strongly than normal epithelium.     

Characterizing the glycocalyx of poultry spermatozoa: II. In vitro storage of Turkey semen and mobility phenotype affects the carbohydrate component of sperm membrane glycoconjugates

The turkey sperm glycocalyx is known to contain residues of sialic acid, alpha-mannose/alpha-glucose, alpha- and beta-galactose, alpha-fucose, alpha- and beta-N-acetyl-galactosamine, monomers and dimers of N-acetyl-glucosamine, and N-acetyl-lactosamine. Potential changes in these carbohydrates during in vitro semen storage at 4 degrees C were evaluated using males of both high- and low-sperm-mobility phenotypes. Changes in carbohydrate residues were quantified by flow cytometry analysis using a battery of 14 fluorescein isothiocyanate-labeled lectins in combination with control (sialylated) or neuraminidase-treated (nonsialylated) sperm. Sperm were evaluated at 0, 2, 4, 8, 12, and 24 hours of storage. For control sperm, 4 different patterns of lectin binding were observed over time: 1) increased mean fluorescence intensity (MnFI) at 2 hours (Griffonia simplicifolia lectin-I [GS-I]) and 8 hours (Ricinus communis lectin-I [RCA-I]) that remained elevated during storage; 2) increased MnFI at specific time points (Limax flavus lectin [LFA], 2 hours; Artocarpus integrifolia lectin [jacalin] and succinyl Triticum vulgare lectin [sWGA], 8 hours; Galanthus nivalis lectin [GNA], 12 hours) followed by decreasing MnFI during the remainder of the 24-hour storage period; 3) increased MnFI only at the 24-hour time point (Lotus tetragonolobus lectin [lotus] and Arachis hypogaea lectin [PNA]); and 4) no changes in MnFI during the 24-hour storage period (Erythrina cristagalli lectin [ECA], GS-II, Pisum sativum lectin [PSA], Glycine max lectin [SBA], and Wisteria floribunda lectin [WFA]). For nonsialylated sperm, increased binding of ECA, GS-II, SBA, and WFA was observed at variable time points; only Canavalia ensiformis lectin (Con A) and PSA remained unchanged during storage. Differences between mobility phenotypes existed for lectins Con A, GS-II, LFA, PSA, SBA, and sWGA, with sperm from low-mobility males exhibiting higher MnFI than high-mobility males throughout 24 hours of storage. We concluded that the observed increases in lectin binding during semen storage indicate an augmentation of nonsialylated terminal residues, which could alter sperm antigenicity and negatively impact fertility. Further, spermatozoa from low-mobility males may have higher antigenicity even before semen storage. Other possible functional implications are discussed.     

Localization of specific carbohydrate configurations in the hemophilic synovial membrane

Fluorescein isothiocyanate-conjugated lectins include: concanavalin A (Con-A), Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia-I (GS-I), Griffonia simplicifolia-II (GS-II), Arachis hypogaea agglutinin (PNA), Maclura pomifera agglutinin (MPA), Ricinus communis agglutinin-I (RCA-I), Glycine max agglutinin (SBA), Ulex europaeus agglutinin-I (UEA-I), and wheat germ agglutinin (WGA). These lectins were used to histochemically demonstrate lectin bindings on hemophilic synovial membrane. GS-I (galactose and galactosamine specific) and SBA (galactosamine specific) was shown to bind strongly in the cytoplasm of the lining cells. Lymphocytes and mast cells were largely bound by Con-A (mannose and glucose specific) and GS-II (glucosamine specific) positive. UEA-1 (fucose specific) was shown to bind specifically to synovial vascular endothelial cells. Lectin histochemistry is a useful method for classification of the cells on the synovial membrane.     

Distribution of Glycoconjugates in Human Testis A Histochemical Study Using Fluorescein- and Rhodamine-conjugated Lectins

Differentiation in the seminiferous epithelium involves the orderly transformation of germ cells into spermatozoa. We have employed ten fluorescein- and rhodamine-labeled lectins to visualize distinctive changes in the distribution of carbohydrate containing compounds during spermatogenesis and noticed the increase in RCA I, PNA, SBA and HPA binding sites during germ cell differentiation, suggesting the appearance of certain galactose and N-acetylgalactosamine containing glycoconjugates. Besides, in the cytoplasm of all germ cell types the positive reactions with Con A, LCA, WGA, LPA and UEA I indicate the presence of mannose, N-acetylglucosamine, sialic acid and fucose containing glycosubstances. Developing acrosomes demonstrated binding sites for most lectins, and particular HPA binding glycoconjugates were expressed in the equatorial segment region of late spermatids and testicular spermatozoa. In addition, the characteristic staining patterns of other testicular compartments are described. Our results suggest that human germ cells are rich in various carbohydrate containing compounds and there are specific alterations in cellular glycoconjugates during germ cell differentiation.     

Papillary serous cystadenoma of the testis

A case is presented of a 50-year old man with a unilocular cystic intratesticular tumour exhibiting the morphological features demanded from WHO for the diagnosis of serous papillary cystadenoma of the ovary. Keratin filaments could be demonstrated in the cyst lining and papillae covering cells by means of PAP-technique; AFP and SP-1 were lacking. The epithelial cells of the tumour showed a lectin binding pattern (WGA, UEA-1, PNA, Con A, PSA, LCA, RCA) different from the epithelium of rete testis and epididymis. We intend to classify our tumour as the male analogue of the respective ovarian growth.     

Profiling of glycan alterations in regrowing limb tissues of Cynops orientalis

Glycans are known to play important roles in molecular recognition, cell–cell adhesion, molecular trafficking, receptor activation, and signal transduction during development and regeneration. Despite numerous investigations of regenerating salamander limbs, global analysis of the precise variation of glycans during the limb regeneration process has received little attention. Here, we have used lectin microarrays and lectin histochemistry to analyze the alterations and distribution of glycans during the early stages leading to blastema formation during Cynops orientalis limb regeneration in response to limb amputation. Compared with the control group, analysis at several time points (3, 7, and 14 days postamputation) using microarrays containing 37 lectins showed that limb tissues expressed significantly different complements of glycans recognized by 9 different lectins. Postamputation limb tissues showed higher expression of two glycan structures recognized by the lectins STL and LTL and lower expression of seven glycan structures recognized by PHA‐E, MAL‐I, SNA, UEA‐I, PHA‐E + L, VVA, and GNA. We also observed significant changes in glycans specifically at 7 days postamputation, including higher binding capacity by WFA, GSL‐I, and NPA and lower binding capacity by PNA, HHL, ConA, LCA, GSL‐II, and PWM. Next, we validated our lectin microarray data using lectin histochemistry in limb tissues. Glycans recognized by STL and GNA showed similar changes in signal intensity to those found in the lectin microarrays, with STL staining in the cytoplasm and GNA in the cytoplasm and nucleus. Our findings are the first report of significant postamputation changes in glycans in limb tissues and suggest that those glycans perform potentially important functions during the early stages of C. orientalis limb regeneration.