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.     

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.     

Testicular Lectinhistochemistry in the Sertoli-Cell-Only-Syndrome/Testikuläre Lectinhistochemie beim Sertolizell-Syndrom

Glycoconjugate expression was studied in tissue specimens of the Sertoli-cell-only-syndrome by lectinhistochemistry. Both ConA and WGA marked the cell surface of Sertoli cells fainty. UEA I labelled their nucleoli. PNA and RCA failed to stain Sertoli cells. These staining patterns resembled those of normal human testis. The thickened tubular walls in the Sertoli-cell-only-syndrome were abundant in binding sites for WGA, PNA, UEA I, and RCA. ConA stained the outer part of the wall heavily, but failed to react with the adluminal part. It is suggested, that the Sertoli-cell-only-syndrome is accompanied by a selective loss of mannosyl residues in the adluminal part of thickened tubular walls.     

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.     

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。     

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.     

Effect of lectins on KK-47 bladder cancer cell line

The effect of three lectins, Ricinus communis agglutinin (RCA II), concanavalin agglutinin (ConA), and wheat germ agglutinin (WGA), on KK-47 bladder cancer cell line was studied, RCA II showed effective inhibition of H3-uridine and H3-thymidine uptake by KK-47. ConA showed a stimulatory effect in all three concentrations used. WGA also showed stimulatory effect, but it was less pronounced than ConA.     

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.     

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.     

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.     

Immunohistochemical localization of calmodulin

The localization of calmodulin in the normal human testis was studied by the indirect immunoperoxidase method. Three different types of fixative were used: phosphate-buffered formalin, Bouin's solution or Carnoy's solution. Immunoreactivity specific for calmodulin was not detectable in the testis fixed in Carnoy's solution. The specimens fixed in phosphate-buffered formalin and in Bouin's solution were stained. The immunostaining for calmodulin was observed in pachytene spermatocytes, secondary spermatocytes and round spermatids but not in spermatogonia, or in pre-leptotene, leptotene and zygotene spermatocytes, elongated spermatids, spermatozoa or Leydig cells. Sertoli cells were not stained or were stained slightly. Among pachytene spermatocytes, the cells at the early stage were barely stained but those at the middle and late stages were slightly and intensely stained, respectively. The present report provides the first confirmation of the localization of calmodulin in the human testis.     

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 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.     

Changes in the lectin binding sites on the testicular, epididymal, vas, and ejaculated spermatozoon surface of dog

Summary. Changes in the localization of sperm surface glycocomponents of testicular, epididymal, vas deferens, and ejaculated spermatozoa of dog ( Canis domesticus ) were studied employing fluorescein isothiocyanate conjugated lectins viz., Concanavalin A (ConA), Triticum vulgaris (WGA), Maclura pomifera (MPA), and Arachis hypogaea (PNA) agglutinins. The plasma membrane clothing the acrosome of the testicular, epididymal, and vas deferens spermatozoa shows reactivity with all the lectins used. However, in the ejaculated spermatozoa, the entire sperm surface shows reactive sites for ConA, WGA, and PNA. Variation in the labelling of the cytoplasmic droplet in different stages of spermatozoon transit in the epididymis has been discussed.     

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.     

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.     

Age-dependent changes in the carbohydrate pattern of human prostatic epithelium as determined by peroxidase-labeled lectins

Peroxidase-conjugated lectins (peanut agglutinin: PNA; Ricinus communis agglutinin: RCA) were used for histochemical demonstration of the respective carbohydrate binders (PNA: β-D-galactosyl-(1-3)-N-acetyl-galactosamine; RCA: D-galactose). The study of prostatic specimens from four different age groups showed marked differences in cytoplasmic PNA binding of prostatic epithelium. The basal cells of the glandular epithelium displayed cytoplasmic PNA binding only in the infantile stage and during early puberty. With the onset of puberty a number increasing with age of secretory cells developed cytoplasmic binding of PNA, mostly appearing as apically located vacuoles. Intraluminal secretion, however, was never stained. During senile involution the prostatic epithelium lost rapidly its PNA binding capacity. Since no RCA binding of the epithelium was observered, the carbohydrate moiety responsible for PNA binding presumably represents a galactosyl-N-acetyl-galactosamine containing molecule, most likely a glycoprotein, which is present in actively functioning pro-static epithelial cells. It is uncertain whether or not it represents a structural or a secretory component of the prostatic cell.     

Identification of N- and O-linked oligosaccharides in human seminal vesicles

The main oligosaccharide residues and the saccharide linkage in infantile and adult human seminal vesicles were studied by means of lectin histochemistry at light and electron microscopy levels. In adult glands, the epithelial cell cytoplasm and luminal content reacted positively to the following residues: (GlcNAc)n (WGA), Galbeta1,3GalNAc (PNA), GalNAcalpha1,3Gal (SBA), GalNAcalpha1,3GalNAc (HPA), Fucalpha1,2Galbeta1,4GlcNAc (UEA-I), and alphaL-Fuc1,6DGlcNAc-O-Melibiosc (AAA). The presence of intense staining in the luminal content suggest that glycoproteins containing these oligosaccharide moieties are secreted by epithelial cells. Adult epithelial cells also reacted to Neu5Acalpha2,6Gal (SNA), Neu5Acalphaa2,3Galbeta1,4GlcNAc (MAA), Galbeta1,4GlcNAc (DSA), branched mannose chains (ConA), Man1,3Man (GNA), and Fucalpha1,2Galbeta1,4GlcNAcFucalpha1,3GlcNAc (LTA) but reaction to these residues was weak (MAA, DSA, ConA, and LTA) or absent (SNA and GNA) in the gland lumen, which suggests that they belong to intracytoplasmic proteins. The chemical and enzymatic treatments used suggest that the residues recognized by SNA, MAA, PNA, DSA, HPA, and SBA belong to O-linked oligosaccharides; those residues localized by ConA and GNA have an N-glycosidic linkage, and those bound by WGA, LTA, UEA-I, and AAA are linked to both N- and O-oligosaccharides. In prepubertal seminal vesicles, reaction in the epithelial cell cytoplasm was similar to that observed in adults, except for GNA and HPA, which showed a weaker reaction. However, the lumen of prepubertal seminal vesicles showed intense reaction to WGA and SBA only. The chemical and enzymatic treatments suggest that the scanty glycoproteins secreted by the prepubertal glands belong to the mucin-type.     

Lectin binding in immunohistologically AFP/HCG positive and negative testicular carcinoma

The morphologic display of testicular cancer is a heterogenous cellular pattern. A biological heterogeneity is also true for the expression of tumor markers. The biosynthesis of tumor marker proteins alpha-fetoprotein (AFP), ferritin, Schwangerschaftsprotein (SP 1) glycoprotein, tissue polypeptide antigen and of hormones (beta-human chorionic gonadotropin (HCG) = significantly present in nonseminoma germ cell tumors--does, however, define only a small number of cancer cells. To better visualize the majority of cancer cells, lectin binding was studied. During the oncogenic transformation a distinct change of cell membrane glycoproteins has been observed. Reactions of WGA/PNA lectins which get attached to glycoproteins with cancer tissue sample from seminomas (n = 20) and nonseminomas (n = 20) were analyzed. The results were correlated to AFP/beta-HCG positive (negative) immunohistology to establish further subgroups of biological homogeneity. The binding of WGA lectin appears relatively more frequent in both seminoma and nonseminoma than that of PNA. Lectin binding of WGA and/or PNA can be stained in 3/11 AFP- and beta-HCG-negative nonseminoma tissues while lectin staining is positive in 7/18 beta-HCG-negative seminomas. The fact that lectin binding is dependent on the spermatozoogenesis and on androgens in normal testis tissues asks for more detailed studies in this field.     

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.