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.     

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.     

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 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.     

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.     

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.     

Characterization and expression of a stage specific antigen by monoclonal antibody TRA 54 in testicular germ cells

To study the mechanism of spermatogenesis, we have isolated many monoclonal antibodies (mAb) which recognize specific steps of mouse germ cell differentiation and then have evaluated the specific expression and characterization of antigenic molecules using immunohistochemistry and Western blotting. Monoclonal antibody TRA 54 recognized specific organelles in germ cell cytoplasm from spermatocytes to spermatids; that is, a large granule was stained in mid–late pachytene, diplotene and secondary spermatocytes and in round spermatids at stage I while the acrosome of spermatids at steps 2–3 to step 12 were also positive. Thereafter, the antigens disappeared from spermatids at more advanced stages of differentiation. Western blots using TRA 54 revealed broad bands with approximate molecular weights of >200, 190 and 85 kDa in the testis. The expression of these antigens during testicular germ cell development should be of interest in relation to the biogenesis of organelles such as the chromatoid body and acrosome and will be a useful stage-specific molecular marker for the study of spermatogenesis.     

Phosphorylation of mitogen-activated protein kinase 8 (MAPK8) is associated with germ cell apoptosis and redistribution of the Bcl2-modifying factor (BMF)

Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids. When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.     

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.     

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.     

A possible direct action of oxytocin on spermatogenesis and steroidogenesis in pre‐pubertal mouse

The aim of this study was to evaluate the effects of in vivo and in vitro treatments of oxytocin (OT) on the testis of pre‐pubertal mice. The OT treatment produced significant changes in the spermatogenic and steroidogenic activity by increasing expression of OT‐receptor in the testis of pre‐pubertal mice. Treatment with OT showed increased proliferation of germ cells as indicated by increased number of spermatocytes and round spermatids. Dose‐dependent increase in expression of PCNA, Bcl‐2 and AR proteins was observed in the testis of OT‐treated mice as compared with the control and further supports the role of OT in germ cell proliferation and survival. The pre‐pubertal mice treated with increasing dose of OT showed significant increase in testosterone synthesis due to dose‐dependent stimulatory effects on 3β‐HSD activity and increased expression of STAR, LH‐receptor (LH‐R) and gonadotrophin‐releasing hormone receptor (GnRH‐R) proteins in the testis. The in vitro study has confirmed in vivo finding showing direct action of OT on testicular steroidogenesis. Thus, OT stimulates testicular spermatogenesis and steroidogenesis by directly acting on testis in mice.     

Lectin Histochemical Study of Lipopigments Present in the Cerebellum of Solanum fastigiatum var. fastigiatum Intoxicated Cattle

This study was carried out to investigate the pattern of lectin binding in the cerebellum of calves poisoned with Solanum fastigiatum var. fastigiatum. For the experimental reproduction of the illness, S. fastigiatum var. fastigiatum was collected from farms where the intoxication occurs. The dried ground plant was administered to two 1‐year‐old cattle by a ruminal cannula. The animals received 5 g/kg b.w. daily, 5 days a week, during periods of 107 and 140 days. After these periods the animals were bled to death. For the histological study, transverse sections of the cerebellum were used. Paraffin‐embedded sections were incubated with the following biotinylated lectins with different specificity: Concanavalia ensiformis (Con‐A), Glycine max (SBA), Dolichos biflorus (DBA), Ulex europeus‐I (UEA‐I), Triticum vulgaris (WGA), succynyl‐WGA (sWGA), Arachis hypogaea (PNA), Ricinus communis‐I (RCA‐I) and Bandeirea simplicifolia‐I (BS‐I). Avidin–biotin–peroxidase complex was applied as a detection system. Purkinje cells showed vacuolation in the pericaryon. The stored material present in the cells reacted strongly with the following lectins: Con‐A, sWGA, WGA and RCA‐I. An irregular affinity was observed with PNA and DBA. The lectin‐binding pattern was compatible with a glycolipid storage disease.     

Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated

The Y-chromosomal DAZ (deleted in azoospermia) gene and the autosomal Dazl (deleted in azoospermia-like) gene are two crucial factors for the achievement and maintenance of spermatogenesis. Whereas Y-chromosomal DAZ is present in certain primates, it is lacking in rodents and other species. We have investigated the expression of Dazl protein during spermatogenesis in the adult rat testis using immunohistochemistry. Dazl immunoreactivity was found predominantly in the cytosol of primary pachytene spermatocytes. A weaker but clearly detectable signal was present in intermediate and B spermatogonia and in early spermatocytes from preleptotene to zygotene. The highest expression patterns were observed between stages IV and VIII during the spermatogenic cycle when spermatocytes prepare for the first meiotic division. Specific staining could also be observed in step 11-19 elongating spermatids in the acrosome region. Treatment for 42 days with a potent GnRH-antagonist abolished gonadotrophin secretion and led to a regressed testis, lacking most of the advanced germ cell types such as spermatids but still bearing spermatogonia and spermatocytes. No difference in staining pattern for Dazl protein was observed in GnRH antagonist-treated rats despite the lack of gonadotrophins and substantial impairment of the spermatogenic process, indicating that Dazl expression is clearly hormone-independent. The localization and level of Dazl expression suggests an important role in the regulation of the first meiotic stages of spermatogenesis. The hormone independent onset of expression points to an autonomous cell-cycle event in which Dazl seems to be essential for the entry into meiosis. The presence of Dazl in the acrosome region of elongating spermatids might reflect an unknown role of Dazl as a morphogenetic factor during spermiogenesis.     

Changes in the expression of P-cadherin in the normal, cryptorchid and busulphan-treated rat testis

Adhesion between Sertoli cells and germ cells is important for spermatogenesis. Cadherins are Ca(2+)-dependent transmembrane proteins that mediate cell-cell adhesion. The aim of this study was to compare the expression of P-cadherin in unilaterally cryptorchid and busulphan-treated rat testes using immunohistochemistry. The pattern of expression of P-cadherin in the seminiferous epithelium changed with the stage of the seminiferous epithelium. The membranes of round spermatids and membranes and cytoplasm of spermatocytes were strongly positive. Our experiments revealed that busulphan treatment (2 doses - 10 mg/kg of body weight - 21 days apart) and cryptorchism led to destructive changes in the structure of seminiferous tubules, together with the decrease in P-cadherin expression. The expression of P-cadherin disappeared in the spermatids segregated from the epithelium while segregated spermatocytes remained still positive for P-cadherin during the 3- to 11-day cryptorchid period. In busulphan-treated animals, the expression of P-cadherin was dependent on the presence or absence of the spermatocytes and spermatids in the tubules. Strong positivity for P-cadherin was observed in the spermatocytes that re-appeared in the regenerating seminiferous epithelium. We suggest that P-cadherin participates in the architecture of adherens junctions in testis, plays an important role in maintaining normal spermatogenesis and that cryptorchism and busulphan treatment lead to adherens junction disintegration.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

The length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts

Xenografting of immature mammalian testis tissue into mice can accelerate sperm production. To determine whether this shortened time to sperm production is because of reduced length of the spermatogenic cycle, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Small testis fragments from newborn pigs and sheep were ectopically grafted into mice. Once complete spermatogenesis was present in grafted tissue, mice were injected with BrdU and grafts were recovered at different time points thereafter. In porcine grafts, the most advanced germ cells labeled 1 hour, 9 days, 12.3 days, and 18 days after BrdU injection were stage 1 preleptotene/leptotene primary spermatocytes, stage 1 pachytene primary spermatocytes, stage 5 newly-formed round spermatids, and late stage 2 elongating spermatids, respectively. In ovine grafts, the most advanced labeled germ cells at 1 hour, 11 days, and 22 days post-BrdU injection were stage 2 preleptotene/leptotene primary spermatocytes, late stage 1 pachytene primary spermatocytes, and stage 2 elongating spermatids, respectively. These results indicate that each spermatogenic cycle in porcine and ovine xenografts lasts approximately 9 and 11 days, respectively, which is similar to their durations in situ. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts. This is consistent with earlier reports showing that the cycle length is inherent to the germ cell genotype. The shortened time to sperm production in xenografts therefore appears attributable to accelerated maturation of the testicular somatic compartments. Our results suggest that testis xenografts provide a useful model to study the timing of testicular maturation and spermatogenesis in different mammalian species.     

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.     

Chloroform extract of Carica papaya seeds induces long—term reversible azoospermia in langur monkey

AIM: To evaluate the antifertility activity of the chloroform extract of Carica papaya seeds by oral administration in langur monkey, Presbytis entellus entellus. METHODS: The chloroform extract of Carica papaya seeds, 50 mg/kg/day, was administered orally for 360 days to adult male langur monkeys. The sperm characteristics by light and electron microscopy, the sperm functional tests, the semen biochemistry, the serum testosterone level, the Leydig cell function, and the histology and ultrastructure of testis were determined to evaluate the antifertility activity and the blood biochemistry and hematology, to evaluate the toxicology. RESULTS: The extract gradually decreased the sperm concentration since days 30-60 of treatment with a total inhibition of sperm motility, a decrease in sperm viability and increase in sperm abnormality. Azoospermia was observed after day 90 of treatment and continued during the whole treatment period. Treatment withdrawal resulted in a gradual recovery in these parameters and 150 days later they reverted to nearly the pretreatment values. Morphological observation of the ejaculated sperm by light and scanning electron microscopy showed deleterious changes, particularly on the mid-piece. Sperm functional tests, viz., sperm mitochondrial activity index, acrosome intactness test and hypo-osmotic swelling test scored in the infertile range during treatment and returned to the fertile values 150 days after drug withdrawal. Histology of the testis revealed shrunken tubules, germ cell atrophy and normal Leydig cells. Ultrastructure of the testis showed vacuolization in the cytoplasm of Sertoli cells and germ cells. Loss of cytoplasmic organelles were evident in the spermatocytes and spermatids. Round spermatids showed loss of Golgi bodies, peripheral mitochondria and vacuolated cytoplasm, indicating maturational arrest. Leydig cell functional test indicated a mild inhibition of steroidogenic function. Haematology and serum biochemistry study disclosed no significant toxicological effect and the serum testosterone level was not affected. CONCLUSION: Carica papaya seed extract may selectively act on the developing germ cells, possibly mediated via Sertoli cells, leading to azoospermia.     

HtrA2 is up-regulated in the rat testis after experimental cryptorchidism

AIM: The aim of the present study was to elucidate the role of high temperature requirement A2 (HtrA2) in germ cell loss in the heat-stressed testis. METHODS: We examined the expression of HtrA2, caspase-9 activity and proteolytic activity of HtrA2 in the rat testis, and their in vivo responses to experimental cryptorchid treatment. RESULTS: Northern analysis revealed the expression of HtrA2 mRNA peaked at days 1 and 7 after cryptorchid treatment. While expression of HtrA2 mRNA was seen in the spermatogonium, spermatocytes and some spermatids in normal adult rat testis, experimental cryptorchidism treatment resulted in a marked increase in its signal intensity in spermatocytes and some spermatids, and the layers of spermatogonium and early primary spermatocytes became negative at days 1 and 7 after the treatment. However, the spermatogonium, Sertoli cells and interstitial cells appeared to have strong intensities at days 14, 28 and 56 after the treatment. Western analysis revealed the expression of HtrA2 protein peaked at day 2 coinciding with the increase of positive spermatogonium, the appearance of protein-positive interstitial cells, and day 28 coinciding with the reappearance of protein-positive interstitial cells. Caspase-9 activity peaked at day 2 and HtrA2 proteolytic activity peaked at day 28. Consequently, the first peak of HtrA2 mRNA expression was followed by the peak of caspase-9 activity and the second peak was followed by the peak of proteolytic activity; however, the second peak of mRNA expression had considerable chronological difference from that of the protein. CONCLUSION: These findings suggest the probabilities that the heat stress results in germ cell death by a caspase-independent manner with the elevation of HtrA2 proteolytic activity, as well as a caspase-dependent manner with the elevation of caspase-9 activity.