Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation

The transit of spermatozoa in the genital tract of the male mouse was investigated by quantitative light microscopic autoradiography after intraperitoneal injection of tritiated thymidine. Transit duration in the caput and the corpus of the epididymis was shown to be 3 days; the total duration of transit in the genital tract was 5 days. These findings indicate that the time required for the transit of spermatozoa in the epididymal caput and corpus was comparable to that calculated in other mammals studied. However, the duration of sperm storage in the epididymal cauda appeared to be shorter than that previously reported for rodents.     

Antibodies to sperm surface fertilization antigen (FA-1): their specificities and site of interaction with sperm in male genital tract

The fertilization antigen (FA-1) isolated from murine testes demonstrated its dimeric form of 49,000 +/- 2,000 molecular weight (M.W.) or a monomer of 23,000 M.W. on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The FA-1 was immunogenic in all three female rabbits tested and raised a high-titer antisera [enzyme-linked immunosorbent assay (ELISA) titers; 1:1,024 to 1:4,096]. The rabbit anti-FA-1 antisera predominantly recognized the dimeric form of 49,000 +/- 2,000 M.W. on the Western blot of lithium diiodosalicylate (LIS)-solubilized murine testes. None of the antisera reacted with any somatic tissue, indicating germ-cell specificity of FA-1. To determine the cellular localization of the immunoreactive FA-1, a novel ultrasensitive immunogold-silver staining (IGSS) procedure was developed. The anti-FA-1-IgG showed intense staining in the luminal region of the seminiferous tubules containing spermatids and spermatozoa. No reaction was observed in the peripheral area of the tubules containing Sertoli cells, spermatogonia, leptotene, and zygotene spermatocytes. The biodistribution studies of 125I-labeled anti-FA-1 IgG in mice revealed that the antibodies do not bind to somatic tissues such as blood cell, liver, heart, kidney, muscle, and gastrointestinal tissue and do not transudate into testes and seminal vesicle. However, the antibodies preferentially transudate into epididymis (especially corpus or cauda regions) and vas deferens to bind to sperm cells. In conclusion, our data indicate that FA-1 can induce an immune response that is germ cell-specific, directed against later stages of spermatogenesis. The antibodies to FA-1 interact with sperm after penetration through epididymis (especially corpus and cauda regions) and vas deferens rather than through testes and seminal vesicles.     

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.     

A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida

Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha- lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta- N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.     

Plasminogen activator: Morphological evidence of binding, internalization and delivery to lysosomes in 3T3 mouse fibroblasts

Summary Using a direct conjugate of urokinase and ferritin, the binding has been followed at the plasma membrane and the internalization of urokinase into BALB/C-3T3 fibroblasts, cultured in plasminogen-free conditions. At 0° C, the conjugate was observed bound on both coated and uncoated cell surface regions as singlets, and small and large clusters. No binding was observed in the presence of excess native urokinase. The binding was impaired by preincubation of the conjugate with a competitive inhibitor of the catalytic site, suggesting an interaction between the receptor and the catalytic site of the enzyme.Within 1 min at 37° C, urokinase clustered on coated regions of the plasma membrane. At 5 min after warming, ferritin was found on deeply indented coated pits and in both coated and uncoated vesicles close to the cell surface. By 10 min at 37° C, ferritin particles were present in uncoated endosomes and in multivesicular bodies in the Golgi area. Within 10 min, the receptors on the surface strongly decreased. New receptors were observed on the membrane after 20 min at 37° C. At this time, ferritin was observed both in endosomes or multivesicular bodies and in vesicles close to the plasma membrane.     

Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: colocalization of cell surface urokinase with vinculin

Several cell types display binding sites for [125I]urokinase (Vassalli, J.-D., D. Baccino, D. Belin. 1985. J. Cell Biol. 100:86-92) which in certain cases are occupied with endogenous urokinase. These sites appear to focus urokinase at cell surfaces and hence may participate in tissue matrix destruction and cell invasion. Recently Pollanen et al. (1987) demonstrated that the cell surface urokinase of human fibroblasts and fibrosarcoma cells is deposited underneath the cells in strands, apparently at sites of cell-to-substratum contact. Here, using immunofluorescence double labeling, we show that the urokinase strands present on human foreskin fibroblasts are colocalized with strands of vinculin, an intracellular actin-binding protein that is deposited at cell-to-substratum focal adhesion sites. Thus, this indicates linkage of the plasminogen/plasmin system both to sites of cell adhesion and to the cytoskeleton. The urokinase strands on HT 1080 fibrosarcoma cells are more numerous and have shapes that are more tortuous than those on normal fibroblasts. In intact HT 1080 cells, colocalized vinculin strands are obscured by an intense background of soluble vinculin but are apparent on isolated ventral plasma membranes. Certain properties of the urokinase strands suggest that they are related to the [125I]urokinase-binding sites that have been described by several groups: (a) incubating fibroblasts with dexamethasone for 48 h or at pH 3 at 5 degrees C for 10 min greatly decreases the number and intensity of the urokinase strands; (b) strands reappear when glucocorticoid- treated cells are incubated with exogenous 54-kD (but not 35-kD) urokinase, and this process is inhibited by a previously described 16- amino acid peptide that blocks [125I]urokinase binding to the cells.     

Molecular differentiation of the mouse genital tract: altered protein synthesis following prenatal exposure to diethylstilbestrol

Exposure in utero to the synthetic estrogen diethylstilbestrol (DES) has been associated with the subsequent development of reproductive tract lesions in both women and experimental animals. Using the techniques of organ culture and two-dimensional (2-D) gel electrophoresis, the effects of DES on protein synthetic patterns were studied during fetal and neonatal development of the CD-1 mouse. The protein patterns, analyzed by comparing 2-D fluorograms after [35S] methionine incorporation at different developmental stages, were correlated with the histology at the same age. Several qualitative and quantitative changes in protein synthesis were observed after prenatal DES exposure. A protein, apparent by Day 14 of gestation, with molecular weight approximately 70,000 and pI of 5.8, was observed to be greatly diminished in all reproductive tract tissues exposed to DES during prenatal development. This alteration, induced in utero, persists through the early postnatal differentiation of the genital tract (17 days old). This protein may provide an early marker for alterations in normal reproductive tract function.     

Geometry of GPPE binding to picrate and to the urokinase type plasminogen activator

Crystal structure of 2-(4-guanidynephenyl)-1-phenyl-ethanone (GPPE) in two different environments was determined in order to compare the binding geometry of these compound to a simple picrate anion and to protein, urokinase-type plasminogen activator (uPA), which may be treated as a target for anti-cancer drugs. It was shown that the conformation and the hydrogen-bonding formation by GPPE molecule are similar in both environments, but several important differences were discovered and described.     

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.     

Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin

Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.     

Hypochlorous acid-induced stress on human spermatozoa. A model for inflammation in the male genital tract

The fertilising ability of human spermatozoa may be impaired by inflammations of the genital tract, although details of these processes are still unknown. Hypochlorous acid (HOCl), an important product of myeloperoxidase released from stimulated neutrophils, induces a concentration-dependent increase in externalisation of phosphatidylserine in ejaculated human spermatozoa as revealed by fluorescence-activated cell sorting (FACS) analysis. The increase of annexin-V binding cells starts already at about 10(-5) mol/l HOCl, while a formation of lysophosphatidylcholines as detected by matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is only found at HOCl concentrations higher than 10(-4) mol/l. Thus, changes in lipid composition of spermatozoa are unlikely responsible for the phosphatidylcholine (PS)-externalisation. These data gave concomitant evidence that HOCl itself leads to a dramatic damage of the cell membrane. Thus, the neutrophil-derived HOCl contributes to the deterioration of spermatozoa leading to diminished fertilisation ability.     

Ultrastructure of the male genital tract,spermatogenesis and spermatozoa of hattena cometis domrow (Acari: Gamasida: Ameroseiidae)

The ameroseiid mite Hattena cometis has a male genital system that consists of an unpaired, u‐shaped testis and paired deferent ducts leading into an unpaired accessory genital gland and ejaculatory duct. The genital opening is located anteriorly immediately in front of the sternal shield. Spermatogenesis is simple, probably due to the haploid nature of the male. Eight stages of spermatogenesis could be roughly distinguished. Mature spermatozoa as found in the deferent duct lumen are peculiar in having a bisected nucleus and numerous peripheral flat chambers, which were formed from indentations of the plasmalemma. In inseminated females, spermatozoa were observed in the syncytial tissue of the sperm access system and in the somatic cells of the ovary. These spermatozoa have achieved a new structure, i.e., an electron‐dense plate dividing the cell into two unequal halves. The dense plate has an intricate substructure. Its function is unknown. These sperm cells are considered to represent capacitated spermatozoa. The peripheral chambers are reduced in number inside the female. Similar sperm cells, containing a dense plate, were seen in vacuoles within the epithelium of the deferent duct of one male. These cells are evidently under destruction, but before being completely dissolved had undergone a development leading beyond that of the mature sperm cells found in the deferent duct. Apparently, entering the cell of the deferent duct epithelium or the syncytium tissue triggers the production of the dense plate (or the capacitation process). Our observations are compared with results obtained from other anactinotrichid Acari, mainly Gamasida, and confirm and complete the interpretation of the correlated evolution of components of gamasid reproductive systems. J. Morphol. 274:1010–1025, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro

In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.     

Immunolocalization of angiotensin 1 converting enzyme in the human male genital tract by the avidin-biotin-complex method

Summary Immunoreactivity for Angiotensin 1 Converting Enzyme was investigated in a series of 12 fixed and paraffinembedded normal human genital tract specimens. The Avidin-Biotin-Complex immunoperoxidase method was used with overnight (12 h) incubation with a polyclonal antihuman kidney Angiotensin 1 Converting Enzyme antiserum. All tissues, including testis, different parts of epididymis, ductus deferens, prostate and seminal vesicles, demonstrated a staining pattern. Immunoreactivity was observed on the luminal surface of these epithelia especially on nonmotile stereocilia. An intracellular positivity was only observed in spermatids on the acrosomal cap. Besides, an immunologic identity of Angiotensin 1 Converting Enzyme located on the different epithelia of the human male genital tract, on the endothelial cells of vessels and on the proximal tubule brush border of the kidney was observed.     

Immunolocalization of angiotensin 1 converting enzyme in the human male genital tract by the avidin-biotin-complex method

Immunoreactivity for Angiotensin 1 Converting Enzyme was investigated in a series of 12 fixed and paraffin-embedded normal human genital tract specimens. The Avidin-Biotin-Complex immunoperoxidase method was used with overnight (12 h) incubation with a polyclonal antihuman kidney Angiotensin 1 Converting Enzyme antiserum. All tissues, including testis, different parts of epididymis, ductus deferens, prostate and seminal vesicles, demonstrated a staining pattern. Immunoreactivity was observed on the luminal surface of these epithelia especially on non-motile stereocilia. An intracellular positivity was only observed in spermatids on the acrosomal cap. Besides, an immunologic identity of Angiotensin 1 Converting Enzyme located on the different epithelia of the human male genital tract, on the endothelial cells of vessels and on the proximal tubule brush border of the kidney was observed.     

Fate of superfluous sperm products after vasectomy and in the normal male tract of the mouse

The progression of 3H-labelled spermatozoa (thymidine or arginine) was followed through the tracts of unilaterally vasectomized, bilaterally vasectomized, oligozoospermic (t6/tw5) and normal mice; the regional lymph nodes were also investigated. The same rate of sperm production and transport was found in normal and in vasectomized tracts, down to the corpus epididymidis; there was some delay in spermatozoa entering the cauda in the vasectomized tracts. In the mouse, therefore, vasectomy does not affect the rates of sperm production or transport until just before the blockage in the swollen cauda epididymidis. Radioactivity appeared in the caudal and 'para-aortic' lymph nodes as the radioactive spermatozoa passed from the corpus, showing that this is one route of disposal of spermatozoa, or of sperm products, after vasectomy. Naturally oligozoospermic and normal mice gave similar results; again the caudal, iliac and renal lymph nodes received radioactive spermatozoa/sperm products. Some loss of (by definition) superfluous spermatozoa in the normal male tract therefore occurs naturally by this route, and we suggest that vasectomy further exploits this physiological pathway. This would account for the finding that many males do not make antisperm antibodies after vasectomy, just as normal males do not, even though their lymph nodes normally receive spermatozoa/sperm products.     

Relationship between two types of mouse sperm surface sites that mediate binding of sperm to the zona pellucida

There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.     

Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding

We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.