scholarly journals Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner

Reproduction ◽  
2012 ◽  
Vol 144 (2) ◽  
pp. 209-219 ◽  
Author(s):  
Hongbin Xu ◽  
Fang Liu ◽  
Nopparat Srakaew ◽  
Chaitanya Koppisetty ◽  
Per-Georg Nyholm ◽  
...  
Keyword(s):  
Active Site ◽  
Acrosome Reaction ◽  
Size Exclusion ◽  
Arylsulfatase A ◽  
Dot Blot ◽  
Active Site Pocket ◽  
Acrosomal Membrane ◽  
Sperm Penetration ◽  
Cross Linker ◽  
Inner Acrosomal Membrane

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm–egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.

The histochemical localization of acrosin in guinea-pig sperm after the acrosome reaction

1978 ◽  
Vol 32 (1) ◽  
pp. 177-184
Author(s):  
D.P. Green ◽  
A.R. Hockaday
Keyword(s):  
Electron Microscopy ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Sperm Head ◽  
Ultrastructural Evidence ◽  
Acrosomal Membrane ◽  
Sperm Penetration ◽  
Inner Acrosomal Membrane ◽  
Dense Marker

The protease acrosin is widely considered to be an essential component of a zona lysin which enables sperm to penetrate the zona pellucida of the egg. Sperm form a characteristic penetration slit little wider than the sperm head itself and this has long suggested that any zona lysin is attached to the sperm surface after an acrosome reaction. This paper provides the first ultrastructural evidence that this is the case. The protein acrosin inhibitor, Kunitz soybean trypsin inhibitor, has been covalently attached to the electron-dense marker, ferritin, and the conjugate incubated with guinea-pig sperm which have undergone an A23187-induced acrosome reaction. Electron microscopy shows that ferritin is distributed unevenly over the outer surface of the newly exposed inner acrosomal membrane but does not extend to the equatorial segment. This is further evidence that acrosin can be considered as a candidate for the role of zona lysin. The mechanism of sperm penetration of the zona is discussed in the light of these observations.

Capacity of goat epididymal spermatozoa to undergo the acrosome reaction and subsequent fusion with the egg plasma membrane

1993 ◽  
Vol 5 (3) ◽  
pp. 239 ◽  
Author(s):  
H Harayama ◽  
H Kusunoki ◽  
S Kato
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Morphological Changes ◽  
Glass Tube ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Acrosomal Membrane ◽  
Sperm Penetration ◽  
Epididymal Spermatozoa ◽  
Caput Epididymidis

The capacity to undergo the acrosome reaction and subsequent fusion with the egg plasma membrane was examined in goat epididymal spermatozoa. Spermatozoa from the proximal and distal caput and distal cauda were preincubated in a sealed glass tube for induction of the acrosome reaction, and their viability, acrosome morphology and penetrability into zona-free hamster eggs were determined. A simplified triple-stain technique revealed that most of the preincubated live spermatozoa in the samples from the distal caput and distal cauda epididymides underwent morphological changes that indicated the occurrence of the acrosome reaction. Electron microscopic examination revealed that the outer acrosomal membrane of many spermatozoa in these samples showed fusion at multiple sites to the plasma membrane. However, the rates of acrosome-reacted cells in the proximal caput spermatozoa were still lower. The sperm penetration assay demonstrated that the penetration rates of distal caput and distal cauda spermatozoa preincubated for 2 h were 93% and 74% respectively, whereas proximal caput spermatozoa scarcely penetrated into eggs. These results indicate that increasing numbers of goat spermatozoa improve in the functions related to the acrosome reaction and subsequent fusion with the egg plasma membrane during their transit through the caput epididymidis.

Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 105-111 ◽  
Author(s):  
R. D. Moreno ◽  
M. Hoshi ◽  
C. Barros
Keyword(s):  
Zona Pellucida ◽  
Active Site ◽  
Acrosome Reaction ◽  
Penetration Rate ◽  
Limited Proteolysis ◽  
Sulphated Polysaccharides ◽  
Functional Interactions ◽  
Sperm Binding ◽  
Sperm Penetration ◽  
Sperm Acrosome Reaction

Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARIS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.

How does sperm meet egg?--in a marsupial

1994 ◽  
Vol 6 (4) ◽  
pp. 485 ◽  
Author(s):  
WG Breed
Keyword(s):  
Acrosome Reaction ◽  
Small Region ◽  
Sperm Head ◽  
Sminthopsis Crassicaudata ◽  
Acrosomal Membrane ◽  
Wide Range ◽  
A Cell ◽  
Inner Acrosomal Membrane ◽  
Head Morphology ◽  
Range Of Variation

Australian marsupials exhibit a wide range of variation in sperm head morphology, and in thickness of the zona pellucida around the oocyte, suggesting interspecfic differences in the processes of sperm-egg interaction. The observations described here are largely based on the dasyurid Sminthopsis crassicaudata. They show that in oestrous females, after mating, a coagulum forms in the lateral vaginae and, within an hour of insemination, numerous spermatozoa congregate in the isthmus of the oviduct in which the vanguard population undergoes transformation with the head rotating on its axis with the tail to form a T-shape. Once oocytes are released, a few spermatozoa migrate to the higher reaches of the oviduct where sperm-zona binding occurs by way of the plasmalemma over the acrosomal region. The acrosome reaction takes place here and, as the egg rotates, the tail of the spermatozoon becomes parallel to the head. A small region of acrosome sometimes appears to remain intact at this time because spermatozoa with partly intact acrosomes have been found within the zona matrix. In some of these, electron-dense bridges between part of the inner and outer acrosomal membranes which may act as stabilizing structures, were also seen. The zona matrix is tightly packed around the penetrating spermatozoon, but that close to the acrosomal region becomes less electron-dense and more filamentous. Once incorporated into the egg, the spermatozoon lacks a cell membrane around the tail but vesicles close to the sperm head may, at least in part, be remnants of an inner acrosomal membrane. How generally applicable these observations are to other Australian marsupials remains to be determined.

scholarly journals Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 126-126
Author(s):  
Daniël Verhoef ◽  
Mark Schreuder ◽  
Ka Lei Cheung ◽  
Pieter H. Reitsma ◽  
Mettine H.A. Bos
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Factor Xa ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Bleeding Events ◽  
Surgical Interventions ◽  
Anionic Phospholipids ◽  
Active Site Pocket

Abstract The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG. Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography. Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket. We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma. Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. Disclosures Bos: Bayer Hemophilia Awards: Research Funding.

Ultrastructural observations on in vivo fertilisation in the brushtail possum, Trichosurus vulpecula, following PMSG/LH superovulation and artificial insemination

Zygote ◽  
1999 ◽  
Vol 7 (4) ◽  
pp. 307-320 ◽  
Author(s):  
M.K. Jungnickel ◽  
A.J. Harman ◽  
J.C. Rodger
Keyword(s):  
Artificial Insemination ◽  
Acrosome Reaction ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Chromatin Decondensation ◽  
Large Hole ◽  
Enzymatic Action ◽  
Acrosomal Membrane ◽  
Sperm Penetration

Information on the dynamics of gamete interaction in marsupials is very limited and not available for any species from the major Australian Order Diprotodontia which includes most of the more familiar animals such as kangaroos, possums and the koala. This study addressed this deficiency by examining the ultrastructure of in vivo fertilised eggs from common brushtail possums (Trichosurus vulpecula). Females were superovulated by treatment with 15 IU PMSG and then 4 mg porcine LH 3 days later, and inseminations were performed 910-13 h after LH) using epididymal spermatozoa. Between 33 and 39 h after LH injection females were killed, reproductive tracts excised and the oviduct ampulla segment flushed for eggs. Three of the six eggs examined were fertilised as judged by the presence of sperm remnants in the cytoplasm. On the basis of these eggs it was found that sperm penetration left a large hole in the zona pellucida (ZP), suggesting that sperm zona penetration occurs primarily by the enzymatic action of acrosomal enzymes. Sperm lying within the perivitelline space were lacking both an outer acrosomal membrane and the associated acrosomal contents, while both these structures were found on sperm embedded within the mucoid layer, which is consistent with induction of the acrosome reaction by binding to the ZP. Once inside the egg cytoplasm, the sperm head travelled only a short distance before chromatin decondensation occurred. Fertilised eggs showed signs of cytoplasmic activation including cytoskeleton association with apparently dividing mitochondria and prominent rough endoplasmic reticulum. Unfertilised eggs appeared to be undergoing degenerative changes and lacked any evidence of activation. This study has demonstrated that superovulation and laparoscopic intravaginal artificial insemination provide a system through which perifertilisation events in the possum and other monovular Australian marsupials can be examined experimentally.

scholarly journals Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm

2003 ◽  
Vol 23 (7) ◽  
pp. 2614-2622 ◽  
Author(s):  
Naokazu Inoue ◽  
Masahito Ikawa ◽  
Tomoko Nakanishi ◽  
Misako Matsumoto ◽  
Midori Nomura ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Acrosome Reaction ◽  
Null Mutation ◽  
Membrane Cofactor Protein ◽  
Wild Type ◽  
Acrosomal Membrane ◽  
Fertilizing Ability ◽  
Inner Acrosomal Membrane ◽  
Sperm Acrosome Reaction ◽  
Sperm Acrosome

ABSTRACT Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46−/− males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous acrosome reaction doubled in CD46−/− sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46−/− sperm. These data suggest that CD46 may have some role in regulating sperm acrosome reaction.

scholarly journals PACAP-mediated sperm–cumulus cell interaction promotes fertilization

Reproduction ◽  
2011 ◽  
Vol 141 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Ichiro Tanii ◽  
Tadashi Aradate ◽  
Kouhei Matsuda ◽  
Akira Komiya ◽  
Hideki Fuse
Keyword(s):  
Sperm Motility ◽  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Interaction ◽  
Fertilization Rate ◽  
Type I ◽  
Dependent Manner ◽  
Sperm Penetration

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus–oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus–oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.

Sign in / Sign up

Export Citation Format

Share Document