Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction

2015 ◽  
Vol 84 (8) ◽  
pp. 1378-1386 ◽  
Author(s):  
J. Beek ◽  
H. Nauwynck ◽  
R. Appeltant ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Zona Pellucida ◽  
Serine Proteases ◽  
Acrosome Reaction ◽  
Fertilization In Vitro ◽  
Sperm Binding ◽  
Sperm Penetration

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.

scholarly journals Expression and putative function of fibronectin and its receptor (integrin α5β1) in male and female gametes during bovine fertilization in vitro

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Mirjan Thys ◽  
Hans Nauwynck ◽  
Dominiek Maes ◽  
Maarten Hoogewijs ◽  
Dries Vercauteren ◽  
...  
Keyword(s):  
Sperm Cell ◽  
Acrosome Reaction ◽  
Male Gamete ◽  
Inhibitory Effect ◽  
Fertilization In Vitro ◽  
Integrin Receptor ◽  
Male And Female ◽  
Sperm Penetration ◽  
Bovine Spermatozoa

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in human. Recently, it has been demonstrated that Fn – when present during bovine IVF – strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (α5β1) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin α5 on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin α5 at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm–oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin α5β1 receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin α5 expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a ‘velcro’ interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm–egg binding.

scholarly journals Role of Tetraspanin CD9 Molecule in Fertilization of Mammals

2015 ◽  
pp. 279-293 ◽  
Author(s):  
J. JANKOVIČOVÁ ◽  
M. SIMON ◽  
J. ANTALÍKOVÁ ◽  
P. CUPPEROVÁ ◽  
K. MICHALKOVÁ
Keyword(s):  
Acrosome Reaction ◽  
Vitro Fertilization ◽  
Fertilization Process ◽  
Sperm Binding ◽  
Sperm Penetration ◽  
Ambiguous Data ◽  
Precise Role ◽  
Candidate Molecule

Fertilization process is a very clever and unique process comprising some essential steps resulting in formation of zygote. Tetraspanin CD9 is considered to be a serious candidate molecule participating in these events. The importance of CD9 has been discussed in relation to acrosome reaction, sperm-binding, sperm-penetration, sperm-egg fusion and eventually, egg activation. The abundant expression of CD9 oocyte plasma membrane and the presence of CD9-containing vesicles in the perivitelline space of intact oocytes have been confirmed. Despite the fact that majority of authors analyzed CD9 expressed on oocytes, several studies considered the function of sperm CD9, too. To understand CD9 involvement, various conditions of in vitro fertilization (IVF) assays using polyclonal as well as monoclonal antibodies or knockout mice were carried out. However, ambiguous data have been obtained about the importance of CD9 in sperm-egg binding or fusion. Although the current findings did not prove any hypothesis, the indispensable role of CD9 in fertilization process was not excluded and the precise role of CD9 remains unexplained.

scholarly journals Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction.

1979 ◽  
Vol 83 (3) ◽  
pp. 544-555 ◽  
Author(s):  
P M Saling ◽  
B T Storey
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Probe ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Sperm Head ◽  
Fertilization In Vitro ◽  
Ionophore A23187 ◽  
Mouse Sperm ◽  
Sperm Acrosome

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.

scholarly journals ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction

2021 ◽  
Vol 8 ◽  
Author(s):  
Mª José Izquierdo-Rico ◽  
Carla Moros-Nicolás ◽  
Míriam Pérez-Crespo ◽  
Ricardo Laguna-Barraza ◽  
Alfonso Gutiérrez-Adán ◽  
...  
Keyword(s):  
Animal Model ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Functional Roles ◽  
Sperm Binding ◽  
The Status ◽  
Murine Rodents ◽  
Mouse Species ◽  
Mammalian Eggs

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1–ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

294 THE EFFECT OF PLASMIN ON THE IN VITRO FERTILIZATION ABILITY OF PORCINE GAMETES

2006 ◽  
Vol 18 (2) ◽  
pp. 254
Author(s):  
H.-H. Rhee ◽  
S.-J. Sa ◽  
H.-T. Cheong ◽  
B.-K. Yang ◽  
C.-K. Park
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Proteolytic Enzymes ◽  
Extracellular Proteins ◽  
Control Group ◽  
Sperm Viability ◽  
Vitro Fertilization ◽  
Sperm Binding

Plasminogen activators (PAs) are specific proteolytic enzymes that convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a nonspecific, potent protease that cleaves blood fibrin clots and several other extracellular proteins. The purposes of the present study were (1) to assess the effect of plamin on sperm viability and acrosome reaction (AR), (2) to examine the effect of plasmin on zona pellucida (ZP) solubility and the binding of sperm to ZP, and (3) to evaluate the effect of plasmin on fertilization responses, including penetration and incidence of polyspermy during in vitro fertilization in the pig. Ejaculated semen was collected from three mature Duroc boars by artificial vagina. The same three boars were used for all experiments. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG; Sigma). Porcine spermatozoa, which were washed in Dulbecco PBS (Sigma), were resuspended and incubated in fertilization medium (mTBM) containing 0, 0.1, 1.0, 10.0, or 100.0 ng/mL plasmin (Sigma). Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The present study suggests that sperm viability was not affected by plasmin treatment. Also, addition of plasmin in doses ranging between 0.1 and 100.0 ng/mL for 2, 4, or 6 h to washed boar spermatozoa resulted in enhancement of acrosome reaction (AR), compared with untreated cells. Concentrations of 0 and 0.1 ng/mL plasmin (83 � 15 and 95 � 18 sperm/oocyte, respectively) had no effect on sperm binding, whereas 1.0 (123 � 21 sperm/oocyte), 10.0 (124 � 16 sperm/oocyte), and 100 ng/mL (124 � 15 sperm/oocyte) plasmin increased (P < 0.05) sperm binding, compared with the control. The zona pellucida solubility (zona digestion time) was significantly (P < 0.05) lower in medium with 1.0 (123 � 24 s), 10.0 (99 � 15 s), or 100.0 ng/mL (95 � 19 s) plasmin, compared with control (176 � 27 s). When porcine oocytes and spermatozoa were co-incubated in various concentrations of plasmin for 6 h, the penetration rate was significantly (P < 0.05) higher in medium with 1.0 ng/mL plasmin (77.5 � 3.1%), compared with control. However, there were no significant differences in the polyspermic rates and mean numbers of sperm (MNS)/oocyte among the groups treated with plasmin and the control group. We found that addition of plasmin to fertilization medium increases the percentage of acrosome-reacted spermatozoa and the sperm-binding ability of the pig ZP. These results suggest that plasmin may play a role in events related to fertilization in the pig.

317 ANTI-HYALURONIDASE ACTION OF ELLAGIC ACID EFFECTIVELY PREVENTS POLYSPERMY THROUGH SUPPRESSION OF THE ACROSOME REACTION INDUCED BY THE SPERM - ZONA INTERACTION DURING PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 274
Author(s):  
I. Tokeshi ◽  
H. Tatemoto ◽  
N. Muto ◽  
T. Yoshimoto ◽  
S. Nakamura ◽  
...  
Keyword(s):  
Ellagic Acid ◽  
Acrosome Reaction ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Sperm Binding ◽  
Sperm Penetration

We previously reported that the anti-hyaluronidase agents oligosaccharide and tannic acid (TA) were efficient probes for promoting the normal fertilization process in terms of an effective decrease in the incidence of polyspermy, not only in cumulus-enclosed but also in denuded oocytes in pigs. It was unclear, however, why the polyspermic penetration into the zona pellucida (ZP) was directly prevented by the anti-hyaluronidase action. The present study was conducted to examine the effects of 3 tannin relatives [TA, gallic acid (GA), and ellagic acid (EA)] on IVF parameters and the acrosome reaction induced by the sperm–ZP interaction. The anti-hyaluronidase and radical-scavenging activities of tannin relatives were measured by the colorimetric and the DPPH methods, respectively. Porcine cumulus–oocyte complexes (COCs) were cultured for 44 h in 0.1 mL of TCM-199 supplemented with 0.6 mM cysteine, 40 mU mL-1 of FSH, 20 mU mL-1 of LH, and 10% porcine follicular fluid. After in vitro maturation (IVM), the COCs were freed from their cumulus cells and inseminated by frozen-thawed ejaculated sperm in modified Tris-buffered medium (IVF medium) containing 0 (control) or 5 �g mL-1 of tannin relatives. After 2 h of co-incubation, the oocytes were gently pipetted to remove loosely bound sperm and stained with Hoechst 33342 to count the number of ZP-bound sperm, or stained with fluorescein isothiocyanate (FITC)-PNA, PI, and 422,6-diamidino-2-phenylindole to evaluate the acrosomal status. At 10 h post-insemination, IVF parameters were examined by lacmoid staining. The data were analyzed by ANOVA and the Tukey-Kramer test. None of the tannin relatives caused a protective proteolytic modification of the ZP matrix or a reduction of the acrosomal proteolytic activity or the number of ZP-bound sperm. There was no difference in the sperm penetration rate even in the presence of tannin relatives (73-82%). However, the incidence of polyspermy was remarkably prevented by TA (32%; 31/98) and EA (21%; 20/94) compared with the control (58%; 58/100; P &lt; 0.05), resulting from their strong anti-hyaluronidase actions, whereas GA without the anti-hyaluronidase action had no effect on the prevention of polyspermy (51%; 43/84). The rate of acrosome reaction induced by the sperm–ZP interaction was decreased by TA (15%; 123/833) and EA (16%; 110/708) compared with the control (25%; 238/939; P &lt; 0.05), and a similar result was found in sperm binding to the pretreated ZP with 500 U of hyaluronidase for 2 h (18%; 351/1959). Interestingly, when sperm were incubated in IVF medium with 10 �g mL-1 of progesterone for 0.5 h to induce a compulsory acrosome reaction instead of the ZP, EA never disturbed the acrosome reaction (23%; 98/424) as control (23%; 102/437), although this reaction was blocked by TA (13%; 57/427) and GA (13%; 50/375), which possessed higher levels of radical-scavenging activity than EA (P &lt; 0.05). These results indicate that the anti-hyaluronidase action of TA and EA effectively prevented polyspermy during porcine IVF as a consequence of suppression of the acrosome reaction functionally induced by the sperm–ZP interaction requiring the hyaluronidase intervention.

Use of oocytes that failed to be fertilized in vitro to study human sperm-oocyte interactions: comparison of sperm-oolemma and sperm-zona pellucida binding, and relationship with results of IVF

1990 ◽  
Vol 2 (6) ◽  
pp. 641 ◽  
Author(s):  
DY Liu ◽  
A Lopata ◽  
HW Baker
Keyword(s):  
Zona Pellucida ◽  
Logistic Regression Model ◽  
Sperm Concentration ◽  
Human Sperm ◽  
Fertilization In Vitro ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
Binding Ratio ◽  
Sperm Binding

A test for human sperm binding to the oolemma was developed with oocytes that failed to be fertilized in vitro. The zonae pellucidae of the oocytes were removed under a dissecting microscope by brief exposure to dilute HCl (pH 2.5-3.0) in 0.9% NaCl. The zona-free oocytes (ZFOs) were incubated with a mixture of equal numbers of motile sperm from men to be tested and fertile donors. The sperm was differentially labelled with fluorescein or rhodamine and the results expressed as a ratio of the number of test to control sperm bound to several ZFOs in order to control for variability in the ability of the oolemma to bind sperm. The number of sperm bound to the oolemma increased with time and sperm concentration. The sperm-oolemma binding ratio determined for 32 patients undergoing in vitro fertilization (IVF) was significantly correlated with the sperm-zona pellucida (ZP) binding ratio but was not correlated with other sperm tests. The sperm-oolemma binding ratio was also related to the IVF rate, but this was not significant if the sperm-ZP binding ratio was included in the logistic regression model. Only four of the 32 patients had failure of fertilization in vitro. The human sperm-oolemma binding test may be useful for studying the interaction between gametes, but the test is unlikely to be as useful clinically as the sperm-ZP binding test for predicting fertilization in vitro.

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