scholarly journals Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro

Reproduction ◽  
1992 ◽  
Vol 95 (2) ◽  
pp. 431-440 ◽  
Author(s):  
Y. Hatanaka ◽  
T. Nagai ◽  
T. Tobita ◽  
M. Nakano
Keyword(s):  
Zona Pellucida ◽  
Fertilization In Vitro

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro

Reproduction ◽  
2000 ◽  
pp. 127-135 ◽  
Author(s):  
W Bone ◽  
NG Jones ◽  
G Kamp ◽  
CH Yeung ◽  
TG Cooper
Keyword(s):  
Zona Pellucida ◽  
Glucose Utilization ◽  
Cumulus Cells ◽  
Glycolytic Enzyme ◽  
Male Rats ◽  
Fertilization In Vitro ◽  
Swimming Pattern ◽  
Principal Piece ◽  
Free Media

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

319 PROTEIN GENE PRODUCT 9.5 REGULATES SPERM PENETRATION DURING PORCINE FERTILIZATION IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 275
Author(s):  
Y. J. Yi ◽  
G. Manandhar ◽  
M. Sutovsky ◽  
C. S. Park ◽  
P. Sutovsky
Keyword(s):  
Gene Product ◽  
Zona Pellucida ◽  
Protein Gene ◽  
Cumulus Cells ◽  
Fertilization In Vitro ◽  
Protein Gene Product 9.5 ◽  
Dapi Staining ◽  
Protein Gene Product ◽  
Oviductal Fluid

The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCHs), which regenerate monoubiquitin from ubiquitin–protein complexes or polyubiquitin chains by cleaving the amide linkage next to the C-terminal glycine of ubiquitin. Identified in the acrosome of boar spermatozoa, we hypothesized that PGP9.5 might regulate sperm–zona pellucida interactions during porcine IVF. The cumulus–oocyte complexes isolated from slaughterhouse ovaries were cultured in TCM-199 media for 44 h at 38.5�C, 5% CO2 in air. After completion of in vitro maturation (IVM), cumulus cells were removed by 0.1% hyaluronidase, and metaphase II (MII) oocytes were used for IVF. In Experiment 1, oocytes were co-incubated with different sperm concentrations (1 � 106, 5 � 105, and 1 � 105 sperm mL-1) in TBM medium with or without anti-PGP9.5 antibody (1 : 50 dilution). In Experiment 2, oocytes were inseminated with 1 � 106 sperm mL-1 in TBM medium containing different concentrations of extracted oviductal fluids (0, 0.1, 0.5, 1, 2, and 3 �g mL-1) for 6 h. After IVF, oocytes were transferred into NCSU23 medium containing 0.4% BSA for further culture. The fertilization rates were evaluated by DAPI staining at 13 to 19 h. Data were analyzed by ANOVA and Duncan's multiple range test using the SAS program. Polyspermy was increased by the addition of anti-PGP9.5 antibody to the IVF medium (56.5–60.2% at polyspermy). This PGP9.5-antibody-induced polyspermy increase was sustained even with decreasing sperm concentrations. The polyspermy rates were reduced by the addition of isolated porcine oviductal fluid to IVF medium (50.4, 44.8, 28.0, 31.1, 1.6, and 0.0% at oviductal fluid concentrations of 0, 0.1, 0.5, 1, 2, and 3 µg mL−1, respectively). Biochemical analysis by Western blotting detected the appropriate 24-kDa PGP9.5 band in porcine oviductal fluid used for these experiments. Enzymatic UCH activity comparable to activity of recombinant UCH-L3 was detected in sperm extract, whole spermatozoa, and isolated oviductal fluid by fluorometric assay using fluorogenic UCH-substrate ubiquitin-AMC. This UCH activity was not reduced by the general protease inhibitor phenyl methyl sylfonyl fluoride, but it was reduced in a statistically significant manner (P < 0.05) by the specific UCH-inhibitor ubiquitin aldehyde. In conclusion, the polyspermy increased with different concentrations of sperm in the anti-PGP9.5 antibody, and PGP9.5 was detected in oviductal fluids, suggesting that PGP9.5 is involved in the sperm–zona pellucida interaction during porcine fertilization. This work was supported by the US Department of Agriculture (USDA-NRI grant #2002-35203-12237 to P.S.), the F21C Program of The University of Missouri–Columbia (P.S.), and the ERC Program of the Korea Science and Engineering Foundation (KOSEF grant no. R11-2002-100-00000-0 to Y.J. Yi and C.S. Park).

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.

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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