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

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.

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Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa

Biochemical Journal ◽

10.1042/bj2590397 ◽

1989 ◽

Vol 259 (2) ◽

pp. 397-406 ◽

Cited By ~ 76

Author(s):

E R S Roldan ◽

R A P Harrison

Keyword(s):

Acrosome Reaction ◽

Fertilization In Vitro ◽

Ionophore A23187 ◽

Bivalent Cation ◽

Phosphoinositide Breakdown ◽

Phosphoinositide Cycle ◽

Phosphatidylinositol 4 ◽

Sperm Acrosome Reaction ◽

Sperm Acrosome

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the ‘sperm acrosome reaction’. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.

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ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

PLoS ONE ◽

10.1371/journal.pone.0251973 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0251973

Author(s):

Yoku Kato ◽

Satheesh Kumar ◽

Christian Lessard ◽

Janice L. Bailey

Keyword(s):

Endoplasmic Reticulum ◽

Zona Pellucida ◽

Acrosome Reaction ◽

Porcine Model ◽

Sperm Head ◽

Mouse Sperm ◽

Phosphorylated Proteins ◽

Boar Sperm

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine.

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Neurotensin stimulates the sperm acrosome reaction and alters percentages of fertilization in vitro

Fertility and Sterility ◽

10.1016/j.fertnstert.2019.07.643 ◽

2019 ◽

Vol 112 (3) ◽

pp. e202

Author(s):

Genevieve E. Campbell ◽

Estella L. Jones ◽

Pierre Comizzoli ◽

Diane M. Duffy

Keyword(s):

Acrosome Reaction ◽

Fertilization In Vitro ◽

Sperm Acrosome Reaction ◽

Sperm Acrosome

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

Theriogenology ◽

10.1016/j.theriogenology.2015.07.022 ◽

2015 ◽

Vol 84 (8) ◽

pp. 1378-1386 ◽

Cited By ~ 3

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

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In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zonae pellucidae

Developmental Biology ◽

10.1016/0012-1606(86)90388-x ◽

1986 ◽

Vol 114 (1) ◽

pp. 119-131 ◽

Cited By ~ 107

Author(s):

Gary N. Cherr ◽

Hovey Lambert ◽

Stanley Meizel ◽

David F. Katz

Keyword(s):

Zona Pellucida ◽

Golden Hamster ◽

Acrosome Reaction ◽

In Vitro Studies ◽

Reaction Completion ◽

Sperm Acrosome Reaction ◽

Sperm Acrosome

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Matrine Inhibits Mouse Sperm Function by Reducing Sperm [Ca2+]i and Phospho-ERK1/2

Cellular Physiology and Biochemistry ◽

10.1159/000369703 ◽

2015 ◽

Vol 35 (1) ◽

pp. 374-385 ◽

Cited By ~ 7

Author(s):

Tao Luo ◽

Qian-xing Zou ◽

Yuan-qiao He ◽

Hua-feng Wang ◽

Na Li ◽

...

Keyword(s):

Acrosome Reaction ◽

Male Reproduction ◽

In Vitro Toxicity ◽

Sperm Function ◽

Ionophore A23187 ◽

Epididymal Sperm ◽

Mouse Sperm ◽

Extracellular Signal ◽

Mature Mouse

Background: Matrine is a bioactive alkaloid that has a variety of pharmacological effects and is widely used in Chinese medicine. However, its effects on male reproduction are not well known. In this study, we aimed to investigate the in vitro toxicity of matrine on mature mouse sperm. Methods: Mouse cauda epididymal sperm were exposed to matrine (10-200 µM) in vitro. The viability, motility, capacitation, acrosome reaction and fertilization ability of the mouse sperm were examined. Furthermore, the intracellular calcium concentration ([Ca2+]i), calcium (Catsper) and potassium (Ksper) currents, and phosphorylation of extracellular signal regulated kinases 1/2 (p-ERK1/2) of the sperm were analyzed. Results: After exposure to 100 µM or more of matrine, mouse cauda epididymal sperm exhibited a significant reduction in total motility, progressive motility, linear velocity and acrosome reaction rate induced by Ca2+ ionophore A23187. As a result, the fertilization ability of mouse sperm was remarkably decreased by matrine. Our data further demonstrated that matrine significantly reduced sperm [Ca2+]i and [Ca2+]i-related p-ERK1/2; however, both the CatSper and KSper currents, which are thought to interactively regulate Ca2+ influx in sperm, were not affected by matrine. Conclusion: Our findings indicate that matrine inhibits mouse sperm function by reducing sperm [Ca2+]i and suppressing the phosphorylation of ERK1/2.

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