Identification of the Bovine Follicular Fluid Protein Involved in the in vitro Induction of the Hamster Sperm Acrosome Reaction

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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301 THE EFFECT OF EQUINE FOLICULLAR FLUID ON IN VITRO INDUCTION OF THE ACROSOME REACTION IN FROZEN - THAWED STALLION SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab301 ◽

2010 ◽

Vol 22 (1) ◽

pp. 307

Author(s):

D. S. Silva ◽

P. Rodriguez ◽

N. S. Arruda ◽

R. Rodrigues ◽

J. L. Rodrigues

Keyword(s):

Contact Area ◽

Follicular Fluid ◽

Acrosome Reaction ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Heparin Group ◽

Fluorescent Stain ◽

In Vitro Induction

The capacitation process occurs in vivo upon exposure of the spermatozoa through the female reproductive tract, but can be induced in vitro in the presence of several compounds. This study was conducted to assess the effect of heparin or equine follicular fluid on hyperactivated motility and in vitro induction acrosome reaction swim-up method with frozen-thawed stallion semen. Two hundred microliters of frozen-thawed equine semen was placed in a tube (45°C) to increase contact area and incubated at 37°C for 1 h. After incubation 800 μL of the supernatant was collected by centrifugation (500 × g, 10 min) to collect spermatozoa. The resulting pellet was resuspended in capacitation medium Fert-TALP supplemented with 5.0 μg mL-1 heparin or 100% follicular fluid and incubated for different times (1, 2, 3, 4, and 5 h) at 37°C. After incubation the hyperactivated motility and acrosome-reacted spermatozoa were evaluated. Hoechst stain was used to differentiate live and dead spermatozoa, and chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm; data were analyzed by ANOVA. The effect of equine follicular fluid resulted in improved percentage of spermatozoa with acrosome reaction at all times of incubation (60, 63, 57, 52, and 58%) but immediately after 3 h of incubation, the hyperactivated motility decreased in heparin group and follicular fluid (42 and 30%, respectively).

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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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Dynamics of human sperm acrosome reaction: relation with in vitro fertilization

Fertility and Sterility ◽

10.1016/s0015-0282(16)54312-x ◽

1991 ◽

Vol 55 (5) ◽

pp. 994-999 ◽

Cited By ~ 58

Author(s):

Patrick Fénichel ◽

Michèle Donzeau ◽

Dariush Farahifar ◽

Bernard Basteris ◽

Noël Ayraud ◽

...

Keyword(s):

In Vitro Fertilization ◽

Acrosome Reaction ◽

Human Sperm ◽

Vitro Fertilization ◽

Sperm Acrosome Reaction ◽

Sperm Acrosome

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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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Potassium ion influx and Na+,K+-ATPase activity are required for the hamster sperm acrosome reaction.

The Journal of Cell Biology ◽

10.1083/jcb.91.1.77 ◽

1981 ◽

Vol 91 (1) ◽

pp. 77-82 ◽

Cited By ~ 28

Author(s):

R J Mrsny ◽

S Meizel

Keyword(s):

Atpase Activity ◽

Acrosome Reaction ◽

Atpase Inhibitor ◽

Mammalian Sperm ◽

Ion Influx ◽

K 12 ◽

Low K ◽

Sperm Acrosome Reaction ◽

Sperm Acrosome

The role of a K+ ion influx and Na+,K+-ATPase activity in the hamster sperm acrosome reaction (AR) was examined, using a range of concentrations of K+,K+ ionophores and a Na+,K+-ATPase inhibitor. Washed epididymal hamster sperm, capacitated in vitro in an artificial medium containing 2 mM Ca2+, 147 mM Na+, and 3, 6, 12, 18, or 24 mM K+, began undergoing the AR after 3 h of incubation. Sperm incubated in low K+ (0.9 mM) failed to undergo the AR even after 5 h of incubation. Sperm in 0.9 mM K+ could be induced to undergo the AR when either K+ (12 mM) alone or K+ (12 mM) with 0.1 microM nigericin was added after 3.5 h of incubation. The addition of K+ alone stimulated the AR in 30 min, whereas nigericin plus K+ stimulated the AR 15 min after addition. Neither nigericin added alone (0.9 mM K+) nor nigericin plus 12 mM K+ added to a low Ca2+ (0.35 mM) system resulted in acrosome reactions. Valinomycin (1 nM) did not stimulate the AR when added together with K+ (3-24 mM) to sperm incubated in 0.9 mM K+ for 3.5 h but markedly decreased sperm motility. Micromolar levels of ouabain blocked the AR when added between t = 0--3 h to sperm incubated with 3-24 mM K+. Inhibition of AR by the addition of 1 microM ouabain to sperm incubated with 3 mM K+ was completely reversed by the addition of 0.1 microM nigericin at t = 3.5 h. These results suggest that Na+,K+-ATPase activity and the resulting K+ influx are important for the mammalian sperm AR. Some similarities between requirements for the hamster sperm AR and secretory granule exocytosis are discussed.

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