Unlike in lower animals, ejaculated sperm from mammals must undergo capacitation in order to fertilize an oocyte. Sperm become "fertilization competent" as they reside in the female reproductive tract through a series of physiological changes known as capacitation. Capacitation has been referred to as changes that enable the sperm to undergo both the acrosome reaction and hyperactivation. Capacitation has been correlated with changes in sperm intracellular ion concentration, plasmic membrane fluidity, metabolism and motility. Changes in sperm cyclic nucleotide metabolism and protein phosphorylation have also been implicated in sperm functions such as capacitation. However, the molecular basis behind these events has been poorly understood (Visconti et al., 1995).
It appears that the media in which the sperm are incubated play an integral role in many sperm processes. Lee and Storey (1986); Kopf and Gertion (1991); Yanagimachii (1994) have suggested that Ca2+ and NaHCO3 are required for the induction of the acrosome reaction by the zona pellucida.
Visconti et al. investigated the need for CaCl2, bovine serum albumin and NaHCO3 to induce the capacitation process in mouse caudal epididymal sperm. They also investigated the relationship between the capacitation state of sperm and protein tyrosine phosphorylation and proposed a possible mechanism by which this may occur via a cAMP pathway.
They hypothesized that Ca2+, HCO3- and bovine serum albumin are required for the capacitation process and that capacitation is accompanied by a time dependent increase in protein tyrosine phosphorylation.
Visconti et al. performed five different tests and assays to determine which factors were necessary in sperm capacitation, acrosomal reaction and ability to form pronuclei. They performed an experiment to test how much BSA, if any, was required for sperm capacitation. In the absence of BSA, they discovered that protein tyrosine phosphorylation was restricted to p95/116 hexokinase. As BSA concentrations were increased, degrees of protein tyrosine phosphorylation increased as well, displaying a maximum at about 3mg/ml of BSA.
The correlation of capacitation with the increase in protein tyrosine phosphorylation could be demonstrated if the sperm now possessed the ability to undergo ZP induced acrosomal reaction as determined by the Chlortetracycline (CTC) assay and to form pronuclei.
In a similar way, the researchers performed experiments to test the role of calcium ion and the bicarbonate ion. In both cases, they found that a sufficient concentration of the ions produced protein tyrosine phosphorylation as determined by probing the fixed proteins with an anti-phosphotyrosine antibody. It should be noted that the sodium bicarbonate was not merely a pH affect, and the researchers performed tests to establish this.
In their accompanying report, Visconti et. al. (1995b) continued their work and showed that protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Subsequent investigations revealed that sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin could be induced to undergo capacitation by the addition of cyclic AMP analogues.
The role of bovine serum albumins may involve accelerating the loss of
cholesterol from the sperm plasma membrane. Changes in membrane composition
led to an increased permeability of the membrane to calcium and bicarbonate
ions. Ca2+ and HCO3- subsequently activate the adenylyl cyclase or as in
the case of calcium ion stimulate the activity of phosphodiesterases. A
rise in cAMP then results in the activation of protein kinase A, which in
turn leads to the activation of sperm tyrosine kinases and/or inactivation
of phosphoprotein phosphatases. The result is an increase in protein tyrosine
Visconti et al. performed experiments to determine the necessity of BSA, calcium ion and sodium bicarbonate in protein tyrosine phosphorylation. They then correlated this phosphorylation with the CTC assay, showing that it was linked to sperm capacitation. Their further experiments provided a theoretical mechanism for the role of the phosphotyrosine in a cyclic AMP dependent pathway.
Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and G.S. Kopf . 1995a. Capacitation of mouse spermatozoa: Correlation between the capacitation state and protein tyrosine phosphorylation. Development 121: 1129-1150.
Visconti, P.E., Moore, G.D., Bailey, J.L., Leclerc, P., Connors, S.A., Pan, D., Olds-Clarke, P. and Kopf, G.S. 1995b. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121: 1139-1150.
Copyright © Stacey Beaudin, Donna Kipta and Annamarie Orr.1996 This material may be reproduced for educational purposes only provided credit is given to the original source.
October 9, 1996