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The phosphorylation of a putative sperm microtubule-associated protein 2 (MAP2) is uniquely sensitive to regulation
1Department of Ophthalmology, Oregon Health Sciences University, Portland 97201.
Abstract:
We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule-associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes.
Insights
Researchers identified a bovine sperm phosphoprotein, pp255, similar to microtubule-associated protein 2 (MAP2). Its unique phosphorylation is sensitive to pH, calcium, and inhibitors, potentially regulating sperm capacitation and acrosome reaction.
Area of Science:
- Reproductive Biology
- Molecular Cell Biology
- Biochemistry
Background:
- Microtubule-associated protein 2 (MAP2) is crucial in neuronal development.
- Sperm function relies on complex regulatory mechanisms involving protein phosphorylation.
Purpose of the Study:
- To identify and characterize a novel bovine sperm phosphoprotein.
- To investigate the regulatory mechanisms of this phosphoprotein's phosphorylation state.
- To explore the potential role of this phosphoprotein in sperm function.
Main Methods:
- Immunological identification using antibodies against rat brain MAP2.
- Subcellular fractionation to determine protein localization.
- Two-dimensional gel electrophoresis to analyze phosphorylation.
- Phosphorylation analysis under various conditions (pH, calcium, inhibitors).
Main Results:
- A bovine sperm phosphoprotein (pp255) was identified, cross-reacting with anti-MAP2 antibodies.
- Sperm pp255 phosphorylation is uniquely sensitive to intracellular pH, calcium, MIX, H-8, and fluoride.
- This phosphoprotein is localized to sperm heads and exhibits high specific radioactivity.
- Phosphorylation state can be modulated independently of sperm motility.
Conclusions:
- Bovine sperm pp255 is a novel MAP2-like phosphoprotein.
- Its phosphorylation is tightly regulated by factors influencing sperm capacitation and acrosome reaction.
- Sperm MAP2 phosphorylation may play a key regulatory role in these essential sperm processes.