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Dephosphorylation of sperm midpiece antigens initiates aster formation in rabbit oocytes
C Pinto-Correia1, D L Poccia, T Chang
1Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst 01003.
Abstract:
During fertilization in most mammals, the penetrating sperm organizes an aster of microtubules. We have investigated the mechanisms underlying this function of the sperm by a series of experiments based on microinjection of isolated sperm midpieces into unfertilized oocytes. These midpieces contain antigens recognized by the MPM-2 antibody. These antigens, which are absent from the rest of the tail fraction, correspond to three phosphorylated polypeptides of 77, 81, and 85 kDa. Dephosphorylation with alkaline phosphatase abolishes antigenicity on blots and in whole sperm. Reactivity to the antibody disappears between 1 and 3 hr after calcium stimulation of oocytes, following the decline in H1 kinase activity and coincident with aster formation. In unactivated oocytes, no aster forms and the antigen remains unchanged. MPM-2 treatment of midpieces prior to injection blocks their ability to form asters in oocytes activated by calcium stimulation. The epitope also disappears in 6-methyl-aminopurine-treated oocytes, implying that maintenance of the phosphorylated state requires kinase activity. A result that confirms this view is that sperm midpieces dephosphorylated by alkaline phosphatase can be rephosphorylated after injection into oocytes or by exposure in vitro to a Xenopus oocyte cytoplasmic fraction high in H1 kinase activity. We suggest that the microtubule nucleation activity of sperm midpieces after fertilization is triggered by the calcium-induced decrease in maturation promoting factor, which results in dephosphorylation of specific sperm centrosomal proteins.
Insights
Sperm midpieces organize microtubule asters during fertilization. This function is linked to specific phosphorylated proteins, identified using the MPM-2 antibody, which are dephosphorylated upon oocyte activation.
Area of Science:
- Cell Biology
- Reproductive Biology
- Microtubule Dynamics
Background:
- Sperm play a crucial role in fertilization by organizing microtubule asters.
- The molecular mechanisms governing sperm's microtubule-organizing ability remain incompletely understood.
Purpose of the Study:
- To investigate the mechanisms by which sperm midpieces organize microtubule asters after fertilization.
- To identify specific sperm proteins involved in aster formation and their post-translational modifications.
Main Methods:
- Microinjection of isolated sperm midpieces into unfertilized oocytes.
- Immunological detection of phosphorylated antigens using the MPM-2 antibody.
- Biochemical assays including dephosphorylation with alkaline phosphatase and kinase activity measurements.
Main Results:
- Isolated sperm midpieces contain specific phosphorylated antigens (77, 81, 85 kDa) recognized by MPM-2.
- Dephosphorylation abolishes antigenicity and blocks aster formation.
- Antigenicity disappears post-calcium stimulation, correlating with decreased H1 kinase activity and aster formation.
- Kinase activity is required to maintain the phosphorylated state of these sperm proteins.
Conclusions:
- Sperm microtubule nucleation activity is triggered by a decrease in maturation promoting factor after fertilization.
- This decrease leads to the dephosphorylation of specific sperm centrosomal proteins, enabling aster formation.
- The MPM-2 recognized antigens are key phosphorylated sperm proteins involved in microtubule organization during fertilization.