26 Jan

Isoforms of the Mitochondrial Capsule Protein: DISCUSSION

Protein tyrosine phosphorylation is one of the important biochemical mechanisms regulating sperm capacitation. In the present study, we found the time-dependent increase in protein tyrosine phosphorylation of hamster spermatozoa in media that supports capacitation. It has been proposed that the tyrosine phosphorylation of an 80-kDa hamster sperm protein, localized to the principal piece of the flagellum, is associated with the acquisition of hyperactivation of sperm motility. Visconti et al. also observed a capacitation-associated increase in protein (Mr 30 000-150 000) tyrosine phosphorylation in hamster spermatozoa. However, their figures also show a capacitation-dependent tyrosine-phosphorylated band less than 24 kDa, which may represent PHGPx. Differences in staining intensity could be due to the different source of phosphotyrosine monoclonal antibody used in this study. The signaling cascade and the tyrosine kinase(s) that regulate the phosphorylation of these substrates have yet to be identified. Kalab et al. demonstrated that the 20-kDa tyrosine-phos-phorylated boar sperm polypeptide was the only polypeptide that can be extracted completely in Triton X-100 among the tyrosine phosphorylated polypeptides (Mr 20 000-230 000). However, the localization and the biochemical characterization of the 20-kDa boar sperm polypeptide remain to be elucidated. Our data reveal several new findings, including the localization of capacitation-de-pendent tyrosine-phosphorylated polypeptides to both the midpiece and the principal piece of hamster spermatozoa and the biochemical characterization of the 19-kDa tyro-sine-phosphorylated polypeptide in the Triton X-100 plus DTT-soluble fraction demonstrates that the 19-kDa polypeptide is a disulfide cross-linked, membrane-anchored component.

The molecular mechanism of mammalian sperm capac-itation is still poorly understood. Recently, work from several laboratories regarding the identification of tyrosine-phosphorylated polypeptides during capacitation is contributing to our understanding of the molecular basis of sperm capacitation. A calcium-binding polypeptide (CABYR) localized to the fibrous sheath of human sperm flagellum becomes tyrosine phosphorylated during capacitation. Two other protein components of the fibrous sheath (AKAP4 and AKAP3) were previously shown to be phosphorylated during in vitro capacitation. Ficarro et al. demonstrated that the tyrosine phosphorylation of a valosin-containing protein (VCP) and the translocation of VCP from the neck to the anterior head occurred during capacitation of human spermatozoa. In the present study, the proteomic identification of the 19-kDa tyrosine-phos-phorylated polypeptide purified from the Triton X-100 plus DTT-soluble fraction reveals that the polypeptide is PHGPx, suggesting that the tyrosine phosphorylation of the structural protein of the sperm mitochondrial capsule occurs during capacitation in a time-dependent manner. At present, the molecular characterization of the 19-kDa tyrosine-phos-phorylated protein present in the Triton X-100-soluble fraction is not known. Future studies will address this issue.

It is well established that the mammalian testis possesses the highest activity of PHGPx of all mammalian tissues investigated and PHGPx is expressed specifically by spermatids. During differentiation of spermatids into mature spermatozoa, the enzymatically active PHGPx switches to an enzymatically inactive, disulfide crosslinked, insoluble structural protein of the sperm mitochondrial capsule. In the present study, we found that hamster sperm PHGPx is solubilized by 32 mM DTT (Fig. 5B, lane 3). Ursini et al. used a different disulfide-cleavable compound (mercaptoethanol) and guanidine hydrochloride (a denaturing agent) for the solubilization of rat sperm PHGPx. Both studies confirm that the PHGPx of mammalian spermatozoa from the cauda epididymis is a disulfide cross-linked structural protein of the mitochondrial capsule. It is believed that male infertility in selenium-deficient animals, characterized by impaired sperm motility and structural abnormalities of sperm tails, is due to insufficient PHGPx content. Tyrosine 130 of PHGPx is the potential tyrosine-phosphorylation consensus motif as determined by the phosphorylation site prediction program (NetPhos 2.0 Server). At present, the downstream effect(s) of tyrosine phosphorylation of PHGPx is not known. One possibility is that tyrosine phosphorylation can reactivate the catalytic activity of PHGPx. A second potential function is that tyrosine-phosphorylated PHGPx may interact with other signaling protein(s) that could potentially affect mitochondrial function and/or participate in one of the pathways regulating the hyperactivation of sperm motility. Finally, sperm mitochondria degenerate in the egg shortly after fertilization and tyrosine phosphorylation of PHGPx may regulate the stability of the mitochondrial capsule. This is analogous to somatic cells, where phosphorylation of the nuclear lamins regulates the disassembly of the nuclear envelope during mitosis. Additional studies are needed to resolve these issues.

In the present study, we have demonstrated posttransla-tional modifications of PHGPx during capacitation of hamster spermatozoa. Tyrosine phosphorylation contributes to the generation of a complex charge-variant pattern of PHGPx with multiple isoforms. Whether other posttrans-lational modifications, such as serine/threonine phosphorylation or tyrosine nitration, also contribute to the charge heterogeneity of PHGPx remains to be investigated. In conclusion, our data suggest that the tyrosine phosphorylation of PHGPx may represent an important event in the signaling cascade(s) associated with capacitation that may impact the regulation of hyperactivation of sperm motility and/or mitochondrial function.

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