Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein

Abstract

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction is comprised of three major (54, 50, and 45kDa) and several minor (38–19kDa) polypeptides. The set of minor polypeptides (38–19kDa) termed “OMCrpf polypeptides” is selectively solubilized by high-pH extraction (pH 10.5) while the three major polypeptides (55, 50 and 45kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38–19kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment; whereas, IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidyl choline (LPC)-induced acrosome reaction; whereas, the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction.

Changes in the Cellular Distribution of Tyrosine Phosphorylation and Its Relationship with the Acrosomal Exocytosis and Plasma Membrane Integrity during In Vitro Capacitation of Frozen/Thawed Bull Spermatozoa

“…Herein, we aimed to reveal the link between the dynamics of PTP during capacitation with the AE and the PMI. In our experiment, the incubation for 3 h under capacitating conditions induced the occurrence of capacitation-related events known to occur in bull spermatozoa such as an increase of the BCF [41] (Table 1), and enrichment of the PTP of the sperm head as well as of the flagellum [42] ( Table 2). Furthermore, we confirmed successful capacitation by and increased susceptibility to induced AE by calcium ionophore after 3 h of incubation when compared to non-capacitated control (sample at the beginning of the incubation) [36] (Table 2).…”

Compartmentalization of the proteasome-interacting proteins during sperm capacitation

“…The redistribution of lactadherin MFGE8 (aliases p47, SED1) from the apical ridge region of the ejaculated spermatozoa to the entire acrosomal region of capacitated spermatozoa was observed in accordance with previously reported results as well as the presence of two different forms of lactadherin in spermatozoa 34,44–47 . Furthermore, MFGE8 can be isolated from OAM-matrix complex of bovine spermatozoa after IVC 45 . We found that the proteasome participates in events linked to the redistribution and degradation of MFGE8, which was hindered by proteasomal inhibitors causing the accumulation of the 48 kDa isoform (Fig.…”

Biochemical Characterization of Two Major Proteins of the Hamster Sperm Acrosomal Matrix

“…We have observed an identical architect of related proteins in the bovine sperm acrosomal matrix [20,62] reflecting the presence of a similar acrosomal distribution pattern of SP-10 related proteins in nonhuman spermatozoa. Recently, we have shown that proteomic identification of the OMC32 polypeptide (one of the bovine sperm acrosomal proteins), by MALDI-TOF-TOF analysis, yielded 2 peptides that matched the NCBI database sequence of acrosin-binding protein (Bos Taurus; GI: 194666681) [46]. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19kDa range with isoelectric points ranging between 4.0 and 5.1 which are moderately acidic [46].…”

“…Recently, we have shown that proteomic identification of the OMC32 polypeptide (one of the bovine sperm acrosomal proteins), by MALDI-TOF-TOF analysis, yielded 2 peptides that matched the NCBI database sequence of acrosin-binding protein (Bos Taurus; GI: 194666681) [46]. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19kDa range with isoelectric points ranging between 4.0 and 5.1 which are moderately acidic [46]. Human sperm SP-10 peptides from 24-34kDa have isoelectric points of approximately 4.9; whereas, the isoelectric point 17.5kDa peptide is basic [55].…”

“…For example, while some acrosomal proteins such as hyaluronidase, dipeptidylpeptidase I, and AA 1 do not appear to be associated with the acrosomal matrix and are freely soluble and rapidly released upon cell disruption or during the acrosome reaction, other components, such as acrosin, are associated with a particular sperm function and are released slowly during the acrosome reaction [37][38][39][40][42][43][44][45]. Currently, we have shown that a significant portion of SPACA3 was released after the lysophosphatidyl choline (LPC)-induced acrosome reaction; whereas, the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction [46].…”