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Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa

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Abstract

Defective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64–65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55–68 kDa, consistent with the original spot used for immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with an anti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with anti-ubiquitin antibodies and affinity purified with matrix-bound, recombinant ubiquitin-binding UBA domain. These bands did not show the typical pattern of ARSA glycosylation but overlapped with bands preferentially binding the Lens culinaris agglutinin (LCA) lectin. By both epifluorescence microscopy and IBFC, the LCA binding was increased in the ubiquitinated spermatozoa with diminished ARSA immunoreactivity. ARSA was also found in the epididymal fluid suggesting that in addition to intrinsic ARSA expression in the testis, epididymal spermatozoa take up ARSA on their surface during the epididymal passage. We conclude that sperm surface ARSA is one of the ubiquitinated sperm surface glycoproteins in defective bull spermatozoa. Defective sperm surface thus differs from normal sperm surface by increased ubiquitination, reduced ARSA binding, and altered glycosylation.

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Acknowledgments

We thank Dr. Francis Pau (Oregon National Primate Research Center, Beaverton, OR) for help with rabbit immunization, Dr. Jan Pohl (Microchemical Facility, Emory University School of Medicine, Atlanta, GA) for peptide sequencing, Ms. Kathryn Craighead for manuscript editing, and Mr. Heinz Leigh and Ms. Nicole Leitman for technical assistance.

Funding

This work was in part supported grant #13324-2007 from The Missouri Life Sciences Research Board, National Research Initiative Competitive Grants no. 2007-35203-18274 and no. 2011-67015-20025 from the USDA National Institute of Food and Agriculture, Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23231 from the USDA NIFA, and seed funding from the F21C program of the University of Missouri-Columbia.

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Authors and Affiliations

  1. Division of Animal Sciences, University of Missouri, Columbia, MO, 65211-5300, USA

    Kathleen M. Kelsey, Michal Zigo, Karl Kerns, Gaurishankar Manandhar, Miriam Sutovsky & Peter Sutovsky

  2. Departments of Obstetrics & Gynecology and Reproductive Health Program, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA, 30310, USA

    Winston E. Thompson

  3. Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal

    Gaurishankar Manandhar

  4. Departments of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO, 65211, USA

    Peter Sutovsky

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  1. Kathleen M. Kelsey
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Correspondence to Michal Zigo.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All studies involving vertebrate animals were completed under the strict guidance of an Animal Care and Use protocol, and Antibody Production Protocol (included in the Supplementary data file) approved by the Animal Care and Use Committee (ACUC) of the University of Missouri. This article does not contain any studies with human participants performed by any of the authors.

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Kelsey, K.M., Zigo, M., Thompson, W.E. et al. Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa. Cell Tissue Res 379, 561–576 (2020). https://doi.org/10.1007/s00441-019-03144-1

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