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Odorant Receptors and Desensitization Proteins Colocalize in Mammalian Sperm

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Abstract

Background

The identification of transcripts encoding putative olfactory receptors in mammalian germ cells (1) has generated the hypothesis that olfactory receptors may serve a chemosensory role in sperm Chemotaxis during fertilization. We have sought to identify and localize these receptors and their regulatory machinery in rat sperm in order to gain further insight into mammalian sperm Chemotaxis and odorant receptor physiology.

Materials and Methods

We conducted reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers directed against sequences conserved across members of the known odorant receptor family to identify transcripts from testis and round spermatids. Western analysis and immunohistochemistry were performed using antibodies raised against two peptide sequences conserved among odorant receptors and using fusion protein antibodies to G-protein receptor kinase 3 (GRK3/βARK2) and β-arrestin2.

Results

We detected transcripts encoding putative odorant receptors in both testis and round spermatids of the adult rat. Restriction digests of the PCR products demonstrated the existence of multiple gene products. Two anti-odorant receptor antibodies specifically recognized a 64 kD band in rat sperm preparations by Western blot. The proteins GRK3 and β-arrestin2, implicated in olfactory desensitization, were detected in sperm cytosolic extracts using Western analysis. Immunohistochemistry colocalized putative odorant receptors, GRK3 and β-arrestin2 to elongating spermatids in the testis and to the midpiece of mature sperm.

Conclusions The specific localization of odorant receptors to the respiratory center of mature sperm is consistent with a role for these proteins in transducing chemotactic signals. Based on the colocalization, it is plausible that GRK3 and β-arrestin2 function in sperm to regulate putative chemoreceptor responses

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Acknowledgments

LDW is supported by NIH Medical Scientist Training Program Grant GM 07309; AJR is supported by USPHS Grant F32 DC00121-02; SHS is supported by USPHS Grant DA-00266 and Research Scientist Award DA-00074; and GVR is supported by a McKnight Scholars Award, a grant from the W. M. Keck Foundation, and the Whitehall Foundation, International Flavors and Fragrances, and USPHS Grant 1 R29 DC 00872-01. The authors thank Dr. Osamu Matsuzaki and Robert Bakin for their assistance in antibody production and affinity purification. Dr. William Wright for assistance in spermatid purification and for critically reading the manuscript, Michael Delannoy for his expert technical guidance with confocal microscopy, and Dr. Thomas S. K. Chang, Noam Cohen, and David Sabatini for helpful discussions.

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Loren D. Walensky & Solomon H. Snyder

  2. Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    A. Jane Roskams, Solomon H. Snyder & Gabriele V. Ronnett

  3. Department of Psychiatry and Behavioral Sciences and Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Solomon H. Snyder

  4. Departments of Medicine and Biochemistry and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA

    Robert J. Lefkowitz

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  1. Loren D. Walensky
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Walensky, L.D., Roskams, A.J., Lefkowitz, R.J. et al. Odorant Receptors and Desensitization Proteins Colocalize in Mammalian Sperm. Mol Med 1, 130–141 (1995). https://doi.org/10.1007/BF03401561

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