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ORIGINAL RESEARCH ARTICLE

Fourier Transform Infrared Spectroscopic Analysis of the Intact Zona Pellucida of the Mammalian Egg: Changes in the Secondary Structure of Bovine Zona Pellucida Proteins During Fertilization

Masayuki Nara^*, Naoto Yonezawa^,, Takeshi Shimada, Kazuya Takahashi, Masaru Tanokura, Fumiaki Yumoto, Hiroyuki Nakagawa, Kazuyo Ohashi^,||, Seizo Hamano^¶ and Minoru Nakano^,,1

^* Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Chiba 272-0827, Japan; Department of Chemistry, Faculty of Science; and Graduate School of Science and Technology, Chiba University, Chiba 263-8522, Japan; Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; ^|| Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan; and ^¶ Livestock Improvement Association, Tokyo 140-0002, Japan

To whom requests for reprints should be addressed at ¹ Department of Chemistry, Faculty of Science, Chiba University, 1–33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan. E-mail: mnakano{at}faculty.chiba-u.jp

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and preimplantation. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of ß-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. -Helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs indicated that the zona proteins in the native zona pellucida also have ß-structure as the main constituent. Attenuated total reflection-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the ß-structure content increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.

Key Words: zona pellucida • FTIR • ATR-FTIR • mammalian fertilization • zona hardening • secondary structure

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