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Chizuru Ito, Kenji Yamatoya, Keiichi Yoshida, Lisa Fujimura, Hajime Sugiyama, Akiko Suganami, Yutaka Tamura, Masahiko Hatano, Kenji Miyado and Kiyotaka Toshimori

A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn / ) mouse model and found that the EQTN-KO males have reduced fertility and sperm–egg adhesion, while the EQTN-KO females are fertile. Eqtn / sperm were normal in morphology and motility. Eqtn / -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn / with Eqtn +/+-Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn / males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn / sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn / sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 / ) was more severe compared with that of Eqtn / males. Eqtn / -Tg (Eqtn) males, which were generated to rescue Eqtn / males, restored the reduced fertility.

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Keiichi Yoshida, Chizuru Ito, Kenji Yamatoya, Mamiko Maekawa, Yoshiro Toyama, Fumie Suzuki-Toyota and Kiyotaka Toshimori

It is important to establish a reliable and progressive model of the acrosome reaction. Here, we present a progression model of the acrosome reaction centering around the acrosomal membrane-anchored protein equatorin (MN9), comparing the staining pattern traced by MN9 antibody immunofluorescence with that traced by Arachis hypogaea agglutinin (PNA)–FITC. Prior to the acrosome reaction, equatorin was present in both the anterior acrosome and the equatorial segment. Since sperm on zona pellucida showed various staining patterns, MN9-immunostaining patterns were classified into four stages: initial, early, advanced, and final. As the acrosome reaction progressed from the initial to the early stage, equatorin spread from the peripheral region of the anterior acrosome toward the center of the equatorial segment, gradually over the entire region of the equatorial segment during the advanced stage, and finally uniformly at the equatorial segment at the final stage. In contrast, the PNA–FITC signals spread more quickly from the peripheral region of the acrosome toward the entire equatorial segment, while decreasing in staining intensity, and finally became weak at the final stage. MN9-immunogold electron microscopy showed equatorin on the hybrid vesicles surrounded by amorphous substances at advanced stage of acrosome reaction. Equatorin decreased in molecular mass from 40–60 to 35 kDa, and the signal intensity of 35 kDa equatorin increased as the acrosome reaction progressed. Thus, the established equatorin-based progression model will be useful for analyzing not only the behavior of equatorin but also of other molecules of interest involved in the acrosome reaction.

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Risako Oda-Sakurai, Hiroshi Yoshitake, Yoshiki Miura, Saiko Kazuno, Takashi Ueno, Akiko Hasegawa, Kenji Yamatoya, Kenji Takamori, Atsuo Itakura, Hiroshi Fujiwara, Satoru Takeda and Yoshihiko Araki

Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.