During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.

Bioresource Engineering Division RIKEN BioResource Research Center 305 0074 Ibaraki Japan

Bioresource Engineering Division RIKEN BioResource Research Center 305 0074 Ibaraki Japan;

Bioresource Engineering Laboratory RIKEN Cluster for Pioneering Research 351 0198 Saitama Japan

Communal Laboratory National Center for Global Health and Medicine 162 8655 Tokyo Japan

Cooperative Division of Veterinary Sciences Tokyo University of Agriculture and Technology 183 8509 Tokyo Japan

Department of Anatomy Biochemistry and Physiology Institute for Biogenesis Research John A Burns School of Medicine University of Hawaii Honolulu HI 96822;

Department of Developmental Biology Institute of Experimental Medicine The Czech Academy of Sciences Prague 142 20 Czech Republic

Faculty of Life and Environmental Sciences University of Tsukuba 305 8572 Ibaraki Japan

Faculty of Life and Environmental Sciences University of Tsukuba 305 8572 Ibaraki Japan;

Institute of Laboratory Animals Kyoto University Graduate School of Medicine 606 8501 Kyoto Japan

Laboratory of Epigenetic Regulations Institute of Molecular Genetics of the ASCR the Czech Academy of Sciences Prague 142 20 Czech Republic

School of Medicine Tokai University Isehara 259 1193 Kanagawa Japan

Proc Natl Acad Sci U S A. 2020 Sep 29;117(39):24601. doi: 10.1073/pnas.2018022117. PubMed

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Formation of spermatogonia and fertile oocytes in golden hamsters requires piRNAs

Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals