CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†

Lu, Yonggang; Oura, Seiya; Matsumura, Takafumi; Oji, Asami; Sakurai, Nobuyuki; Fujihara, Yoshitaka; Shimada, Keisuke; Miyata, Haruhiko; Tobita, Tomohiro; Noda, Taichi; Castaneda, Julio M; Kiyozumi, Daiji; Zhang, Qian; Larasati, Tamara; Young, Sam; Kodani, Mayo; Huddleston, Caitlin A; Robertson, Matthew J.; Coarfa, Cristian; Isotani, Ayako; Aitken, R. John; Okabe, Masaru; Matzuk, Martin M.; Garcia, Thomas; Ikawa, Masahito

doi:10.1093/biolre/ioz103

Cited by 90 publications

(61 citation statements)

References 44 publications

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“…The CRISPR-KO approach efficiently screens male fertility genes in vivo. In fact, we revealed that more than 90 genes are dispensable (22)(23)(24)(25)(26), while 10 genes and two clusters are required (21,(27)(28)(29)(30) for male fertility. Recently, we reported that a testis-specific gene, 4930451I11Rik (renamed as Fertilization Influencing Membrane Protein [Fimp]), is another sperm factor critical for sperm−oocyte fusion (31).…”

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confidence: 91%

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

Noda

Fujihara

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Sperm−oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm−oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm−oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm−oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouseSof1,Tmem95,andSpaca6rescued the sterility ofSof1,Tmem95, andSpaca6KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm−oocyte fusion.

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mentioning

confidence: 91%

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

Noda

Fujihara

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

117

162

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“…In the past, it was assumed that evolutionarily conserved genes must be important for the reproductive organ system they are expressed in, especially since they continue to persist in all organisms. Unfortunately, gene-editing technology reports the contrary [30][31][32]. Perhaps these genes had a purpose once individually, but many of the genes we study are not essential for the survival of the organism.…”

Section: Discussionmentioning

confidence: 94%

CRISPR/Cas9-Mediated Genome Editing Reveals Oosp Family Genes are Dispensable for Female Fertility in Mice

Abbasi

Kodani

Emori

et al. 2020

Cells

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There are over 200 genes that are predicted to be solely expressed in the oocyte and ovary, and thousands more that have expression patterns in the female reproductive tract. Unfortunately, many of their physiological functions, such as their roles in oogenesis or fertilization, have yet to be elucidated. Previous knockout (KO) mice studies have proven that many of the genes that were once thought to be essential for fertility are dispensable in vivo. Therefore, it is extremely important to confirm the roles of all genes before spending immense time studying them in vitro. To do this, our laboratory analyzes the functions of ovary and oocyte-enriched genes in vivo through generating CRISPR/Cas9 KO mice and examining their fertility. In this study, we have knocked out three Oosp family genes (Oosp1, Oosp2, and Oosp3) that have expression patterns linked to the female reproductive system and found that the triple KO (TKO) mutant mice generated exhibited decreased prolificacy but were not infertile; thus, these genes may potentially be dispensable for fertility. We also generated Cd160 and Egfl6 KO mice and found these genes are individually dispensable for female fertility. KO mice with no phenotypic data are seldom published, but we believe that this information must be shared to prevent unnecessary experimentation by other laboratories.

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“…Redundancy is the phenomenon that two or more gene products are performing the same or overlapping function in a given physiogical or cellular context and the inactivation of one of the redundant genes will be compensated by other genes with similar function and has little or no effect on the organism itself (Nowak et al, 1997). Till now, many testis-specific-genes are reported the redundant gene and the knocking out of these genes do not impair male fertility in mouse models (Iwamori et al, 2011;Lu et al, 2019;Ozturk et al, 2014). Due to the lack of evidence of domains or sequence motifs in Tex33, it's still hard to explain which gene replaces the Tex33 in vivo yet.…”

Section: Discussionmentioning

confidence: 99%

Spermatogenesis is normal in Tex33 knockout mice

Zhu

Zhang

Zeng

et al. 2020

PeerJ

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Testis expressed gene 33 (Tex33) is a recently reported testis-specific gene and it is evolutionarily conserved in vertebrates. The Tex33 expression is found in cytoplasm of round spermatids in Mus musculus. However, the in vivo function of Tex33 remains unknown. In this study, we made a 62bp in frame deletion on Exon2 of Tex33 gene by CRISPR/Cas9 in C57B/L6 mouse, which cause frame shift mutation of Tex33 gene. Tex33-/-adult male were fertile, and there is no significant change on litter size compared with male wildtype (Tex33+/+) adult. Besides, no overt differences were found in testis/body weight ratios, testicular/epididymal tissue morphology, sperm counts, sperm morphology and spermatozoa motility in adult Tex33-/-male mice (N = 3), in comparison with Tex33+/+ adult (N = 3). TUNEL assay also indicates the germ cells apoptosis ratio was not significantly changed in adult Tex33-/- adult male mouse testis (N = 3), compared with adult Tex33+/+ male (N = 3). Importantly, the first wave of elongating spermatids formation happens in 5w old mice. We find that the first wave of spermiogenesis is not disrupted in both 5-week-old Tex33+/+ and Tex33-/-male mouse testes and three hallmarks of spermatogenesis, PLZF,γ-H2AX and TNP1, are all detectable in seminiferous tubule. All results indicate that Tex33 is a redundant gene to spermatogenesis. This study can help other researchers avoid repetitive works on redundant genes.

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CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†

Cited by 90 publications

References 44 publications

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

CRISPR/Cas9-Mediated Genome Editing Reveals Oosp Family Genes are Dispensable for Female Fertility in Mice

Spermatogenesis is normal in Tex33 knockout mice

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