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Sandeep Goel Laboratory of Reproductive Biology, Centre for Cellular and Molecular Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and
Laboratory of Reproductive Biology, Centre for Cellular and Molecular Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and

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Mayako Fujihara Laboratory of Reproductive Biology, Centre for Cellular and Molecular Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and

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Naojiro Minami Laboratory of Reproductive Biology, Centre for Cellular and Molecular Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and

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Masayasu Yamada Laboratory of Reproductive Biology, Centre for Cellular and Molecular Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and

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Hiroshi Imai Laboratory of Reproductive Biology, Centre for Cellular and Molecular Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and

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Gonocytes are primitive germ cells that are present in the neonatal testis and are committed to male germline development. Gonocytes differentiate to spermatogonia, which establish and maintain spermatogenesis in the postnatal testis. However, it is unknown whether large animal species have pluripotency-specific proteins in the testis. Nanog and Pou5f1 (Oct3/4) have been identified as transcription factors essential for maintaining pluripotency of embryonic stem cells in mice. Here, we show that NANOG protein was expressed in the germ cells of neonatal pig testes, but was progressively lost with age. NANOG was expressed in most of the lectin Dolichos biflorus agglutinin- and ZBTB16-positive gonocytes, which are known gonocyte-specific markers in pigs. NANOG was also expressed in Sertoli and interstitial cells of neonatal testes. Interestingly, POU5F1 expression was not detected at either the transcript or the protein level in neonatal pig testis. In the prepubertal testis, NANOG and POU5F1 proteins were primarily detected in differentiated germ cells, such as spermatocytes and spermatids, and rarely in undifferentiated spermatogonia. By using a testis transplantation assay, we found that germ cells from 2- to 4-day-old pigs could colonize and proliferate in the testes of the recipient mice, suggesting that primitive germ cells from neonatal pig testes have stem cell potential.

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Shinnosuke Suzuki
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Yusuke Nozawa
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Satoshi Tsukamoto Laboratory of Reproductive Biology, Laboratory of Animal and Genome Sciences Section, Graduate School of Medicine, Graduate School of Agriculture, Kyoto University, Kyoto 606-8052, Japan

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Takehito Kaneko Laboratory of Reproductive Biology, Laboratory of Animal and Genome Sciences Section, Graduate School of Medicine, Graduate School of Agriculture, Kyoto University, Kyoto 606-8052, Japan

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Hiroshi Imai
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Naojiro Minami
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SET and MYND domain-containing protein 3 (Smyd3) is a histone H3 lysine 4 (H3K4) di- and tri-methyltransferase that forms a transcriptional complex with RNA polymerase II and activates the transcription of oncogenes and cell cycle genes in human cancer cells. However, the study of Smyd3 in mammalian early embryonic development has not yet been addressed. In the present study, we investigated the expression pattern of Smyd3 in mouse preimplantation embryos and the effects of RNA interference (RNAi)-mediated Smyd3 repression on the development of mouse embryos. We showed that Smyd3 mRNA levels increased after the two-cell stage, peaked at the four-cell stage, and gradually decreased thereafter. Moreover, in two-cell to eight-cell embryos, SMYD3 staining was more intense in the nuclei than it was in the cytoplasm. In Smyd3-knockdown embryos, the percentage of inner cell mass (ICM)-derived colony formation and trophectoderm (TE)-derived cell attachment were significantly decreased, which resulted in a reduction in the number of viable offspring. Furthermore, the expression of Oct4 and Cdx2 during mid-preimplantation gene activation was significantly decreased in Smyd3-knockdown embryos. In addition, the transcription levels of ICM and epiblast markers, such as Oct4, Nanog, and Sox2, the transcription levels of primitive endoderm markers, such as Gata6, and the transcription levels of TE markers, such as Cdx2 and Eomes, were significantly decreased in Smyd3-knockdown blastocysts. These findings indicate that SMYD3 plays an important role in early embryonic lineage commitment and peri-implantation development through the activation of lineage-specific genes.

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Dan Liu Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

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Takuto Yamamoto Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

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Haoxue Wang Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

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Naojiro Minami Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

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Shinnosuke Honda Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

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Shuntaro Ikeda Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

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In brief

Proper early embryonic development in mammals relies on precise cellular signaling pathways. This study reveals that NSUN5 is crucial for the regulation of the Hippo pathway, ensuring normal proliferation and differentiation in mouse preimplantation embryos.

Abstract

NOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. Our data revealed an upregulation of Nsun5 at the two-cell stage of mouse preimplantation development, suggesting its significance in early embryonic development. Given m5C’s important role in stabilizing rRNA and mRNA and the Hippo signaling pathway’s critical function in lineage segregation during embryogenesis, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. To examine this hypothesis, we employed Nsun5-specific small interfering RNAs for targeted gene silencing in mouse preimplantation embryos. Nsun5 knockdown resulted in significant developmental impairments including reduced blastocyst formation, smaller size of blastocysts, and impaired hatching from the zona pellucida. Nsun5 knockdown also led to decreased cell numbers and increased apoptosis in embryos. We also observed diminished nuclear translocation of yes-associated protein 1 (YAP1) in Nsun5 knockdown embryos at the morula stage, indicating disrupted cell differentiation. This disruption was further evidenced by an altered ratio of CDX2-positive to OCT4-positive cells. Furthermore, Nsun5 depletion was found to upregulate the Hippo signaling-related key genes, Lats1 and Lats2 at the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway.

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