Glycine Receptor Α4 Subunit Facilitates the Early Embryonic Development in Mice

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REPRODUCTIONRESEARCH

Glycine receptor α4 subunit facilitates the early embryonic development in mice

Hirofumi Nishizono1,2,3, Mohamed Darwish4,5, Takaho A Endo6, Kyosuke Uno7, Hiroyuki Abe3 and Ryohei Yasuda1 1Max Planck Florida Institute for Neuroscience, Jupiter, Florida, USA, 2Life Science Research Center, University of Toyama, Toyama, Japan, 3Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa, Japan, 4Department of Behavioral Physiology, Graduate School of Innovative Life Science, University of Toyama, Toyama, Japan, 5Department of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt, 6Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan and 7Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Science, Setsunan University, Hirakata, Japan Correspondence should be addressed to H Nishizono; Email: [email protected]

Abstract

Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the β subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg. Reproduction (2020) 159 41–48

Introduction important role of glycine in the development of fertilized eggs, the addition of glycine to a medium containing Mammalian oocytes are fertilized in the oviduct and bovine fertilized oocytes facilitates its development to are developed while traveling inside the fallopian tube the blastocyst stage (Van Winkle et al. 1990, Dawson toward the uterus. Oviduct fluid is the environment et al. 1998, Takahashi & Kanagawa 1998, Steeves & first experienced by oocytes and actively regulates Baltz 2005, Herrick et al. 2016). fertilization and subsequent preimplantation One potential mechanism by which glycine may exert development (Rizos et al. 2016, Pérez-Cerezales et al. its effect on the fertilized egg is through glycine receptors 2017). Chemical composition of oviduct fluids contains (GlyRs). GlyRs are ligand-gated chloride channels that a variety of molecules including simple and complex mediate inhibitory neurotransmission in the spinal cord carbohydrates, inorganic ions, lipids, phospholipids and brain (Moss & Smart 2001). They are pentameric lactate, pyruvate, glucose, proteins and free amino acids channels consisting of α and β subunits: four α-subunits (Leese et al. 2001, Avilés et al. 2010, Rizos et al. 2016, (α1 to α4) and one β-subunit have been identified in Pérez-Cerezales et al. 2017). Among the amino acids many species, including human (Miyazawa et al. 2003). found in oviduct fluid, the non-essential amino acid Of the GlyRs subunits, the function of the α4 subunit is glycine is one of the most abundant amino acids reaching not well characterized since it has been considered as a up to few millimolar levels in many mammalian species, pseudogene in humans due to the lack of transmembrane including bovine (Hugentobler et al. 2007), rabbits domains (Simon et al. 2004). Recent studies, however, (Leese et al. 1979, Miller & Schultz 1987), and rats suggested that the GlyRα4 subunit may have important (Nakamura et al. 2016). Glycine has been recognized as functions, as its deletion is related to human disease an essential component in culture medium for in vitro (Labonne et al. 2016), and it is required for startle and fertilized eggs such as potassium-supplemented simplex escape responses in zebrafish (Leacock et al. 2018). It has optimization medium (KSOM) (Moore & Bondioli 1993, been reported that GlyRs play a vital role in the fertilization Lee & Fukui 1996, Summers et al. 2000). Consistent with process and are involved in zona pellucida-mediated

© 2020 Society for Reproduction and Fertility https://doi.org/10.1530/REP -19-0312 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via https://rep.bioscientifica.com Downloaded from Bioscientifica.com at 09/27/2021 06:00:56AM via free access

-19-0312 42 H Nishizono and others acrosome reaction in mice, pigs, and hamsters (Sato were cultured in KSOM (ARK Resource) to remove extra sperm et al. 2000, Llanos et al. 2001, Bray et al. 2002, Meizel and cumulus cells. Fertilized eggs were used for GlyR inhibition & Son 2005). However, it is unknown whether GlyRs are experiments and the generation of knockout mice. involved in glycine-induced molecular processes in the oocytes after fertilization. GlyR inhibition experiments in fertilized eggs Here, we examined the roles of GlyRs in preimplantation development of fertilized eggs. Our results Two inhibitors, strychnine nitrate 5H2O (FUJIFILM Wako Pure showed that (1) GlyR inhibitors reduce fertilization rate Chemical Corp., Osaka, Japan) and ω-phosphono-α-amino and preimplantation development of mouse oocytes, (2) acid (PMBA, Sigma-Aldrich), were used for GlyR inhibition experiments in fertilized eggs. Each was dissolved in water mRNA and proteins of α4 subunit of GlyR is expressed at the MII stage to four-cell stage in mouse oocytes and to prepare a highly concentrated stock solution, which was fertilized eggs, and (3) knockout of the gene encoding then added to the embryo culture medium for use. The final concentration in the embryo culture medium was adjusted to 1 Glra4 impairs the developmental rate and cell number mM strychnine and 100 M PMBA regarding previous reports of blastocysts in mouse embryos during preimplantation μ (Saitoh et al. 1994, Jensen 2005). In the vehicle group, an equal development and also reduces resulting litter size. Thus, volume of H2O was added to the embryo culture medium. our results suggest that glycine in oviduct activates GlyRs Fertilized eggs were divided into groups of 20–50 and cultured on the fertilized eggs to facilitate the preimplantation at 37°C and 5% CO2 in KSOM with inhibitor or vehicle. Culture development of eggs. experiments were conducted five times or more for each experimental group. We monitored embryo development under Materials and methods a stereoscopic microscope every 24 h after insemination. The developmental rate was calculated as the number of four-cell Reagents embryos, morulae, or blastocysts per two-cell embryo ×100. All reagents were purchased from Sigma-Aldrich unless otherwise stated. The human fallopian tube fluid (HTF) medium Transcriptomic analysis before and after fertilization and potassium-supplemented simplex optimized medium (KSOM) used for in vitro fertilization and embryo culture were Gene expression data were collected from the NCBI GEO purchased from ARK Resource Co. Ltd. (Kumamoto, Japan). database. RNA-seq datasets from murine embryos (GSE66390) were used. Mouse RNA-seq data were aligned against the corresponding RefSeq mRNAs using kallisto (Bray et al. Animals 2016) (https://pachterlab.github.io/kallisto/). C57BL/6J male mice (8–18 weeks of age) and female mice (4–16 weeks of age) were purchased from Japan SLC (Hamamatsu, Immunohistochemical staining Japan). We used 4- 8-week-old female mice for making gene- modified mice, because mice in this age range generally Immunohistochemical staining of fertilized mouse oocytes ovulate more oocyte. For studies of embryonic development was based on the method of Sauvegarde et al. (2016). For such as in vitro experiment (Figs 1 and 2) or phenotypic GLRA4 detection, an anti-GLRA4 rabbit polyclonal IgG analysis of Glra4 KO embryos (Figs 3 and 4), we used 12–16 antibody (HPA044759, Sigma-Aldrich), and as a negative weeks of age. Control and experimental groups are littermates control, a rabbit IgG polyclonal isotype control antibody and thus always of the same ages. The number of animals used (ab37415, Abcam plc) was used. Anti-rabbit IgG-FITC (F0382, in each experiment is summarized in Supplementary Table 1 Sigma-Aldrich) was used as the secondary antibody. After (see section on supplementary materials given at the end of the secondary antibody reaction, embryos were washed, this article). Animals were maintained under a 12:12-h light: the solution was replaced with 40% glycerol/PBS, and then dark cycle at 22 ± 2°C and relative humidity of 40–60% with embryos were mounted on a 35-mm glass-bottom dish (3960- access to chow (CE2, CLEA Japan Inc., Tokyo, Japan) and 035, AGC TECHNO GLASS Co. Ltd., Shizuoka, Japan) and water ad libitum. All experiments were undertaken according examined using a confocal laser microscope (LSM 780, Zeiss). to the rules and regulations of the Guide for the Care and Embryo fluorescence was observed using ZEN software (Zeiss). Use of Laboratory Animals (https://grants.nih.gov/grants/ olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf). Generation of Glra4-knockout mice using the The experimental protocol was approved by the Animal Care CRISPR/Cas9 system Committee of Toyama University. For the design of gRNA, we used CRISPR direct (https://crispr. dbcls.jp), which is a publicly available program (Naito et al. Preparation of mouse embryos by in vitro fertilization 2015). To reduce off-target effects, the sequences suggested Fertilized eggs were prepared by in vitro fertilization as described by CRISPR direct were confirmed by BLAST, and the gRNA in our previous report (Nishizono et al. 2017). In brief, the with the fewest predicted off-target effects was adopted. collection of C57BL/6J mouse oocytes, preculture of C57BL/6J Specific gRNA sequences and primers for DNA sequencing mouse spermatozoa, and in vitro fertilization were carried out analysis are shown in Supplementary Table 2. The crRNA, in HTF medium (ARK Resource). Oocytes and fertilized eggs tracrRNA, and Cas9 protein were all purchased from IDT Co.

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Figure 1 Glycine receptor (GlyR) inhibitors inhibit the development of mouse early embryos from the four-cell to morula stage. (A) Experimental scheme. In the first experiment (B), the inhibitor was added immediately after insemination (0-h protocol), and in subsequent experiments (C, D, E and F), inhibitors were added 3 h after insemination (3-h protocol). (B) Fertilization rate with vehicle (white box: n = 9 replicates, 18 animals, 273 oocytes) and strychnine (gray box: n = 7 replicates, 14 animals, 269 oocytes) using 0-h protocol. The right panel is fertilized eggs of vehicle group and strychnine group after 24-h insemination. (C) Fertilization rate with vehicle (white box: n = 9 replicates, 18 animals, 266 oocytes) and strychnine (gray box: n = 5 replicates, 10 animals, 130 oocytes) using 3-h protocol. The right panel is fertilized egg of vehicle group and strychnine group after 6 h and 24 h insemination. (D) Blastocyst developmental rate with vehicle (white box: n = 9 replicates, 18 animals, 159 embryos), strychnine (gray box: n = 5 replicates, 10 animals, 93 embryos) and ω-phosphono-α-amino acid (PMBA) (black box: n = 7 replicates, 14 animals, 191 embryos) after 96-h insemination. (E) Time course of developmental rate every 24 h after inhibitor addition. Vehicle group (blue: n = 9 replicates, 18 animals, 159 embryos), strychnine group (red: n = 5 replicates, 10 animals, 93 embryos) and PMBA group (orange circle: n = 7 replicates, 14 animals, 191 embryos). (F) Embryo morphology with vehicle, strychnine, and PMBA after 96-h insemination. *P < 0.05, **P < 0.01, the difference between the vehicle group and each inhibitor group was tested by Brunner–Munzel test (B and C) and post hoc test after Kruskal–Wallis test (D and E). Size bars: B, C, and F, 100 µm. https://rep.bioscientifica.com Reproduction (2020) 159 41–48

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Figure 2 Glycine receptor alpha 4 subunit (Glra4) is expressed from the MII oocyte to the four-cell stage. (A) Expression level of Glycine receptor alpha 1 subunit (Glra1), Glycine receptor alpha 2 subunit (Glra2), Glycine receptor alpha 3 subunit (Glra3), Glra4, and Glycine receptor beta subunit (Glrb) by developmental stage. The RNA-seq data from MII oocyte to eight-cell stage mouse embryo in public database is reanalyzed. Each line indicates the expression level calculated by RPKM. (B) Immunohistochemical staining of Glra4 protein (GLRA4) from MII stage oocyte to blastocyst stage embryo (n = 50 animals, 407 embryos). Green indicates GLRA4, and blue indicates Hoechst 33342. The lower row shows the result using a negative control antibody instead of the anti-GLRA4 antibody. Size bars: 100 µm.

(Coralville, IA, USA). Delivery of gRNA and Cas9 protein complex into C57BL/6J mouse embryos was performed, such as our previous paper (Darwish et al. 2019). When the founder mice reached 3 weeks of age, genomic DNA was extracted from the ear tissue, and DNA sequences were confirmed by Sanger sequencing. To reduce the influence of off-target effects, the founder mice were backcrossed for two generations, and then N3 mice were generated. Genotyping was performed by sequence analysis using CRISP-ID software (http://crispid. gbiomed.kuleuven.be/) (Dehairs et al. 2016).

In vitro fertilization and embryo culture of fertilized eggs obtained from Glra4-knockout mice To investigate the influence of Glra4 deficiency in oocytes and fertilized eggs, we removed the oocytes from the wild-type and the Glra4-knockout mice by superovulation treatment and performed in vitro fertilization with sperm taken from C57BL/6J male mice. To eliminate the effect of glycine receptors on the sperm, C57BL/6J sperms were used to inseminate WT and Glra4 KO mouse oocytes. After insemination, to remove extra spermatozoa and cumulus cells, oocytes were washed twice and then cultured in KSOM. Fertilized eggs were observed under a stereoscopic microscope every 24 h after insemination. The developmental rate was calculated as the number of four-cell embryos, morulae, or blastocysts per two-cell embryo ×100. To count the number of cells in the Figure 3 Generation of Glra4-knockout mice using the CRISPR/Cas9 blastocyst-stage embryos, nuclei were stained with Hoechst system and confirmation of loss of GLRA4 in fertilized eggs. (A) 33342 as previously reported (Nishizono et al. 2017) and gRNA design scheme. The upper row shows the genome with exons counted using a confocal laser microscope. and introns. The middle section shows the domain structure. The target region and the gRNA sequence are shown at the bottom. (B) DNA sequences of the wild type (WT) and the mutant mice (Mutant). Statistical analysis Dashes (–) indicate nucleotides missing from the original target sequence. (C) Predicted amino acid sequences of the WT and the All variables are reported as means ± s.d. The normality of mutant mice using ExPASy software (https://web.expasy.org/ the data was assessed using the Kolmogorov–Smirnov test translate/). Red letters indicate the open reading frame. (D) for goodness of fit. Statistical differences were determined by Immunohistochemical staining of GLRA4 in the fertilized eggs of the Brunner–Munzel test and nonparametric multiple comparison mutant mice (n = 8 animals, 78 embryos). Green indicates GLRA4, examinations (Brunner–Munzel post hoc after Kruskal–Wallis and blue indicates Hoechst 33342. Size bars: 100 µm.

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Figure 4 Loss of GLRA4 decreases blastocyst development rate and cell number in vitro, and litter size in vivo. (A) Experimental scheme of B–D. To eliminate the effect of glycine receptors on the sperm, C57BL/6J sperms were used to inseminate WT and Glra4 KO mouse oocytes. Blastocyst developmental rate and cell number were measured 96 h after insemination. (B) Comparison of oocyte numbers in the WT (white box: n = 9 animals) and Glra4 KO mice (gray box: n = 6 animals). (C) Blastocyst formation rate of the WT (white box: n = 7 replicates, 7 animals, 191 embryos) and Glra4 KO mice (gray box: n = 7 replicates, 7 animals, 196 embryos) after 96-h insemination. (D) Comparison of cell nuclei numbers of the blastocyst in the WT (white box: n = 7 animals, 107 embryos) and Glra4 KO mice (gray box: n = 5 animals, 116 embryos). After Hoechst 33342 staining, the nuclear number was measured. The right panel shows the fluorescence microscope images. Size bars: 100 µm. (E) Litter size of the WT (white box: n = 12 animals) and Glra4 KO mice (gray box: n = 12 animals) in natural mating. No. of littermate indicates the number of offspring born from one mother in one delivery. The WT and Glra4 KO mice of the same age were used. *P < 0.05 and *****P < 0.000001, the difference between the WT and Glra4 KO mice was tested by Brunner–Munzel test (B, C and D). test). A significance level of P < 0.05 was used for all statistical window between the moment of insemination and 3 h of tests, and two-tailed tests were applied. All analyses were the insemination. For the group in which strychnine was performed using Python3. added at 3 h after insemination, we did not observe any toxic effects on the fertilized eggs; embryos developed to the two-cell stage with normal morphological properties Results (Fig. 1C). These results indicate that our 3-h protocol To investigate the role of glycine during preimplantation is applicable to studies of GlyRs in preimplantation development of mouse embryos, we investigated the development of the fertilized eggs without severe effect of GlyR inhibitors during the preimplantation influence in the fertilization process or sperm function. development of embryos cultured in vitro (Fig. 1). Given that the 3-h protocol does not affect First, to differentiate the effects of GlyR inhibition on fertilization, we used this protocol to further study the preimplantation development from that on fertilization, function of GlyRs on embryonic development using we performed our experiments under two conditions two inhibitors (strychnine and PMBA). We found that where GlyR inhibitor strychnine was added at different the development to blastocysts was strongly inhibited timings, one at the time of insemination (0-h protocol), with a developmental arrest at the time of the four-cell and the other at 3 h following the insemination (3-h to morula transition in both the strychnine-treated and protocol) (Fig. 1A). We found that the 0-h protocol the PMBA-treated groups (Figs 1D and 1E). Notably, reduced the fertilization rate dramatically, as judged the embryos did not undergo apoptosis or necrosis by the formation of pronuclei, without inducing any but showed arrested development in the inhibitor- damage of the cell (Fig. 1B), while the 3-h protocol treated groups (Fig. 1F). Taken together, these data did not influence the fertilization rate judged by the suggest signals downstream of GlyRs are necessary for formation of pronuclei (Fig. 1C). These data indicate that the preimplantation developmental processes of post- GlyRs are required for fertilization only during the time fertilization especially the four-cell to morula transition. https://rep.bioscientifica.com Reproduction (2020) 159 41–48

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Since the inhibitors target all five GlyR subunits (α1–α4 deficient in Glra4 and were used to study the function of and β) (Saitoh et al. 1994, Jensen 2005), these inhibitor Glra4 signal in fertilized eggs. experiments do not reveal which subunits were functional To explore the role of the Glra4 signal in oogenesis and in fertilized eggs. To identify GlyR subunits expressed in early development, we performed in vitro fertilization fertilized mouse eggs, we analyzed RNA-seq data of oocyte and embryo culture using Glra4-knockout mice (Fig. 4A). and early embryo in the public database (NCBI GEO The number of ovulated oocytes obtained from Glra4- database) and found that expression of Glycine receptor knockout mice upon superovulation was not changed beta subunit (Glrb) and Glycine receptor alpha 4 subunit compared to WT mice (Fig. 4B). This data indicates that (Glra4) were transiently expressed during the MII oocyte to Glra4 function is not required for oogenesis and ovulation. two-cell embryos (Fig. 2A). On the other hand, except for We then performed in vitro fertilization and examined an oocyte and early embryo, β subunit was expressed in a the developmental rate to the blastocyst stage after 96 h wide range of organs, whereas α subunits were expressed and the number of cells in the blastocyst. To eliminate only in specific regions, such as the brain (Supplementary the effect of glycine receptors on the sperm, C57BL/6J Fig. 1). Immuno-staining displayed that the expression sperms were used to inseminate WT and Glra4 KO mouse of Glra4 peaked at the two-cell stage, which is became oocytes. Notably, the blastocyst fozrmation rate after 96 h undetectable at the morula and blastocyst stages (Fig. 2B). was significantly reduced in theGlra4 -knockout mice We also observed that the level of expression is maintained (Fig. 4C, P < 0.05). Also, the number of cells at the high until the 4-cell stage. The expression of Glra4 was blastocyst stage, which is an indicator of embryo quality also detected at the MII stage oocyte, probably due to in assisted reproductive technology, was significantly the maternal contribution. Surprisingly, it was found that reduced in the fertilized oocytes of Glra4-knockout the mouse fertilized egg expressed the GlyR α4 subunit, mice (Fig. 4D, P < 0.05). Furthermore, not limited to in whose function is still unclear. Additionally, we analyzed vitro fertilization, the litter size of the knockout mice RNA-seq data available in public databases for each during natural mating was also significantly reduced developmental stage in bovine, and human fertilized eggs. from 9 ± 1 in WT mice to 3 ± 2 in knockout mice (Fig. 4E, The GlyRs were expressed in oocytes and early embryos in P < 0.000001). These results suggest that the Glra4- all species. We found that, while the β subunit is expressed mediated signals from glycine in the oviduct fluid or in the oocytes and embryos in all these species, expression in the culture medium provide crucial support for the of α subunit subtypes differs among animal species. development of a fertilized egg into a blastocyst. Specifically, the α4 subunit was expressed in the fertilized oocytes of mice, 1 was expressed in bovine, and 2 was α α Discussion expressed in humans (Supplementary Fig. 2). The transient expression of Glra4 leads us to hypothesize In this study, we demonstrated that GlyRs are involved that the expressed protein may function as a receptor of in the preimplantation development of mouse embryos. the extracellular glycine in the oviduct fluid. To test this Among the subunits, the β and α4 subunits are expressed hypothesis, we generated mutant mice that lack Glra4 in oocytes and fertilized oocytes in mice. Furthermore, using the CRISPR/Cas9 system. In mice, Glra4 consists of a we revealed that Glra4 facilitates the development of the signal peptide (encoded by exon 1), neurotransmitter-gated blastocyst and maintains embryo quality and litter size ion-channel domain (exons 2–5), and transmembrane in mice, albeit not essential for oogenesis and ovulation. domain (exons 6–9), and there are glycine-binding sites We also clarified that the time point at which GLRA4 encoded in exons 2, 4, and 8 (Fig. 3A). We designed protein exerts its effect is from the start of the fertilization gRNA to introduce insertion/deletion mutations before stage to the four-cell stage, based on the expression the transmembrane domains, which is assumed to lead pattern of Glra4 in oocytes and fertilized eggs. The complete loss of function of this receptor (Fig. 3A, red box). overview of these findings is shown inFig. 5. We obtained a heterozygous founder mouse that carries Our study strongly suggest that GlyRs play a critical role an 11-bp deletion at position 4054–4064 (Fig. 3B). The in early development of the fertilized egg as evident from predicted open reading frame from the DNA sequence of our experiments showing two different inhibitors strongly mutant Glra4 showed that many premature stop codons suppress early development (Fig. 1). GlyRs are expressed are inserted in the mutant due to the frame-shift mutation, not only in mouse fertilized egg but also in fertilized and most of the coding regions are likely untranslated (Fig. eggs of humans and bovine (Supplementary Fig. 2), 3C). Protein structure prediction by I-TASSER software suggesting that the role of GlyRs in early embryonic (Zhang 2008, Roy et al. 2010, Yang et al. 2015) suggests development is conserved across species. We also found that the mutant protein is not only truncated but also has that, while fertilized eggs of mice expressed Glra4, that a different structure from wild type (Supplementary Fig. 3). of other animal species express different subtypes of Glra4 protein, which was expressed from the MII to the α subunits, suggesting that the function of Glra4 can four-cell stage in the fertilized eggs of WT mice, was not be replaced by other alpha subunits. Notably, GLRA2 detected in the fertilized eggs of mutant mice (Fig. 3D). expression in human embryo instead of GLRA4, a Therefore, these mutant mice were considered to be pseudogene in human, highlights the important role

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In conclusion, this study demonstrates that Glra4 is involved in the preimplantation development of mouse embryos; facilitates the development to the blastocyst; maintains the number of cells in blastocysts, and litter size in mice. The study also indicates that glycine, which has been required as a component of embryo culture medium and oviduct fluid, acts on fertilized eggs through GlyRs- mediated signaling pathway. Taken together, these data provide new insights into interactions between oviduct fluid components and mammalian fertilized eggs.

Supplementary materials Figure 5 Graphical abstract of the putative role of GlyR from This is linked to the online version of the paper fertilization to early development. at https://doi.org/10.1530/REP-19-0312. of function GlyRs. GLRA4 has been considered to be a pseudogene in humans since it incorporates a stop Declaration of interest codon in exon 9 before the last transmembrane domain, The authors declare that there is no conflict of interest that thereby encoding a non-functional protein (Simon et al. could be perceived as prejudicing the impartiality of the 2004). However, it is intact and functionally expressed research reported. in other organisms either as a single copy, for example, mouse, rat, and gorilla (Leacock et al. 2018) or duplicated in Zebrafish Imboden( et al. 2001, Hirata et al. 2010). Funding Previous functional analyses suggested that GLRA4 may function in the spinal cord and hindbrain in fish (Leacock This work was supported by the Japan Society for the Promotion of Science (grant numbers 15K20134, 17K11222, and et al. 2018). In this study, we identified a new function of 16H06276) and Hokugin Research Grant (grant numbers H29). Glra4 subunit in mouse fertilized oocytes. Our data clarify an extending role of GLRA4 protein after the fertilization step through facilitating the preimplantation development Author contribution statement of fertilized oocytes. As shown in Fig. 4, the phenotype of Glra4-knockout mice on embryo development was H N performed the majority of experiments. M D performed milder than the result of pharmacological inhibitor some experiments, especially the analysis of Glra4 KO mice’s experiments. We anticipate that this is due to other alpha phenotype. T A E analyzed the RNA-seq data. K U performed subunits of glycine receptors expressed in this stage. A analysis of cell nuclei. H N, M D, K U, H A, and R Y analyzed few evidences are suggesting this: (1) when all GlyRs are the data and wrote the manuscript. H N designed and directed this study. pharmacologically inhibited, almost all of embryos arrest developing in morula (Fig. 1E). (2) In addition to Glra4, a small amount of Glra1 and Glra3 as well as Glrb are Acknowledgment expressed in early mouse embryos, presumably forming functional heteromultimer (Fig. 2A). (3) In bovines and The authors are very grateful to Dr Shinichi Nakagawa for his humans, other subtypes of alpha subunits are expressed advice on writing the paper. instead of Glra4, suggesting that this small amount of alpha subunit other than Glra4 is likely able to compensate References Glra4 function when it is knocked out (Supplementary Avilés M, Gutiérrez-Adán A & Coy P 2010 Oviductal secretions: will they Fig. 2). The glycine receptor is a basic ion channel that be key factors for the future ARTs? Molecular Human Reproduction 16 permeates Cl−, as shown in Fig. 3, and does not itself have 896–906. (https://doi.org/10.1093/molehr/gaq056) Bray C, Son JH, Kumar P, Harris JD & Meizel S 2002 A role for the human a signaling domain (Moss & Smart 2001, Miyazawa et al. sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated 2003). Therefore, it is speculated that signal transduction by recombinant ZP3. Biology of Reproduction 66 91–97. (https://doi. via GlyRs may be through regulating glycine reception org/10.1095/biolreprod66.1.91) by intracellular [Cl−] uptake. This functional coupling Bray NL, Pimentel H, Melsted P & Pachter L 2016 Near-optimal probabilistic RNA-seq quantification.Nature Biotechnology 34 between GlyR and glycine transporters is several known 525–527. (https://doi.org/10.1038/nbt.3519) as signaling pathways that are activated by intracellular Darwish M, Nishizono H, Uosaki H, Sawada H, Sadahiro T, Ieda M & [Cl−] in neurons (Gabernet et al. 2005, Kopec et al. 2010). Takao K 2019 Rapid and high-efficient generation of mutant mice using Taken together, GLRA4 plays a crucial role in both male freeze-thawed embryos of the C57BL/6J strain. Journal of Neuroscience Methods 317 149–156. (https://doi.org/10.1016/j.jneumeth.2019.01.010) and female gametes by supporting fertilization and the Dawson KM, Collins JL & Baltz JM 1998 Osmolarity-dependent glycine early embryonic divisions. accumulation indicates a role for glycine as an organic osmolyte in early https://rep.bioscientifica.com Reproduction (2020) 159 41–48

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