Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro

Yan Zhu A , Ya-Hong Jiang A , Ya-Ping He A , Xuan Zhang A , Zhao-Gui Sun A , Man-Xi Jiang B C D and Jian Wang A D
+ Author Affiliations
- Author Affiliations

A Key Laboratory of Contraceptive Drugs and Devices of National Population and Family Planning Committee, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China.

B Department of Laboratory Animal Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

C Present address: Guangdong No. 2 Provincial People’s Hospital, Guangzhou 510317, China.

D Corresponding authors. Email: manxijiang@yahoo.com; wangjiansippr@126.com

Reproduction, Fertility and Development 27(3) 557-566 https://doi.org/10.1071/RD13269
Submitted: 6 December 2013  Accepted: 16 January 2014   Published: 14 February 2014

Abstract

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription–polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

Additional keywords: ovary, short interfering RNA.


References

Dadi, T. D., Li, M. W., and Lloyd, K. C. (2009). Decreased growth factor expression through RNA interference inhibits development of mouse preimplantation embryos. Comp. Med. 59, 331–338.
| 1:CAS:528:DC%2BD1MXhtlSitbnF&md5=25c8a1dae19cffd1fcffeb33a7ff6651CAS |

Falco, G., Lee, S. L., Stanghellini, I., Bassey, U. C., Hamatani, T., and Ko, M. S. (2007). Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells. Dev. Biol. 307, 539–550.
Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXnsFWlt7c%3D&md5=95130ae0fff899fbc875ca27fcc9bc93CAS |

Feuerstein, P., Puard, V., Chevalier, C., Teusan, R., Cadoret, V., Guerif, F., Houlgatte, R., and Royere, D. (2012). Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors. PLoS One 7, e40449.
Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhtFCgu7rI&md5=0ffd53bf1d53becbf8fa203a09c28a4aCAS |

Grabarek, J. B., Plusa, B., Glover, D. M., and Zernicka-Goetz, M. (2002). Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation. Genesis 32, 269–276.
Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xkt1Sjsrs%3D&md5=25873f0629bde904022a1b13c4c5eb59CAS |

Hamatani, T., Carter, M. G., Sharov, A. A., and Ko, M. S. (2004). Dynamics of global gene expression changes during mouse preimplantation development. Dev. Cell 6, 117–131.
Dynamics of global gene expression changes during mouse preimplantation development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXntlaktA%3D%3D&md5=04cb1bae336ebd15252824098bed696bCAS |

Hamel, M., Dufort, I., Robert, C., Leveille, M. C., Leader, A., and Sirard, M. A. (2010). Genomic assessment of follicular marker genes as pregnancy predictors for human IVF. Mol. Hum. Reprod. 16, 87–96.
Genomic assessment of follicular marker genes as pregnancy predictors for human IVF.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXktlCisA%3D%3D&md5=b0e7af4cd670b768af21aa57369e896cCAS |

Han, J., Mark, M. D., Li, X., Xie, M., Waka, S., Rettig, J., and Herlitze, S. (2006). RGS2 determines short-term synaptic plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca2+ channels. Neuron 51, 575–586.
RGS2 determines short-term synaptic plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca2+ channels.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtVWntL3E&md5=b51e910085ddb172e1fe448b60cfefb5CAS |

Heximer, S. P., Watson, N., Linder, M. E., Blumer, K. J., and Hepler, J. R. (1997). RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proc. Natl Acad. Sci. USA 94, 14 389–14 393.
RGS2/G0S8 is a selective inhibitor of Gqalpha function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXislSnsw%3D%3D&md5=dc0b5dcd6611140600a8090d4e547e72CAS |

Heximer, S. P., Srinivasa, S. P., Bernstein, L. S., Bernard, J. L., Linder, M. E., Hepler, J. R., and Blumer, K. J. (1999). G protein selectivity is a determinant of RGS2 function. J. Biol. Chem. 274, 34 253–34 259.
G protein selectivity is a determinant of RGS2 function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXnslOksbs%3D&md5=568a48ab4abde237fa79ad1e9968d6c9CAS |

Huang, Z. P., Ni, H., Yang, Z. M., Wang, J., Tso, J. K., and Shen, Q. X. (2003). Expression of regulator of G-protein signalling protein 2 (RGS2) in the mouse uterus at implantation sites. Reproduction 126, 309–316.
Expression of regulator of G-protein signalling protein 2 (RGS2) in the mouse uterus at implantation sites.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXnslKksbk%3D&md5=d4dec8998131ef6cbe4cfc43675ef10aCAS |

Kehrl, J. H., and Sinnarajah, S. (2002). RGS2: a multifunctional regulator of G-protein signaling. Int. J. Biochem. Cell Biol. 34, 432–438.
RGS2: a multifunctional regulator of G-protein signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XitFWit7s%3D&md5=de8a5242faeb30252858da34a7cfad02CAS |

Kigami, D., Minami, N., Takayama, H., and Imai, H. (2003). MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos. Biol. Reprod. 68, 651–654.
MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXntVCgsw%3D%3D&md5=5cd8e53be930a942e162b2000663da6aCAS |

Kimple, A. J., Soundararajan, M., Hutsell, S. Q., Roos, A. K., Urban, D. J., Setola, V., Temple, B. R., Roth, B. L., Knapp, S., Willard, F. S., and Siderovski, D. P. (2009). Structural determinants of G-protein alpha subunit selectivity by regulator of G-protein signaling 2 (RGS2). J. Biol. Chem. 284, 19 402–19 411.
Structural determinants of G-protein alpha subunit selectivity by regulator of G-protein signaling 2 (RGS2).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXot12kurw%3D&md5=8379416a2552d862840a6c4da3d4c713CAS |

Ladds, G., Zervou, S., Vatish, M., Thornton, S., and Davey, J. (2009). Regulators of G protein signalling proteins in the human myometrium. Eur. J. Pharmacol. 610, 23–28.
Regulators of G protein signalling proteins in the human myometrium.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXltV2is7w%3D&md5=5324dfdf6542de1ceaa6bda53a2dcb87CAS |

Marcondes, F. K., Bianchi, F. J., and Tanno, A. P. (2002). Determination of the estrous cycle phases of rats: some helpful considerations. Braz. J. Biol. 62, 609–614.
Determination of the estrous cycle phases of rats: some helpful considerations.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3s7ksValsA%3D%3D&md5=2079ebd488edc11b242aafb04c994fd7CAS |

Melamed, P., Savulescu, D., Lim, S., Wijeweera, A., Luo, Z., Luo, M., and Pnueli, L. (2012). Gonadotrophin-releasing hormone signalling downstream of calmodulin. J. Neuroendocrinol. 24, 1463–1475.
Gonadotrophin-releasing hormone signalling downstream of calmodulin.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xhs12ls73L&md5=5866dd33c816215f3da152d73d945fc8CAS |

Melliti, K., Meza, U., and Adams, B. A. (2001). RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line. J. Physiol. 532, 337–347.
RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjtFOnt74%3D&md5=73a924fd42282d7551342eca5e1a7834CAS |

Minami, N., Suzuki, T., and Tsukamoto, S. (2007). Zygotic gene activation and maternal factors in mammals. J. Reprod. Dev. 53, 707–715.
Zygotic gene activation and maternal factors in mammals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFGgs7fI&md5=f773fc66a2230f5725302728d33cb031CAS |

Naor, Z., Harris, D., and Shacham, S. (1998). Mechanism of GnRH receptor signaling: combinatorial cross-talk of Ca2+ and protein kinase C. Front. Neuroendocrinol. 19, 1–19.
Mechanism of GnRH receptor signaling: combinatorial cross-talk of Ca2+ and protein kinase C.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXpsleguw%3D%3D&md5=5373b462d7cec965742092e31d17ab36CAS |

Nguyen, C. H., Ming, H., Zhao, P., Hugendubler, L., Gros, R., Kimball, S. R., and Chidiac, P. (2009). Translational control by RGS2. J. Cell Biol. 186, 755–765.
Translational control by RGS2.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFehsLbF&md5=1110e376298892b9cc095ed70ac931ecCAS |

O’Brien, M., Morrison, J. J., and Smith, T. J. (2008). Upregulation of PSCDBP, TLR2, TWIST1, FLJ35382, EDNRB, and RGS12 gene expression in human myometrium at labor. Reprod. Sci. 15, 382–393.
Upregulation of PSCDBP, TLR2, TWIST1, FLJ35382, EDNRB, and RGS12 gene expression in human myometrium at labor.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXnsFCgu7Y%3D&md5=89d8e8508e1cbd2f5a8f1042da35ca79CAS |

Oliveira-dos-Santos, A. J., Matsumoto, G., Snow, B. E., Bai, D., Houston, F. P., Whishaw, I. Q., Mariathasan, S., Sasaki, T., Wakeham, A., Ohashi, P. S., Roder, J. C., Barnes, C. A., Siderovski, D. P., and Penninger, J. M. (2000). Regulation of T cell activation, anxiety, and male aggression by RGS2. Proc. Natl Acad. Sci. USA 97, 12 272–12 277.
Regulation of T cell activation, anxiety, and male aggression by RGS2.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXnvVSgu7o%3D&md5=bb5cae0995b0306ce0620ee8836c6076CAS |

Paria, B. C., Huet-Hudson, Y. M., and Dey, S. K. (1993). Blastocyst’s state of activity determines the ‘window’ of implantation in the receptive mouse uterus. Proc. Natl Acad. Sci. USA 90, 10 159–10 162.
Blastocyst’s state of activity determines the ‘window’ of implantation in the receptive mouse uterus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXot1Y%3D&md5=4522377748a3e6a89266e446041fed1bCAS |

Park, E. S., Echetebu, C. O., Soloff, S., and Soloff, M. S. (2002). Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells. Am. J. Physiol. Endocrinol. Metab. 282, E580–E584.
| 1:CAS:528:DC%2BD38Xit1Giu7o%3D&md5=6dfba60be511e4703cf3ec7475fbc24bCAS |

Psychoyos, A. (1973). Hormonal control of ovoimplantation. Vitam. Horm. 31, 201–256.
| 1:CAS:528:DyaE2cXksVOktr0%3D&md5=e65d14165d162863709209f5d71e01acCAS |

Segers, I., Adriaenssens, T., and Smitz, J. (2012). Expression patterns of poliovirus receptor, erythrocyte protein band 4.1-like 3, regulator of g-protein signaling 11, and oxytocin receptor in mouse ovarian cells during follicle growth and early luteinization in vitro and in vivo. Biol. Reprod. 86, 1–11.
Expression patterns of poliovirus receptor, erythrocyte protein band 4.1-like 3, regulator of g-protein signaling 11, and oxytocin receptor in mouse ovarian cells during follicle growth and early luteinization in vitro and in vivo.Crossref | GoogleScholarGoogle Scholar |

Sinnarajah, S., Dessauer, C. W., Srikumar, D., Chen, J., Yuen, J., Yilma, S., Dennis, J. C., Morrison, E. E., Vodyanoy, V., and Kehrl, J. H. (2001). RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III. Nature 409, 1051–1055.
RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXhs1GjsrY%3D&md5=ac6c3c58f9a09caa5b5272ca2aa51b91CAS |

Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998). Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 95, 5584–5589.
Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXjtFWktbw%3D&md5=542eb2f6a6ad3ced836a1a702e1fcd9fCAS |

Stein, P., Svoboda, P., Anger, M., and Schultz, R. M. (2003). RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 9, 187–192.
RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXhtVWnu7g%3D&md5=755ea1452fa502c14918a2110c40888cCAS |

Suarez, V. R., Park, E. S., Hankins, G. D., and Soloff, M. S. (2003). Expression of regulator of G protein signaling-2 in rat myometrium during pregnancy and parturition. Am. J. Obstet. Gynecol. 188, 973–977.
Expression of regulator of G protein signaling-2 in rat myometrium during pregnancy and parturition.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXksVWhtLY%3D&md5=7220eab68f3cc59db27d045005c1844eCAS |

Svoboda, P., Stein, P., Hayashi, H., and Schultz, R. M. (2000). Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127, 4147–4156.
| 1:CAS:528:DC%2BD3cXotVWisLY%3D&md5=6384137e6d3294fdf1df4ab9f8800da5CAS |

Ujioka, T., Russell, D. L., Okamura, H., Richards, J. S., and Espey, L. L. (2000). Expression of regulator of G-protein signaling protein-2 gene in the rat ovary at the time of ovulation. Biol. Reprod. 63, 1513–1517.
Expression of regulator of G-protein signaling protein-2 gene in the rat ovary at the time of ovulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXnslCrsb8%3D&md5=b412de09d81b8796a40c2745542a8f65CAS |

Wu, Y. L., Chuang, H. H., Kou, Y. R., Lee, T. S., Lu, S. H., Huang, Y. C., Nishi, Y., and Yanase, T. (2008). Regulation of LH receptor and PGF2alpha receptor signaling by the regulator of G protein signaling 2 (RGS2) in human and mouse granulosa cells. Chin. J. Physiol. 51, 282–291.
| 1:CAS:528:DC%2BD1cXhtl2gsbnE&md5=b3b70f0c9423a63a2c5326d7fc5e3140CAS |

Wurmbach, E., Yuen, T., Ebersole, B. J., and Sealfon, S. C. (2001). Gonadotropin-releasing hormone receptor-coupled gene network organization. J. Biol. Chem. 276, 47 195–47 201.
Gonadotropin-releasing hormone receptor-coupled gene network organization.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXpt1Gntbc%3D&md5=64889f74412a871479e089562e50b75bCAS |

Zeng, F., Baldwin, D. A., and Schultz, R. M. (2004). Transcript profiling during preimplantation mouse development. Dev. Biol. 272, 483–496.
Transcript profiling during preimplantation mouse development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmt1CjtrY%3D&md5=92d00cbac94ec3fbd76bfbd9754d66e7CAS |

Zhang, W., Walker, E., Tamplin, O. J., Rossant, J., Stanford, W. L., and Hughes, T. R. (2006). Zfp206 regulates ES cell gene expression and differentiation. Nucleic Acids Res. 34, 4780–4790.
Zfp206 regulates ES cell gene expression and differentiation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtFeisb7P&md5=b641406b0c12a3d9193581fb0c239478CAS |

Zheng, B., De Vries, L., and Gist Farquhar, M. (1999). Divergence of RGS proteins: evidence for the existence of six mammalian RGS subfamilies. Trends Biochem. Sci. 24, 411–414.
Divergence of RGS proteins: evidence for the existence of six mammalian RGS subfamilies.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXkvVKgtQ%3D%3D&md5=b74cdf0549439bc1937c8b095b0b0f28CAS |

Zhu, Z. Y., Jiang, M. X., Yan, L. Y., Huang, J. C., Lei, Z. L., Jiang, Y., Ouyang, Y. C., Zhang, H. X., Sun, Q. Y., and Chen, D. Y. (2007). Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle. Mol. Reprod. Dev. 74, 655–663.
Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXjs1Kgu7g%3D&md5=f37ad0a9f5278a0aaa89deb0b6dc9801CAS |