Shin Hyejin, Seol Dong-Won, Nam Minyeong, Song Haengseok, Lee Dong Ryul, Lim Hyunjung Jade
Department of Biomedical Science and Technology, Institute of Biomedical Science and Technology, Konkuk University, Seoul 05029, Korea.
Department of Biomedical Science, CHA University, Seongnam 13884, Korea.
Asian-Australas J Anim Sci. 2017 Jun;30(6):781-787. doi: 10.5713/ajas.16.0798. Epub 2016 Dec 17.
The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation.
Expression of in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated (ΔEgr3) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining.
mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-ΔEgr3-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes.
The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.
早期生长反应（Egr）家族由四个成员（Egr1、Egr2、Egr3和Egr4）组成，它们是锌指转录因子。其中，Egr3在肌梭形成和神经突生长过程中参与靶基因的转录调控。我们之前发现，在小鼠减数分裂I期间，免疫反应性Egr3定位于卵母细胞纺锤体并在微管组织中心附近积累。Egr3也被证明定位于精母细胞。我们在此研究Egr3是否在小鼠性腺中表达，以及Egr3阻断是否会导致卵母细胞成熟出现任何缺陷。
通过逆转录 - 聚合酶链反应检测Egr3在小鼠性腺中的表达。将在N端带有Xpress标签、C端带有DsRed2的全长Egr3和截短的Egr3（ΔEgr3）互补RNA（cRNA），以及靶向Egr3的小干扰RNA（siRNA）显微注射到处于生发泡期的小鼠卵母细胞中。通过共聚焦实时成像和免疫荧光染色检查显微注射的Egr3的定位。
在1至4周龄小鼠的卵巢和睾丸中检测到Egr3 mRNA。在3周龄和4周龄的性腺中还检测到一种含有5'非翻译区的未鉴定的较长转录本。在减数分裂过程中，显微注射的Xpress - Egr3 - DsRed2或Xpress - ΔEgr3 - DsRed2定位于细胞核和染色体。在卵母细胞中显微注射这些cRNA或Egr3 siRNA不影响减数分裂成熟。对注射了Xpress - ΔEgr3 - DsRed2的卵母细胞进行Egr3免疫荧光染色，结果显示仅在减数分裂纺锤体上有阳性信号，这表明该抗体无法检测到小鼠卵母细胞中的内源性或外源性Egr3。
结果表明，Egr3在减数分裂过程中定位于染色体，并且某些抗体可能无法如实反映卵母细胞中靶蛋白的定位。Egr3在小鼠卵母细胞成熟过程中似乎是可有可无的。
