Objective: 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 t...
Objective: 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 Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 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-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-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. Conclusion: 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.
Objective: 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 Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 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-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-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. Conclusion: 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.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
In this study, we elaborate further on the expression of Egr3 in gonads and its role in oocyte maturation. Whereas several pieces of information indicate involvement of Egr factors in female reproduction, it is not clear if Egr3 is required for oocyte maturation in mice.
가설 설정
Fifty micrograms of cell lysates were run on 10% SDS-PAGE gels. (B) Microinjection of Egr3 siRNA in GV oocytes does not interfere with meiotic maturation. Confocal live image of meiotic spindle after co-injection of 0.
제안 방법
Figure 1. Diagrams depicting the structure of Egr3 gene and constructs used in this study. (A) The mouse Egr3 gene and the variant detected by RT-PCR.
대상 데이터
Anti-α-tubulin (Sigma-Aldrich, USA) and anti-Egr3 (sc-191; Santa Cruz Biotechnology, USA) antibodies were used at 1:2,000 and 1:500, respectively. Anti-Myc antibody (#631206; Clontech, USA) was used at 1:2,000. Horseradish peroxidase (HRP) -conjugated goat anti-rabbit and goat anti-mouse antibodies (GeneDEPOT, Barker, TX, USA) were used at 1:10,000.
Mice were sacrificed after administration of avertin to minimize suffering. Four-week-old virgin ICR or BDF female mice were purchased from Orient-Bio (Seongnam, Korea).
These constructs were confirmed by in vitro translation by using TNT T7 Coupled Reticulocyte Lysate System kit (Promega, Madison, WI, USA). SMARTpool Egr3 siRNA (#M-046392-01-0005) and non-targeting siRNA (#D-001810-01-05) were purchased from Dharmacon (Lafayette, CO, USA). Knockdown capacity of this siRNA was first confirmed in 293T cells transfected with full-length Egr3 plasmid.
성능/효과
Thus, we herein investigated if functional blockade of Egr3 hinders meiotic maturation of oocytes by microinjecting dominant-negative Egr3 complementary RNA (cRNA) and small interfering RNA (siRNA) specific to Egr3. The results show that Egr3 is dispensable for meiotic maturation in mouse oocytes and that some of the commercially available anti-Egr3 antibodies may not faithfully represent endogenous and exogenous Egr3 proteins in oocytes.
참고문헌 (19)
1 O’Donovan KJ Tourtellotte WG Millbrandt J Baraban JM The EGR family of transcription-regulatory factors: progress at the interface of molecular and systems neuroscience Trends Neurosci 1999 22 167 73 10203854
2 Shin H Kwon S Song H Lim HJ The transcription factor Egr3 is a putative component of the microtubule organizing center in mouse oocytes PLoS One 2014 9 e94708 24722338
3 Lee SL Sadovsky Y Swirnoff AH Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1) Science 1996 273 1219 21 8703054
4 Kim HR Kim YS Yoon JA Egr1 is rapidly and transiently induced by estrogen and bisphenol A via activation of nuclear estrogen receptor-dependent ERK1/2 pathway in the uterus Reprod Toxicol 2014 50 60 7 25461906
5 Tourtellotte WG Milbrandt J Sensory ataxia and muscle spindle agenesis in mice lacking the transcription factor Egr3 Nat Genet 1998 20 87 91 9731539
6 Li L Yun SH Keblesh J Egr3, a synaptic activity regulated transcription factor that is essential for learning and memory Mol Cell Neurosci 2007 35 76 88 17350282
7 Li S Miao T Sebastian M The transcription factors Egr2 and Egr3 are essential for the control of inflammation and antigen-induced proliferation of B and T cells Immunity 2012 37 685 96 23021953
8 Fang F Shangguan AJ Kelly K Early growth response 3 (Egr-3) is induced by transforming growth factor-β and regulates fibrogenic responses Am J Pathol 2013 183 1197 208 23906810
9 Quach DH Oliveira-Fernandes M Gruner KA Tourtellotte WG A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation J Neurosci 2013 33 4570 83 23467373
10 Guerrero-Netro HM Chorfi Y Price CA Effects of the mycotoxin deoxynivalenol on steroidogenesis and apoptosis in granulosa cells Reproduction 2015 149 555 61 25731188
11 Wang J Zhao Y Gu K The novel porcine gene early growth response 4 (Egr4) is differentially expressed in the ovaries of Erhualian and Pietrain pigs Reprod Fertil Dev 2014 26 587 98 23719176
12 Hogarth CA Mitchell D Small C Griswold M EGR4 displays both a cell- and intracellular-specific localization pattern in the developing murine testis Dev Dyn 2010 239 3106 14 20925118
13 Mora GR Olivier KR Cheville JC The cytoskeleton differentially localizes the early growth response gene-1 protein in cancer and benign cells of the prostate Mol Cancer Res 2004 2 115 28 14985468
14 Soubry A Staes K Parthoens E The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material PLoS One 2010 5 e9203 20169156
15 Wei H Wang Q Du J Unique subcellular distribution of RPB1 with a phosphorylated C-terminal domain (CTD) in mouse oocytes during meiotic division and its relationship with chromosome separation J Reprod Dev 2015 61 541 8 26346254
16 Levkovitz Y O’Donovan KJ Baraban JM Blockade of NGF-induced neurite outgrowth by a dominant-negative inhibitor of the egr family of transcription regulatory factors J Neurosci 2001 21 45 52 11150318
17 O’Donovan KJ Baraban JM Major Egr3 isoforms are generated via alternate translation start sites and differ in their abilities to activate transcription Mol Cell Biol 1999 19 4711 8 10373520
18 O’Donovan KJ Levkovitz Y Ahn D Baraban JM Functional comparison of Egr3 transcription factor isoforms: identification of an activation domain in the N-terminal segment absent from Egr3beta, a major isoform expressed in brain J Neurochem 2000 75 1352 7 10987814
19 Baker M Reproducibility crisis: Blame it on the antibodies Nature 2015 521 274 6 25993940
※ AI-Helper는 부적절한 답변을 할 수 있습니다.