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Expression of Egr3 in mouse gonads and its localization and function in oocytes

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 (ΔEgr3) 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-ΔEgr3-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-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. 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.


INTRODUCTION
The early growth response (Egr) family of zinc finger transcription factors regulates critical genetic programs in cellular growth, differentiation, and function [1,2]. There are four Egr mem bers, Egr1, Egr2, Egr3, and Egr4, and each member has specific and overlapping functions in a given physiological setting. Some Egr factors have shown to be involved in reproductive function. Egr1 regulates expression of the luteinizing hormone beta subunit, and Egr1 deficient mice suffer from female infertility resulting from hormone insufficiency [3]. Egr1 is induced by estrogen in the mouse uterus and expressed in stromal cells surrounding implanting blastocysts, suggesting a role for Egr1 in uterine biology [4]. Egr3 is implicated in neurodevelopment, learning and memory, immune response, and fibrogenic response [58], and this list shows functional diversity of this factor. As for Egr3 deficient mice, neurodevelopmental phenotypes are well described [5].
Whether Egr3 deficiency leads to other tissuespecific functions is just beginning to unravel with the availability of the Egr3 floxed mice [9]. In reproduction, expression of Egr3 in mouse oocytes and spermatocytes was reported by us [2]. In bovine granulosa cells, the administration of mycotoxin was shown to induce Egr3 expression [10]. Expression of Egr3 and Egr4 in the pig ovary is also recently reported [11]. In male mice, Egr4 exhibits a dynamic pattern of localization in germ cells and gonadal somatic cells depending on the stage of sexual maturity [12].
Previously, we showed that the immunoreactive Egr3 colo calizes with meiotic spindle and accumulates near cytosolic microtubule organizing centers (MTOCs) in oocytes during meiotic maturation. As a transcription factor, Egr3 was expected to localize to nucleus in cells and tissues, but localization of the immunoreactive Egr3 was noted in the cytoplasmic structures in mouse oocytes, early preimplantation embryos, and spermato cytes [2]. Several transcriptional regulators including Egr1 have been reported to exhibit similar localization on microtubule associated structures [1315].
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. Thus, we herein investigated if functional blockade of Egr3 hinders meiotic maturation of oocytes by micro injecting dominantnegative 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 antiEgr3 antibodies may not faithfully represent endogenous and exoge nous Egr3 proteins in oocytes.

Mice
All mice were maintained in accordance with the policies of the Konkuk University Institutional Animal Care and Use Committee (IACUC). This study was approved by the Konkuk University IACUC (approval number KU15109). Mice were sacrificed after administration of avertin to minimize suffering. Fourweekold virgin ICR or BDF female mice were purchased from OrientBio (Seongnam, Korea).

Collection and culture of oocytes
Fourtofiveweekold virgin CrljOri:CD1 (ICR) female mice were intraperitoneally injected with 7.5 IU of pregnant mare's serum gonadotropin (SigmaAldrich, St. Louis, MO, USA) to induce folliculogenesis. Fully grown follicles were punctured 48 h later and cumulusoocyte complexes were collected. The cumulus cells were then removed by using 300 μg/mL hyaluron idase (SigmaAldrich, USA) and germinal vesicle (GV) stage oocytes were retrieved in Quinn' s Advantage Medium with HEPES (Sage BioPharma Inc., Bedminster, NJ, USA) containing 10% substitute protein serum and 200 μM 3isobutyl1methylxanthine (IBMX) (I5879; SigmaAldrich, USA). Cumulus cells were gently removed from the cumulusoocyte complexes using a Pasteur glass pipette. The oocytes were incubated in microdrops of M16 medium (M7292; SigmaAldrich, USA) containing 200 μM IBMX covered with mineral oil (Sage, USA) at 37°C under 5% CO 2 in atmosphere until microinjection was conducted. After microinjection, these oocytes were placed in drops of M16 medium with 200 μM IBMX for 8 h. For oocyte maturation, these oocytes were placed in IBMXfree M16 medium for 20 h. Rates of GV breakdown (GVBD) and polar body extrusion were monitored.

Reverse transcription-polymerase chain reaction
Reverse transcriptionpolymerase chain reaction (RTPCR) was conducted as described previously [2]. Gonads were individually collected from 1, 2, 3, and 4 weekold female and male ICR mice. Each sample was subjected to RNA preparation by using TRI Reagent (Life Technologies, Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. Isolated RNA (2 μg) was subjected to reverse transcription (RT) using MMLV reverse transcriptase (Beams Biotechnology, Seongnam, Korea). PCR was carried out using Prime Taq Premix (Genet Bio, Daejeon, Korea). Primer sequence are given in Table 1. PCR reaction was also performed for the mouse ribosomal protein L7 (Rpl7), a housekeeping gene. Annealing temperature is 59°C for all reac tions. This experiments were repeated 6 times with independent samples.

Plasmid constructs
Fulllength mouse Egr3 gene (NM_018781.3) was amplified by PCR from the mouse ovary cDNA sample [2]. To generate the dominant negative form of Egr3 [16], the truncated form of Egr3 (missing the first 249 amino acids, ΔEgr3) was generated by PCR from the fulllength mouse Egr3 cDNA. Fulllength and truncated Egr3 DNAs were inserted into BglII and AgeI site of pDsRed2 N1 vector (Clontech, Mountain View, CA, USA) and cloned into KpnI and XhoI site of pcDNA3.1/MycHis vector (Invitrogen, USA). For in vitro transcription, fulllength and truncated Egr3 construct containing Xpress tag at the Nterminus and DsRed2 at the Cterminus were amplified by PCR and cloned into BamHI and HindIII site of pRSETA vector (Invitrogen, USA). The con structs are shown in Figure 1A, 1B.

In vitro transcription and microinjection
For live imaging of Egr3 expression in mouse oocytes, fulllength and truncated Egr3 cRNAs were produced by in vitro transcrip tion using mMessage mMachine T7 transcription kit (Ambion, Austin, TX, USA). To visualize microtubule in some experiments, βtubulingreen fluorescent protein (GFP) cRNA was produced from pRN3βtubulinGFP construct (a gift from MarieHélène Verlhac, CIRB, Collège de France, Paris, France) using T3 trans cription kit. These RNAs were cleaned and concentrated by using RNeasy MinElute Clean up kit (Qiagen, Valencia, CA, USA).
These constructs were confirmed by in vitro translation by using TNT T7 Coupled Reticulocyte Lysate System kit (Promega, Madison, WI, USA). SMARTpool Egr3 siRNA (#M04639201 0005) and nontargeting siRNA (#D0018100105) were purchased from Dharmacon (Lafayette, CO, USA). Knockdown capacity of this siRNA was first confirmed in 293T cells transfected with fulllength Egr3 plasmid. Egr3 siRNA (25 μM) was microinjected into GV oocytes and rates of GVBD and polar body extrusion were monitored.
To visualize Egr3 localization by live imaging, Egr3DsRed2 or truncated Egr3DsRed2 cRNA (0.0034 μM) was microinjected into GV oocytes. Injection volume was 5 to 10 pL, which is about 1% to 3% of total oocyte volume. Mouse oocytes were placed in drops of culture media containing 200 μM IBMX for microin jection. Microinjection of cRNA or siRNA was performed using a Nikon (Nikon Corporation, ShinagawaKu, Tokyo, Japan) mani pulator with picoinjector (Femtojet, Eppendorf, Hamburg, Germany) via pneumatic pressure.

Expression of Egr3 in ovaries and testes in mice
To examine if Egr3 is expressed in mouse gonads at different stages of sexual maturation, we collected ovaries and testes from 1, 2, 3, and 4 weekold mice. The location of primers is depicted in Figure 1A. RTPCR analysis showed that Egr3 mRNA is ex pressed in ovaries and testes in all groups. We also found that an uncharacterized longer transcript of Egr3 (NM_001289925.1) was present in 3 and 4weekold gonads (Figure 2). Whether this form of Egr3 is functional or expressed in other tissues is not known.

Microinjection of full-length and truncated Xpress-Egr3-DsRed2 does not hinder oocyte maturation
We previously showed that the immunoreactive Egr3 is specifi cally localized near MTOCs and on spindle during meiotic maturation of oocytes [2]. We examined the subcellular local ization of Egr3 with live imaging after microinjecting fulllength Egr3 cRNA. This construct has an Xpress tag at Nterminus and DsRed2 at Cterminus ( Figure 1B). We also microinjected trun cated Egr3 RNA (XpressΔEgr3DsRed2) lacking the first 249 amino acids, which is known as a dominantnegative form of Egr3 [16] (Figure 1B). As shown in Figure 3 by confocal live imaging, products of both XpressEgr3DsRed2 and Xpress ΔEgr3DsRed2 cRNAs localize to nucleus and chromosomes during meiotic maturation. We then monitored if microinjection of these Egr3 RNAs affect meiotic maturation rate in oocytes. Microinjection of either construct did not influence the rate of meiotic maturation (Table 2).

Meiotic maturation is not hindered by Egr3 siRNA in oocytes
We next tested if microinjection of Egr3 siRNA has any influence on meiotic maturation. The specificity of Egr3 siRNA was first confirmed by western blotting in 293T cells transfected with Egr3Myc plasmid ( Figure 4A). However, microinjection of Egr3 siRNA to GV oocytes did not affect GVBD or polar body for mation (Table 3). Spindle forms normally in metaphase I (MI) oocytes injected with Egr3 siRNA, as evidenced by normal distri bution of tubulinGFP ( Figure 4B).

Egr3 localization: potential artefact in immunolocalization
The observation that the microinjected XpressΔEgr3DsRed2 constructs are only confined to nuclei and chromosomes con tradicts our previous observation that immunoreactive Egr3 localized to MTOCs and microtubules [2]. Thus, we sidebyside compared localization of Egr3 in microinjected oocytes by confocal live imaging, and antiXpress and antiEgr3 immuno fluorescence staining in fixed oocytes. As shown in Figure 5, DsRed2 signal is clearly confined to chromosomes. AntiXpress antibody detected a diffuse pattern of signal on or near metaphase chromosomes. This pattern is less clear than DsRed2 live imaging, but some signal overlaps with DNA. In contrast, antiEgr3 anti body shows positive signal only on meiotic spindle ( Figure 5). This antiEgr3 antibody does not detect exogenous XpressΔEgr3 DsRed2, because the signal is negative on chromosomes. This Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 cRNA (0.0034 μM) was microinjected into GV oocytes in the presence of 200 μM IBMX and oocytes were kept in M16 containing IBMX for 8 h. They were then washed and cultured in IBMX-free M16 medium for 20 h. Localization of microinjected constructs were monitored by confocal live imaging examining the expression of red fluorescent protein, DsRed2. During oocytes maturation, both Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 proteins are localized in nuclei and chromosomes. Expression of Xpress-Egr3-DsRed2 protein is lower than that of Xpress-ΔEgr3-DsRed2, as translation efficiency of Xpress-Egr3-DsRed2 is lower due to its length (about 750 bp longer than Xpress-ΔEgr3-DsRed2). White arrows indicate chromosomes. PI, prophase I; PMI, prometaphase I; MI, metaphase I; MII, metaphase II; Egr3, early growth response protein 3; GV, germinal vesicle; IBMX, 3-isobutyl-1-methylxanthine. GVBD, germinal vesicle breakdown; MII, metaphase II; IBMX, 3-isobutyl-1-methylxanthine. 1) Microinjection was performed in the presence of IBMX and oocytes were incubated in M16 medium containing 200 μM IBMX for 8 h. They were then cultured in IBMX-free M16 medium for 20 h. GVBD was monitored after 3 to 5 h of culture and polar body extrusion (MII) after 20 h from the beginning of IBMX-free culture. result suggests that spindlepositive signal detected by antiEgr3 antibody seems to be nonspecific.

DISCUSSION
In this study, we show that mouse gonads express Egr3 mRNA, both at immature and sexually maturing stages of age. Herein we present experiments to test functionality of Egr3 in mouse oocytes, providing evidence that Egr3 is dispensable for meiotic maturation in mouse oocytes.
In RTPCR analysis, a bigger, uncharacterized Egr3 isoform was noted in 3 and 4week ovaries. This variant, NM_001289925.1, has another exon inserted between two original exons of the mouse Egr3 gene ( Figure 1A). This form using a downstream start codon is expected to produce a shorter product with distinct Nterminus. Several isoforms of Egr3 have been reported in brain and they seem to have different transcriptional activity [17,18]. Interestingly, the appearance of this variant is associated with sexual maturation of the ovary. Whether this particular variant in mouse gonads is expressed in brain or other tissues and functionally active is currently unknown.
Egr3 without the Nterminal half of the protein is known as a dominantnegative form which blocks activity of Egr factors [16]. Commercially available siRNA specific to Egr3 efficiently downregulates translation of Egr3 ( Figure 4A). Microinjection of either dominantnegative Egr3 or Egr3 siRNA did not influence the rates of GVBD and PB extrusion, suggesting that Egr3 is functionally dispensable during oocyte maturation (Tables 2, 3). During the course of this study, we noticed that XpressEgr3 DsRed2 and XpressΔEgr3DsRed2 proteins are confined only on nuclei and chromosomes after microinjection when observed by live imaging (Figure 3). This is in discrepancy with our previous observation that antiEgr3 antibody produced signal near MTOC and on spindle of maturing oocytes. In our previous  study, we had tested five commercially available antiEgr3 anti bodies and two of them had produced the observed pattern of localization in oocytes [2] (Figure 5). Both antibodies, detecting Cterminal region of Egr3 protein, detect Egr3 in western blotting and recognize exogenous Egr3 in nuclei of PC12 or MCF7 cells. Interestingly, one of these antibodies also detect immunoreactive Egr3 in growing neurites of PC12 cells (data not shown). Thus, certain antiEgr3 antibody recognizes microtubulebased sub cellular structures both in PC12 and mouse oocytes; neurite and meiotic spindle. The similarity between these structure is evident, i.e., they both utilize microtubule. However, our sidebyside comparison in Figure 5 shows that antiEgr3 antibody produces a distinct pattern of subcellular localization compared to live imaging and antiXpress immunofluorescence staining. Anti Xpress antibody detecting Xpress tag on the fusion construct ( Figure 1B) produced a signal much closer to Egr3DsRed2 live imaging ( Figure 5), although the pattern was more diffused than that of DsRed2 live imaging. While we vigorously validated the specificity of antibodies in our previous investigation [2], it is possible that this antiEgr3 antibody might produce artefactual pattern of localization in oocytes. Antibodies are often the most common culprits of problems in biological experiments due to nonspecific binding to other proteins [19]. Whether this parti cular antiEgr3 antibody detects other target(s) or processed form of Egr3 protein is not clear and it requires further validation.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.