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Regular Article |
a Department of Animal Sciences, University of Missouri, 162 Animal Science Research Center, Columbia, Missouri 65211
b Department of Biochemistry, University of Missouri, Medical Sciences Building, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous cellular proteins have been identified that possess such an NES. Cyclin B1 [4] and CDC25 [5], which both play important roles in cell cycle progression, are restricted from the nucleus until the G2 phase of the cell cycle by active export via CRM1. The regulator of transcription, I
B
, which complexes with the transcription factor NF-B and effectively removes NF-B from the nucleus, does so by interaction of its NES with CRM1 [6]. In addition to numerous cellular proteins that possess NESs, some viral proteins also possess NESs that can be recognized by the host cell's endogenous export system. One of the first NESs to be identified was in the Rev protein of human immunodeficiency virus-1 (HIV-1). This protein is required for export of unspliced viral RNA from the nucleus of the host cell, a process that is critical for propagation of the virus. It was found that the NES in Rev interacts with the endogenous CRM1 to achieve export of the protein [3].
Leptomycin B (LMB), an antifungal compound that specifically inhibits CRM1-mediated nuclear export, has been a valuable tool in characterizing the CRM1 export pathway [7]. LMB has been shown to inhibit export of HIV-1 Rev protein [8]. Other groups later demonstrated that LMB could inhibit export of cellular proteins as well [3]. It is now well established that LMB acts as a potent, selective inhibitor of CRM1 function, presumably through covalent modification of a conserved cysteine residue found in CRM1 [9]. Not only does LMB effectively inhibit CRM1-mediated nuclear export, but it has also been shown to arrest cycling cells in both G1 and G2 phases of the cell cycle, demonstrating that CRM1-mediated nuclear export is an essential cellular process [10].
The developmental regulation of CRM1 function was determined in preimplantation porcine embryos in the present experiments. Determining which nuclear trafficking systems are present during early embryogenesis and how they are regulated may provide clues as to what cargoes have access to which cellular compartments at key times during development. Because CRM1 is a highly conserved protein [11] and is tied closely to cell cycle and transcription regulation, we hypothesized that CRM1 would have a critical role in development of the mammalian embryo. Findings from these experiments show that although CRM1 is both present and functional throughout preimplantation development, CRM1 function is not required for development during early cleavage divisions, but becomes necessary as the embryonic genome is activated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals were from Sigma Chemical Company (St. Louis, MO) unless stated otherwise. Porcine ovaries were collected at a local slaughterhouse and transported to the laboratory in 0.9% saline containing 75 µg/ml penicillin and 50 µg/ml streptomycin at 25°C. Antral follicles between 3 and 6 mm in diameter were aspirated manually with a disposable 10-cc syringe and an 18-gauge needle. Follicular fluid was pooled and allowed to settle by gravity. Cumulus-oocyte complexes (COCs) were resuspended in Hepes-buffered medium containing 0.01% polyvinyl alcohol (PVA) [12]. This suspension was examined under a dissecting microscope; COCs with multiple layers of intact cumulus cells were selected for the experiments. For germinal vesicle (GV)-stage oocyte microinjections, COCs were vortexed in 0.1% hyaluronidase in Hepes-buffered medium for 10 min to remove the cumulus cells. Denuded GV-stage oocytes were then rinsed in Hepes-buffered medium containing 3 mg/ml BSA (Fraction V).
In Vitro Maturation, Fertilization, and Embryo Culture
Fifty to 75 COCs were placed in 500 µl of tissue culture medium 199 (TCM-199; Gibco BRL, Grand Island, NY) containing 0.14% PVA, 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 0.5 µg/ml porcine FSH, and 0.5 µg/ml porcine LH [12]. This medium was pre-equilibrated at 39°C and 5% CO2 overnight. COCs were matured for 44 h at 39°C and 5% CO2. COCs were then vortexed in 0.1% hyaluronidase in Hepes-buffered medium containing 0.01% PVA for 3 min to remove the cumulus cells after maturation. Following cumulus removal, denuded oocytes were washed and held in Hepes-buffered medium supplemented with 0.01% PVA. Oocytes were then transferred to a modified Tris-buffered medium (mTBM) and fertilized according to established protocols using frozen-thawed boar semen [13]. Gametes were coincubated for 5 h, then washed and placed in North Carolina State University 23 (NCSU23) embryo culture medium [14]. A total of 50–75 presumptive zygotes were placed in 500 µl of NCSU23 embryo culture medium supplemented with 4 mg/ml BSA under mineral oil in 100% humidity at 39°C with 5% CO2. Embryos were cultured in one of two ways depending on the number of embryos allocated to specific treatments; groups of 3–10 embryos were placed in 50-µl droplets of culture medium, or in groups of 50–75 in 500 µl of culture medium.
Leptomycin B Treatment
Leptomycin B was added to media at a final concentration of 7 nM. This concentration was previously shown to effectively inhibit CRM1-mediated nuclear export of IB/NF-B complexes [15]. A 500x stock solution of LMB in ethanol was used, such that 1 µl of stock solution was added to 500 µl of medium. For control culture groups, an equivalent amount of ethanol was added to the medium.
For oocyte maturation experiments using LMB, oocytes were fixed in 3:1 ethanol:acetic acid for 48 h and subsequently stained with 1% orcein stain to view chromatin. Fertilized oocytes were cultured for 12 h following fertilization, subsequently fixed in 3:1 ethanol:acetic acid for 48 h, and stained with 1% orcein. Slides were examined with a compound microscope using bright field optics.
For all embryo culture experiments using LMB, embryos were kept in NCSU23 embryo culture medium without LMB until they were allocated to treatments. For the first set of embryo culture experiments, from individual cohorts of fertilized eggs, embryos were allocated to treatments 12 h after insemination (the presumptive pronuclear stage), at the 2-cell stage (24–36 h after insemination), and at the 4-cell stage (36–52 h after insemination). In the second set of embryo culture experiments, embryos from individual cohorts of fertilized eggs were allocated to treatment groups at the 8-cell stage (96 h after insemination). All groups of embryos in both sets of culture experiments were cultured until Day 6 (158 h after insemination), when they were stained with Hoechst 33342 (2 µg/ml final concentration) in culture medium, mounted on slides, and viewed on a compound microscope equipped with fluorescence to determine nuclear number.
In the microinjection experiments (see below), LMB and ethanol were added to Hepes-buffered medium containing 3 mg/ml BSA to the same concentrations used in the embryo culture experiments described above. For nuclear microinjection of GV-stage oocytes, oocytes were incubated in the appropriate medium for their respective treatment group for 1 h before microinjection. Immediately before microinjection, GV-stage oocytes were centrifuged at 10 000 x g for 10 min to clear the cytoplasm of lipid, to allow visualization of the peripherally located nucleus with a Nikon inverted diaphot (Melville, NY) using bright field optics. Following microinjection, GV-stage oocytes were returned to Hepes-buffered medium containing 3 mg/ml BSA and either LMB or ethanol for 1 h. After the postinjection incubation, oocytes were stained with Hoechst 33342 as described above and examined with a fluorescent microscope to determine the localization of the nucleus and injected proteins.
Technical barriers prevented us from employing the same microinjection assay in embryos as used in GV-stage oocytes to detect CRM1 function. Pronuclei are typically located in the center of the oocyte, with some variation depending on the precise age of the embryo, whereas GV-stage nuclei are considerably larger and located near the periphery of the oocyte. These two factors, coupled with the opaque cytoplasm of porcine embryos, led us to modify the microinjection assay. Instead of nuclear microinjection, wild type and mutant NES reporter proteins were injected into the cytoplasm of metaphase II oocytes. Both groups of injected oocytes were then allocated to LMB and control treatment groups, fertilized, and fixed after the nuclear envelope formed (see Materials and Methods). Because we had previously demonstrated that our reporter proteins behaved as NES-bearing cargoes should, we hypothesized that if a nuclear envelope formed in space around cytoplasm that possessed an even distribution of our reporter protein, such protein would be actively exported if CRM1 was functional.
For injection of metaphase II oocytes, matured COCs were stripped of their cumulus cells, as described above for the in vitro fertilization procedure. After washing in Hepes-buffered medium containing 3 mg/ml BSA, oocytes were injected with the appropriate proteins (see the Microinjection Assay section below). Following microinjection, oocytes were allocated to mTBM for fertilization. This medium contained either LMB or ethanol in appropriate concentrations. Following gamete coincubation, presumptive zygotes were placed in embryo culture medium containing either LMB or ethanol. Twelve hours after insemination, embryos were centrifuged at 12 000 x g for 10 min to clear the cytoplasm of lipid granules to enable visualization of pronuclei with bright field microscopy. Those embryos possessing pronuclei were fixed at that time or returned to embryo culture medium until cleavage to the 2-cell stage and subsequently fixed. Fixation was performed by incubation in 3.7% paraformaldehyde at 4°C for 2 h. Embryos were mounted on slides in 20 µl of Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL) and directly viewed on a scanning laser confocal microscope.
Protein Production and Labeling
NES reporter proteins were produced as glutathione S-transferase (GST)-fusion proteins using the pGEX-3X vector (Promega, Madison, WI). The NES found in HIV-1 Rev protein, and a mutated version of this NES previously shown to not function as a target for nuclear export by CRM1 [16], were used in our assays. Oligonucleotides flanked with the appropriate restriction sites and that coded for either the wild-type or mutant NES were generated (for HIV-1 Rev NES, 5'-GATCCCCTTACCTCCTTTAGAAAGATTAACTTTAGATTAGTAG-3' and 5'-AA-TT-CTACTAATCTAAAGTTAATCTTTCTAAAGGAGGTAAGGG-3'; for mutant NES, 5'-GATCCCCTTACCTCCTGATTTACGTTTAACTT-TAGA-TTAGTAG-3' and 5'-AATTCTACTAATCTAAAGTTAAACGT-AAATCA-GGAGGTAAGGG-3'). Complimentary oligos were hybridized together and ligated into the pGEX-3X vector, which was previously cut with EcoRI and BamHI. After transformation into DH10b cells, individual clones were sequenced, and those with the proper inserts were used to generate proteins for the microinjection assay.
Vectors were transformed into BL21 cells. One-liter cultures were grown to an optical density reading of 0.4 at 595 nm and induced with isopropylthio-ß-D-galactoside at a final concentration of 0.2 mM for 4 h at 37°C. Cells were pelleted, resuspended in PBS at 4°C, and lysed with a French Press (SLM Instruments, Urbana, IL). GST proteins were purified with a glutathione-agarose column according to the manufacturer's instructions (Sigma). After four washes with cold PBS, but before elution from the column, GST proteins were labeled with fluorescein-5-maleimide (Pierce, Rockford, IL) according to the manufacturer's protocol. Following labeling, the column was washed four additional times with cold PBS. Labeled GST proteins were eluted from the column with a cold solution of 10 mM reduced glutathione in 50 mM Tris-HCl pH 8.0. Protein solutions were diluted to 1 mg/ml with 50 mM Tris-HCl pH 8.0 and glycerol was added to a final concentration of 10%. Protein solutions were divided into 10-µl aliquots and frozen at -80°C. Protein concentrations were determined by the Bradford reaction.
Microinjection Assay
Injection pipettes 1 µm in diameter were attached to a Narishige IM 300 microinjector (Tokyo, Japan). Frozen aliquots of fusion proteins were thawed and centrifuged at 10 000 x g for 3 min. The protein solutions were front-loaded into microinjection pipettes. Oocytes were placed in 50-µl droplets of Hepes-buffered medium containing 0.3% BSA covered with mineral oil. Injection pipettes were driven through the zona pellucida and plasma membrane into the oocyte. Before all microinjection experiments, oocytes were first incubated in Hepes-buffered medium containing LMB or ethanol (as described in the Leptomycin B Treatment section above). For nuclear microinjections, the reporter protein solutions were mixed 1:1 with rhodamine labeled rabbit immunoglobulin G (IgG). Rabbit IgG (Sigma) was labeled with NHS-rhodamine (Pierce) according to manufacturer's protocols. Labeled protein was diluted to 1 mg/ml and frozen in 10-µl aliquots at -80°C. Rhodamine-labeled IgG does not diffuse from the nucleus, and thereby serves as a marker for the site of injection. Only those oocytes that did not lyse and had confirmed nuclear injection (as determined by location of rhodamine-labeled IgG) were included in analyses. For cytoplasmic injections, enough air pressure was applied such that some expansion of the cytoplasm was observed. Only cells that did not lyse after injection were included in the analysis.
Immunocytochemisty
Porcine oocytes and embryos (both those surgically recovered from bred gilts and those produced in vitro) were fixed for 2 h in 3.7% paraformaldehyde at 4°C. Embryos were washed and placed in PBS containing 0.1% Tween-20 for 30 min; this rinse was repeated three times, after which embryos were placed in 500 µl of PBS containing 0.1% PVA and stored at 4°C until further processing, not more than 2 wk.
Unless otherwise stated, the following incubations were carried out at 4°C. Fixed embryos were placed in PBS containing 1% Triton X-100 for 1 h. Embryos were then placed in blocking solution overnight. Blocking solution consisted of 0.1 M glycine, 1% goat serum, 0.01% Triton X-100, 1% powdered nonfat dry milk, 0.5% BSA, and 0.02% sodium azide in PBS [17]. Primary rabbit antibody directed against human CRM1 protein (a gift from Dr. Minoru Yoshida) [9, 11] was diluted 1:500 in PBS containing 0.1% Tween-20 and incubated with embryos for 12 h. This antibody recognizes the C-terminal peptide of human CRM1. Embryos were washed three times for 30 min in PBS containing 0.1% Tween-20. Embryos were placed in secondary antibody (fluorescein isothiocyanate-conjugated anti-rabbit; Sigma), which was diluted 1:500 in PBS containing 0.1% Tween-20, for 12 h. Embryos were washed three times for 30 min in PBS containing 0.1% Tween-20. Embryos were stained with propidium iodide (1 µg/ml final concentration) for 1 h and rinsed once in PBS containing 0.1% Tween-20. Embryos were mounted on slides in 20 µl of Fluoromount-G and examined on a confocal microscope.
Surgical Recovery of Porcine Embryos
Some embryos were surgically recovered from gilts by standard flushing techniques according to a protocol that was approved by our institutional animal care and use committee. Briefly, bred gilts were anesthetized with 30 ml of sodium pentathol i.v. and maintained under general anesthesia with <5% halothane (inhalation). A glass catheter was fed into the oviduct at the infundibulum, and 25 ml of Hepes-buffered medium containing 3 mg/ml BSA was injected into the lumen of the oviduct at the utero-tubal junction. Medium was flushed retrograde and collected in a 50-ml disposable polypropylene tube from the catheter [18].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro maturation (IVM) of porcine oocytes was performed in the presence or absence of LMB to determine the necessity of CRM1 function during oocyte maturation. No statistical difference in progression to metaphase II was detected between LMB (129 of 134 oocytes; 96.3% reached metaphase II) and control groups (131 of 137 oocytes; 96.0% reached metaphase II) over the course of three replicates (chi-square analysis P < 0.79, n = 271). The only detectable difference between LMB and control groups was the complete absence of cumulus cell expansion in LMB treatment groups. Oocytes matured in the presence of LMB were subsequently fertilized and evaluated for pronuclear formation in an attempt to determine the quality of the LMB treated oocytes. No differences in fertilization rates, as determined by pronuclear formation, were detected between LMB (43 of 109; 39.4%) and control groups (50 of 109; 46.0%) over the course of three replicates (chi-square analysis, P < 0.4; n = 218).
CRM1 Function Is Not Required for Development Before the 4-Cell Stage
Porcine embryos were cultured with LMB to determine the time period when the embryo requires CRM1-mediated nuclear export for development. Nuclei numbers were determined on Day 6 (158 h after insemination). Embryos cultured in the presence of LMB arrested in development between the 4-cell and 8-cell stages, whereas control embryos proceeded beyond the 8-cell stage to the blastocyst stage (Table 1), P < 0.001 with the Fisher exact test. This effect was significant regardless of whether LMB was added at the 1-cell, 2-cell, or 4-cell stage. No differences were detected between groups with regard to the time of LMB exposure (n = 77, P < 0.9 with chi-square analysis).
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CRM1 Function Is Required for Development after the 8-Cell Stage
No statistical differences (using ANOVA) in mean cell number were detected between embryos fixed on Day 4 (mean cell number = 8.0) and embryos treated with LMB and examined on Day 6 (mean cell number = 8.3). The mean cell number for the control group (mean cell number = 18.6) of embryos evaluated on Day 6 was significantly higher than either the LMB treatment group or those embryos fixed on Day 4 (n = 106; ANOVA and Fisher least significant difference, P < 0.001).
CRM1 Protein Is Present Throughout Preimplantation Development
Germinal vesicle-stage oocytes and pronuclear, 2-cell, 4-cell, and morula-stage embryos (both in vivo-derived and those produced in vitro) all showed presence of CRM1 protein, as determined by immunodetection with anti-human CRM1 antibody. Staining was predominantly nuclear in all stages examined. Additional staining was observed in a punctate pattern throughout the nucleus and surrounding cytoplasm in pronuclear, 2-cell and 4-cell embryos (Fig. 1). Specificity of the CRM1 antibody was demonstrated previously against both human [9] and Xenopus [11] CRM1, and against porcine CRM1 by Western blot (data not shown).
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CRM1 Activity Exists During the LMB Insensitive Period of Development
Germinal vesicle oocytes were examined for the presence of CRM1 function with a direct microinjection assay. Oocytes treated with ethanol and injected with wild-type NES reporter protein lacked detectable fluorescein staining in their nuclei, whereas oocytes pretreated with LMB and injected with the same protein possessed appreciable amounts of fluorescein staining in their nuclei. All oocytes in both LMB and control groups injected with mutant-NES reporter protein possessed fluorescein staining in their nuclei (Fig. 2).
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After fixation at the pronuclear stage, embryos treated with ethanol and injected with wild-type NES reporter protein lacked detectable fluorescein staining in their nuclei, whereas embryos pretreated with LMB and injected with the same protein possessed fluorescein staining in their nuclei. All embryos injected with mutant NES reporter protein, in both LMB and ethanol groups, possessed fluorescein staining in their nuclei. The same result was found in 2-cell stage embryos (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In an earlier study [20] we showed that LMB, when present from the pronuclear stage onward, caused a developmental arrest during the 4-cell to 8-cell stage in porcine embryos and the 8-cell to 16-cell stage in bovine embryos, corresponding to the time of zygotic genome activation of each species [19]. To determine the nature of the LMB induced arrest, the present experiments expanded on the initial study by initiating LMB treatment at not only the pronuclear stage, but at the 2-cell, 4-cell, and post-8-cell stages as well. The results indicated that porcine embryos arrest at the 4-cell to 8-cell transition, when LMB was added at the 4-cell stage or earlier. In addition, when embryos beyond the 8-cell stage were treated with LMB, an immediate arrest in development occurred.
We also found that CRM1 function is dispensable during oocyte maturation. Although no effect of LMB was evident in terms of progression to metaphase II or pronuclear formation following fertilization, we observed an effect on the surrounding cumulus cells (i.e., a complete lack of cumulus expansion). Normally during porcine oocyte IVM, cumulus cells expand from a dense arrangement closely adhered to the zona pellucida to a loose network extending out from the zona pellucida. During this time, factors produced by the cumulus cells are secreted into the surrounding medium and transferred to the oocyte through cytoplasmic projections through the zona pellucida into the oocyte. The absence of cumulus expansion upon LMB treatment suggests a requirement for CRM1-mediated nuclear export during this process. Although the effects of CRM1 inhibition during IVM may be manifested in a lower quality of matured oocytes, these effects are not immediately detectable as a reduction in fertilization. This could result from inappropriate cytoplasmic maturation, resulting in oocytes that are unable to proceed through development after fertilization. It was not possible to assay this directly, because LMB present during IVM caused embryos to arrest at the four-cell stage of development (data not shown).
A similar group of experiments were recently reported in Xenopus [11]. Instead of causing an arrest at the mid-blastula transition, a time analogous to zygotic genome activation in mammalian embryos, Callanan et al. [11] showed that LMB induced a developmental arrest in Xenopus embryos at the gastrula-to-neurula transition (GNT). In addition, Xenopus CRM1 appeared to adopt an intranuclear localization that was somewhat different than that found in the porcine embryo during corresponding stages. Instead of a punctate pattern that extends throughout the nucleus and into the surrounding cytoplasm, Callanan et al. [11] reported diffuse staining throughout the nucleus with staining organized into a filamentous network running throughout the nucleus during the LMB-insensitive period. The most interesting finding was that while Xenopus CRM1 function appears absent before GNT, CRM1 appears to have a constitutive function in mammalian embryos.
The differences in developmental arrest and intracellular localization of CRM1 reported for these two species strongly suggest regulation of CRM1 may have species-specific differences. Such difference may have arose in response to the specific needs of the species (i.e., the relatively short time required for postfertilization cleavage and gastrulation of Xenopus embryos compared to mammalian embryos).
This is the first report of a mammalian cell type that appears to develop without CRM1 activity. Why would porcine embryos possess CRM1 activity, yet not require it for a defined period of time? This question was addressed indirectly in culture experiments in which cells were only transiently exposed to LMB. In the embryo culture experiments reported here, embryos were cultured in medium containing LMB for several days, thereby rendering all CRM1 protein inactive, including any protein synthesized after LMB treatment was initiated. Addition of LMB at the pronuclear, 2-cell, and 4-cell stages resulted in developmental arrest at the same time period, suggesting inactivation at any time during the LMB-insensitive window results in subsequent developmental arrest. In an unpublished set of experiments, embryos were exposed to LMB for 10 min, then washed repeatedly and allowed to culture. Despite the short exposure time and extensive washing, porcine embryos still arrested at the 4-cell stage. This suggests that CRM1 may indeed be required for development, but that early embryos lack certain check points that cells further down the differentiation pathway possess, and that bypassing these checkpoints, although appearing to have no effect on cleavage, actually have a major effect on development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by U.S. Department of Agriculture National Needs to R.A.C. and Food for the 21st Century at the University of Missouri. 
2 Correspondence. FAX: 573 882 6827; pratherr{at}missouri.edu
Accepted: April 4, 2002.
Received: February 25, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. EMBO J 1999 18:6682-6693[CrossRef][Medline]This article has been cited by other articles:
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