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Embryo |
Department of Biology,2 University of North Carolina at Charlotte, Charlotte, North Carolina 28223
A.R.T. Institute of Washington,3 Washington, D.C
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, early development, embryo, nitric oxide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has previously been demonstrated in our laboratory, using immunocytochemistry, that delayed blastocysts following estrogen activation at 1 h produce both eNOS and iNOS proteins [3]. It has also been shown that an increase in iNOS occurs in both the uterus and the implanting embryo. In pre- and peri-implantation embryos, iNOS was shown to be localized to the inner cell mass. Postimplantation embryos showed decreased iNOS localization [4]. However, it is not known whether all three isoforms are present in earlier stage embryos, specifically preimplantation embryos.
Numerous studies have shown that NO mediates many of its effects through the cyclic guanosine 3',5'-monophosphate (cGMP) signal transduction system [5]. However, NO can also mediate its effects through stimulation of cyclooxygenase enzymes [6], altering phosphodiesterases [7], and activating Gi
1. NO can also inhibit mitochondrial cytochrome oxidase and the activation of NF-kB. In addition, NO can activate calcium-dependent potassium channels, NF-kB, and G-proteins by enhancing the rate of nucleotide exchange [8].
In the mouse, embryo development begins at the one-cell stage, using maternally derived mRNAs. When the embryo progresses to the two-cell stage, it begins to produce embryonically derived mRNAs. It has previously been reported that NG-nitro-L-arginine (L-NA), a nonspecific NOS inhibitor, prevents embryos from developing beyond the two-cell stage [9]. While development was arrested, embryos did not appear to undergo cell death, as determined by visual inspection [3]. Instead, they appeared to be normal two-cell embryos. Sodium nitroprusside (SNP), a NO donor, has been shown to arrest embryo development at high concentrations [9, 10]. Thus, both an excess and a depletion of NO can inhibit embryo development.
We hypothesize that NO levels must be regulated for normal embryo development to occur and thus the mRNA of multiple NOS isoforms must be expressed in preimplantation embryos to support adequate levels of NO production. When concentrations of NO are elevated in cultures with SNP or depleted in L-NA, it is not known whether embryo development is merely arrested or if the embryos are undergoing apoptosis. We predict that excess NO will cause apoptosis of the embryo while a loss of NO will stunt development but not cause cell death. In addition, we hypothesize that NO acts through the cGMP pathway during preimplantation embryo development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twenty- to 25-g virgin female CD-1 mice (Charles River Laboratory, Raleigh, NC) were mated with males of the same strain to produce pregnancy. The presence of a vaginal plug indicated Day 1 of pregnancy. All tissues used for the positive controls (spleen, abdominal aorta, spinal cord) were also obtained from CD-1 females. The animal experiments reported in this study were performed in adherence with the guidelines established in the "Guide for the Care and Use of Laboratory Animals" as adapted and promulgated by the National Institutes of Health.
Embryo Isolation
Embryos were recovered on Day 1 from oviducts, cultured in Whitten media, and harvested at the two-cell, four-cell, and morula stages. Blastocysts were collected on Day 4 from uteri (1000 h for early blastocysts; 1500 h for late blastocysts). Embryos were washed in PBS with 0.1% PVA containing 1 µM RNAse inhibitor (20 IU/µl) (RNA PCR Kit; Perkin Elmer, Branchburg, NJ) and stored at -80°C prior to use.
Embryo Culture
Following recovery from oviducts, one-cell embryos were washed with Whitten medium and then cultured for 48–72 h in either 100 µl Whitten alone or Whitten with one or more of the following: 8-Br-cGMP (from 0.1 to 40 mM), L-NA (from 125 to 500 µM), or SNP (from 0.1 to 500 µM). All cultures were placed in a humidified CO2 chamber at 37°C (95% air/5% CO2). Embryo development was assessed microscopically every 24 h and classified as two-cell, four-cell, morula, or blastocyst. The embryos were transferred to new media following each assessment.
RNA Isolation
RNA was isolated from embryos on Day 2 (two-cell), Day 3 (morula), or Day 4 (early blastocyst, 1000 h, and late blastocyst, 1500 h) of pregnancy using a Stratagene Micro RNA isolation kit (Cedar Creek, TX). Denaturing solution (guanidine isothiocynate, 4 M; sodium citrate, 0.02 M; sarcosyl, 0.5%) was added to the samples according to the manufacturer's recommendations. A fixed amount of exogenous RNA transcribed from the synthetic plasmid pAW109 (Perkin Elmer, Foster City, CA) was added to each sample to serve as a control for RNA recovery and reverse transcription. The pAW109 RNA includes sequences complementary to those present in the plasmid insert. The RNA was precipitated using isopropanol with glycogen as a carrier. Spinal cord, spleen, and abdominal aorta RNAs were isolated to be used as positive controls for nNOS, iNOS, and eNOS, respectively. RNA was isolated using an Ambion RNAqueous kit (Austin, TX) according to the manufacturer's recommendations.
Reverse Transcription
First strand complementary DNA was synthesized by priming with random hexamers. Each RNA pellet was resuspended in 8.5 µl of solution containing 1 µl of 50 µM random hexamers, 0.2 µl of 0.1 M dithiothreitol, 0.05 µl RNase inhibitor (20 IU/µl), and 7.25 µl sterile nuclease free water (RNA PCR Kit; Perkin Elmer, Branchburg, NJ). The hexamers were annealed by incubating the samples at 70°C for 6 min and then setting on ice for 1 min. Reverse transcription was performed by the addition of 4 µl of 25 mM MgCl2, 2 µl of 10x polymerase chain reaction (PCR) buffer II, 4 µl of 2mM dNTP, 1 µl of RNase inhibitor (20 IU/µl), 1.5 µl Moloney murine leukemia virus reverse transcriptase, and 0.5 µl of 50 µM random hexamers (RNA PCR Kit; Perkin Elmer, Branchburg, NJ). The samples were incubated at 37°C for 1 h and 95°C for 5 min and then placed on ice for 1 min. Between 20 ng and 2 µg of cDNA was used in each PCR reaction.
Real-Time PCR
PCR amplification was performed using a Light Cycler (Roche, Indianapolis, IN), a combination microvolume fluorometer, and rapid temperature cycler [11, 12]. The reaction mixture consisted of cDNA, 1 µl of 10x buffer (30 mM MgCl2), 0.5 µl of each primer (80 pg), 1 µl of 10x NTPs, 1 µl Taq DNA polymerase with TaqStart antibody (Clontech, Palo Alto, CA), and 1 µl Sybr Green I solution, diluted 1:3000 of 10 000x solution (Molecular Probes, Eugene, OR). A 10-µl volume was loaded into the capillary reaction vessels. For pAW109, cDNA was denatured by heating to 95°C for 1 min 20 sec. The template was amplified by 40 cycles of 0 sec at 95°C, 0 sec at 55°C, 20 sec at 72°C, and 2 sec at 85°C. For ß-actin, cDNA was denatured by heating to 96°C for 1 min 20 sec. The template was amplified by 45 cycles of 5 sec at 95°C, 20 sec at 58°C, 12 sec at 72°C, and extended at 83°C for 2 sec. For nNOS and eNOS, cDNA was denatured at 94°C for 3 min, then amplified at 94°C for 0 sec, 64°C (-0.5°C for 10 cycles) for 0 sec, and 72°C for 12 sec and then 26 cycles at 94°C for 0 sec, 58°C for 0 sec, and 72°C for 12 sec. For iNOS, cDNA was denatured at 94°C for 1 min and 30 sec, amplified for 35 cycles at 94°C for 0 sec, 55°C for 0 sec, and 72°C for 12 sec. Following amplification, all samples were melted at 94°C for 0 sec, 50°C for 30 sec, and 99°C for 0 sec, and the fluorescence was monitored. The melting curve was drawn as the negative derivative of fluorescence with respect to temperature to generate a melting point. The melting points for pAW109, ß-actin, eNOS, iNOS, and nNOS were 88, 88, 89, 88.5, and 88°C, respectively. Amplified product underwent electrophoresis on agarose/ethidium bromide gels and was visualized under UV light.
Primers
The pAW109 primers were purchased from Perkin Elmer (Foster City, CA). All primers were obtained from Invitrogen (Carlsbad, CA) and were as follows: pAW109: gTCTCTgAATCAgAAATCCTTCTATC: CATgTCAAATTTCACTgCTTCATCC; ß-actin: TgCgTgACATCAAAgAgAAg: CggATgTCAACgTCACACTT; nNOS: TggTTCTggTCTTCgggTgTC: TACCggTTgTCATCCCTCAgC; iNOS: CCggCAAACCCAAggTC: TTgCCCCATAggAAAAgACTg; eNOS: ggAggTTCACCGTgTgCTgTg: gggCCAggCgggTCAAA.
Comet Assay
A Trevigen Comet Assay kit (Gaithersburg, MD) was used to determine whether the cultured embryos had undergone DNA fragmentation. Embryos were cultured in either Whitten or Whitten with one of the following: 500 µM L-NA, 200 µM 8-Br-cGMP, or 250 µM SNP. Following 48 h of culture, embryos were added to 75 µl low melting point agarose (42°C) and pipetted onto a slide. The slide was allowed to solidify at 4°C for 20 min, then was immersed in lysis solution (2.5 M NaCl; 100 mM EDTA, pH 10; 10 mm Tris base; 1% sodium lauryl sarcosinate; 1% Triton X-100) at 4°C for 60 min. The slide was then immersed in alkali solution (0.6 g NaOH, 250 µl 200 mM EDTA, and 49.75 ml deionized water) for 60 min. The slide was washed two times in 1x TBE buffer (108 g Tris base, 55 g boric acid, 9.3 g EDTA) for 5 min. The slide was subjected to an electric field for 10 min at 143 V followed by an ethanol wash and was then dried. Fifty microliters of diluted Sybr green (1 µl Sybr green in 10 ml TE buffer, pH 7) was placed onto each circle of dried agarose and the slide viewed by epifluorescence microscopy. To determine if the minimal tails seen in Whitten were due to the presence of RNA, RNase A (Sigma) was added to the lysis solution (1 µg/ml).
Statistical Analysis
The results of the experiments were analyzed using a chi-square analysis and a sequential Bonferroni analysis. Chi-square analysis (from 2 x 2 contingency tables) was used to test for the difference in proportions of embryos at the appropriate stage of development in either the Whitten versus L-NA, Whitten versus SNP, L-NA versus SNP and SNP/L-NA, Whitten versus 8-Br-cGMP, or L-NA versus 8-Br-cGMP/L-NA. Embryos were cultured in groups in control or experimental media. However, to confirm that culture conditions were stable, several embryos were cultured in control media every few days. Because the number of embryos isolated on each day was variable and the embryos from more than 1 day of culture were pooled as a single experiment, the numbers per group are not identical. The fewest embryos were used in those treatment groups in which all embryos were killed or there was no apparent effect on development, with a minimum of 10 embryos per group used. Statistical significance was reached with a P-value <0.05 and is represented by an asterisk.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amplification of a 340-bp fragment of the nNOS gene was evident in all pooled embryo samples (Fig. 2A). This was also true for expression of a 300-bp fragment of the eNOS gene (Fig. 2B). Amplification of a 264-bp fragment of the iNOS gene was evident in all preimplantation embryos except early blastocysts (Fig. 2C). Melting-point curves shown are representative of three separate reactions run using pooled RNAs from embryos (n = 10 per reaction) or control RNAs (abdominal aorta, spinal cord, or spleen).
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To determine if inhibition of embryo development by L-NA can be reversed by addition of NO, Day 1 embryos were cultured for 48–72 h in either Whitten media alone or Whitten with one or more of the following: L-NA, from 125 to 500 µM, or SNP, from 0.2 nM to 500 µM. Only those embryos that arrested at the two-cell stage were cultured for 72 h to determine if they were simply delayed in the development or were arrested at the two-cell stage. All embryos developed through the two-cell stage (data not shown). The inhibitory effect of L-NA was lost when its concentration was decreased to 125 µM L-NA, and all embryos developed from the two-cell stage to the four-cell stage (Fig. 3A). SNP inhibited normal embryo development past the two-cell stage at all concentrations studied (0.2 nM to 500 µM; P < 0.001) (Fig. 3B). When L-NA was combined with SNP, the inhibitory effects were reversed and a significant number of embryos continued in their development (Fig. 3C). Embryos cultured in L-NA for 48 h were rescued after transfer into Whitten medium. This rescue could not be achieved after L-NA culture for 72 h (Table 1).
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To determine whether NO affects preimplantation embryo development via a cGMP-dependent pathway, embryos were cultured up to 48–72 h in a manner similar to the cultures described above in either Whitten alone or Whitten with 8-Br-cGMP (from 1 nM to 40 mM) (Fig. 4A). Culture with both 8-Br-cGMP and 500 µM L-NA resulted in embryo development rates that were statistically significantly different from 500 µM L-NA alone (P < 0.001) (Fig. 4B).
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To determine whether embryos that remained at the two-cell stage were dying and thus undergoing DNA fragmentation although they appeared visually normal, embryos were cultured for 48 h in either Whitten alone or Whitten with one of the following: 500 µM L-NA, 200 µM 8-Br-cGMP, or 250 µM SNP. Comet assays were run on these embryos. Embryos cultured in Whitten alone had minimal or no tails (Fig. 5A), indicating little or no DNA fragmentation. Embryos cultured in 500 µM L-NA and 200 µM 8-Br-cGMP produced tails that appeared similar in size to those cultured in Whitten and thus had little or no DNA fragmentation (Fig. 5, B and C). All embryos cultured in 250 µM SNP had long comet tails, indicating DNA fragmentation and thus cell death (Figure 5D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gouge et al. demonstrated by immunocytochemistry studies that eNOS and iNOS proteins were present in estrogen-activated blastocysts [3]. Studies on the reproductive ability of single-NOS knockout mice show that, in the absence of each NOS isoform, reproductive abnormalities exist. Such abnormalities include lower ovulatory efficiency, compromised erectile function, shorter and less variable estrous cycles, and reduced ovulation [7, 13–21]. However, no abnormalities in preimplantation embryo development in single knockout mice are seen. Recent studies in our lab indicate that double knockout mice (eNOS/iNOS, eNOS/nNOS, iNOS/nNOS) are preferentially lost during development, although the stage at which embryonic loss occurs is not known (unpublished data).
Gouge et al. also previously demonstrated that, when the NOS inhibitor L-NA (500 µM) was added to culture media with two-cell embryos, development was arrested in 83% of the embryos [3]. In the present study, 100% of one-cell embryos cultured with L-NA (500 µM) were arrested at the two-cell stage. When the NO donor, SNP, was added to Day-1 mouse embryos, embryo development was also completely inhibited past the two-cell stage at concentrations above 1 nM. However, it is important to note that, while 100% of the embryos cultured in 500 µM L-NA remained at the two-cell stage throughout the 4 days of observation, almost half of the embryos cultured in excess SNP (>1 nM) began to fragment and degenerate within 24 h. This could result from the production of free radicals such as superoxide. When NO reacts with oxygen, peroxynitrites (ONOO-) are formed. These peroxynitrites convert to peroxynitrous acid, a powerful oxidant with cytotoxic effects, such as initiation of lipid peroxidation, contributing to cell toxicity [6].
Whereas L-NA (500 µM) inhibited embryo development beyond the two-cell stage, the addition of low concentrations of SNP (
1 nM) resulted in a significant continued normal embryonic development. The addition of SNP (100 nM) at a dose that kills all the embryos when given alone resulted in a significant number of embryos continuing their normal development when added to culture media containing L-NA (500 µM). These data demonstrate that the inhibition of development produced by the lack of NO can be reversed by the addition of a NO donor and that NO production must be regulated within some specific limits. Further experiments utilizing lower concentrations of L-NA (250 µM) with SNP (100 nM) did not rescue a significant number of embryos, providing further evidence that the concentration of NO produced must be controlled within limits. Thus, excess NO leads to degeneration of embryos while insufficient amounts of NO arrests development.
In many tissues, NO can mediate its effects through the cGMP pathway [9]. Our data indicate that NO regulation of embryo development is also working, at least in part, through the cGMP pathway. L-NA inhibits the production of NO, and if NO works through the cGMP pathway, 8-Br-cGMP should be able to reverse the inhibitory effects of L-NA. Embryos cultured in 8-Br-cGMP alone showed a dose-dependent inhibition in embryo development such that, as the concentration was increased to 40 mM, the percentage of embryos developing beyond the two-cell stage decreased to zero. Embryos cultured in 8-Br-cGMP concurrently with 500 µM L-NA showed a significant increase in normal development when compared with those cultured in 500 µM L-NA alone (P < 0.05). Embryos cultured in 1 nM 8-Br-cGMP and 500 µM L-NA showed 63% progression to the four-cell stage, which was significantly greater than for 500 µM L-NA or Whitten alone (P < 0.001). These data indicate that preimplantation embryo development does involve the NO/cGMP pathway.
If, however, NO worked exclusively through the cGMP pathway, we would expect that embryos cultured in 8-Br-cGMP and L-NA would show no difference when compared with those cultured in Whitten. However, it is also possible that the chosen concentrations of 8-Br-cGMP were not optimal to reverse the effects of L-NA. Because 8-Br-cGMP could only partially rescue embryo development, we cannot exclude the possibility that NO works not only through the cGMP pathway but may also involve other pathways. These could include phosphodiesterases, cyclooxygenases, NF-kB, or G proteins, all of which have been shown in other tissues to be regulated by NO [8, 22–24].
Chen et al. cultured embryos in L-NAME, SNP, 8-Br-cGMP, and/or 1H-(1,2,4)oxadiaxlol-(4,3-a)quinoxalin-1 (ODQ), a selective inhibitor of NO-sensitive soluble guanylyl cyclase [10]. Although they did not find that L-NAME (10 µM) inhibited development at the two-cell stage, they did demonstrate a decrease in development to the blastocyst stage. In their culture system, they obtained findings in agreement with our study, showing that SNP could rescue embryo development from the inhibitory effects of L-NAME. Similarly, 8-Br-cGMP could also rescue embryos from L-NAME inhibition, providing the first evidence that NO works through the cGMP pathway in preimplantation embryo development.
Chen et al. also studied whether embryos cultured in excess 8-Br-cGMP or with an 8-Br-cGMP inhibitor, ODQ, underwent apoptosis in their experiments using TUNEL and annexin V staining [10]. These researchers did not determine whether L-NAME alone or 8-Br-cGMP alone caused embryos to undergo apoptosis, even though they found that L-NAME (0.1–10 µM) caused substantial degradation. In comparison, in our study, embryos cultured in 500 µM L-NA appeared normal by visual inspection, albeit arrested in their development. Therefore, we used a comet assay to determine if embryos that appeared visually normal underwent DNA fragmentation and thus were actually undergoing cell death. All embryos cultured in Whitten, L-NA (500 µM), or excess 8-Br-cGMP (200 µM) showed minimal tails, indicating little or no DNA damage. Because 98% of embryos cultured in Whitten media progressed beyond the two-cell stage and developed to the morula stage, we expected to see no DNA damage and therefore no tails. It was possible that the minimal tails were due to RNA degradation. However, when we added Rnase in the procedure, no difference in tail size was seen (data not shown). This indicates that the small tail was not due to RNA and is due perhaps to endogenous DNA damage or damage produced during the procedure. Embryos cultured in excess SNP (250 µM) that fragmented and degenerated upon visual inspection showed sizeable tails, indicating DNA damage and thus cell death. The comet assay results from the SNP and 8-Br-cGMP cultures are in agreement with Chen et al., who found that approximately 77% of embryos cultured in SNP (10 µM) showed apoptotic cell death through annexin V staining. They also found that 8-Br-cGMP (10 µM) concentrations could not induce apoptosis alone, and our comet assay results confirm that the embryos do not have significant DNA damage [10]. These data indicate that, although increased intracellular concentrations of cGMP inhibit mitotic division in preimplantation embryos, as does excess NO, the mechanisms of altered development are not the same.
This study shows, for the first time, the presence of mRNA for all three NOS isoforms in preimplantation mouse embryos, providing further evidence for the importance of NO production in murine embryo development and potential in other mammalian embryos. We also demonstrate, through cultures of embryos in SNP, L-NA, and 8-Br-cGMP, that not only does NO require tight regulation but it also works in part through the cGMP pathway. Further, inhibition of development by lack of NO or excess cGMP does not initiate DNA damage and thus cell death, whereas excess NO does. Thus, embryonic cell death induced by excess NO does not occur through a cGMP-mediated pathway. Future experiments will study regulation of cell-cycle proteins by NO and the cGMP pathway to further our understanding of NO regulation of murine embryo development.
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FOOTNOTES |
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Received: 11 July 2002.
First decision: 9 August 2002.
Accepted: 13 November 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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