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Biology of Reproduction 67, 386-392 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Comparison of Gene Expression During Preimplantation Development Between Diploid and Haploid Mouse Embryos1

Keith E. Latham2,,a,b, Hidenori Akutsuc, Bela Patela, and Ryuzo Yanagimachic

a The Fels Institute for Cancer Research and Molecular Biology and b The Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 c Institute for Biogenesis Research, The John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96822


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Haploid development is a normal part of the life cycle for some animals, but it has not been observed in mammals. Studies in mice have revealed that the preimplantation developmental potential of haploid embryos is significantly impaired relative to diploid embryos. The reasons for the severely limited developmental potential of haploid embryos in mammals have not been discerned. To examine the effects of haploid development on gene expression, and in particular on X-linked gene expression, and to evaluate to what degree newer techniques of producing and culturing such embryos might affect developmental potential, haploid and diploid parthenogenetic and androgenetic embryos were produced and reevaluated for developmental potential, genomic integrity, and relative expression levels of specific autosomal and X-linked gene transcripts. Our data confirm the previously observed restriction in haploid developmental potential, eliminate chromosomal abnormalities as a major factor in this restriction, and reveal subtle alterations in gene expression. Haploid parthenogenones display only very subtle alterations in the expression of most mRNAs but a consistent elevation in X-linked Bex1 mRNA expression. Haploid androgenones seem to lack repression of the Pgk1 gene that is seen in diploid androgenones, but this may reflect ongoing loss of those haploid androgenones that experience X chromosome inactivation. The significance and possible explanations for these differences are discussed.

early development, embryo, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Haploid development is a normal part of the life cycle for some animals (e.g., parasitic wasps) [1], but it is not observed in mammals. Studies in mice reveal that the preimplantation developmental potential of haploid embryos is significantly impaired relative to diploid embryos [2–9]. Embryonic stem (ES) cells derived from haploid parthenogenones only contribute to the development of chimeric animals after they become diploidized [10], and ES cell lines established from haploid parthenogenones likewise become diploid [11]. Development of haploid rabbit parthenogenones is also inefficient [12]. This indicates that the mammalian genome, in contrast to genomes of other organisms, is subject to gene regulatory mechanisms that disrupt the ability of a haploid genome to support early development.

The reasons for the severely limited developmental potential of haploid embryos in mammals have not been discerned. Several interesting possibilities have been examined, but none has provided a likely explanation. For example, earlier studies revealed that aneuploidy can occur in haploid parthenogenones [13], and that this might result in an absence of essential gene functions through chromosome loss. Aneuploidy affects only a small fraction of embryos, however, and is dependent upon the method of oocyte activation employed [13, 14], making this an unlikely explanation. Other studies examined the possible influence of the DNA:cytoplasm ratio on haploid developmental restrictions [2, 8]. Haploid embryos made by zygote bisection, or by pronuclear removal followed by cytoplasmic reduction, showed some improvement in development, but the overall rate of development remained severely reduced relative to diploid embryos. Genomic imprinting offers another possible explanation, as haploid embryos possess exclusively maternal or paternal chromosomes. This is also unlikely by itself to provide a satisfactory explanation, because haploid androgenones, gynogenones, and parthenogenones develop substantially worse than their diploid counterparts [2, 15, 16]. Effects of genomic imprinting on X chromosome function, however, is a possibility that has not been tested. Imprinting of the X chromosome, attributable to differential Xist gene methylation, leads to preferential inactivation of the paternal X chromosome in extraembryonic tissues and preferential expression of the maternal X chromosome during preimplantation development (reviewed in [17]). Imprinting of the X chromosome could thus lead to potentially lethal repression of the single X chromosome in haploid X chromosome-bearing androgenones; the Y chromosome-bearing haploid androgenones would obviously also lack X-linked gene expression. Similarly, dosage compensation by X chromosome inactivation would be unlikely in haploid parthenogenones, because such embryos would have to achieve a 50% expression level of their 1 X chromosome relative to their 1 set of autosomes, and no evidence for such regulation of the X chromosome exists for mammals. Thus haploid parthenogenones may overexpress X-linked genes relative to autosomal genes.

The aim of this study was to examine the effects of haploid development on gene expression, and in particular effects on X-linked gene expression, and to evaluate to what degree newer techniques of producing and culturing such embryos might affect developmental potential. We produced haploid and diploid parthenogenetic and androgenetic embryos, and re-evaluated their developmental potential, their genomic integrity, and their relative expression levels of specific autosomal and X-linked gene transcripts. Our data confirm the previously observed restriction in haploid developmental potential, eliminate chromosomal abnormalities as a major factor in this restriction, and reveal subtle alterations in gene expression. The significance and possible explanations for these differences are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Polyvinyl alcohol (PVA, cold-water-soluble, molecular weight ~10 000) and polyvinyl pyrrolidone (PVP, molecular weight ~360 000) were purchased from Sigma Chemical Co. (St. Louis, MO). Bovine testicular hyaluronidase (200 UPS U/mg) was obtained from ICN Biochemicals (Costa Mesa, CA). BSA (fraction V) was purchased from Calbiochem (La Jolla, CA) and mineral oil from Squibb and Sons (Princeton, NJ). All other reagents were obtained from Sigma unless otherwise stated.

Media

CZB medium [18] supplemented with 5.56 mM D-glucose was used for the culture of mouse oocytes after microsurgery. This was called standard CZB. The medium for collection of oocytes from oviducts and subsequent oocyte treatments, including micromanipulation, was a modified CZB (Hepes-CZB, [19]) containing 20 mM Hepes-HCl, a reduced amount of NaHCO3 (5 mM), and 0.1 mg/ml PVA instead of BSA. Standard CZB was used at 37° under 5% CO2 in air, and Hepes-CZB was used under air.

Animals

Spermatozoa were collected from caudae epididymides of (C57BL/6 x DBA/2)F1 male mice. Females of the same genotype were used as oocyte donors. These animals were maintained in accordance with the guidelines of the Laboratory Animal Service at the University of Hawaii and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources National Research Council (DHEW publication [NIH] 80-23, revised in 1985). The animal handling and treatment protocol was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.

Preparation of Androgenones and Parthenogenones

Haploid and diploid androgenones were prepared using unfertilized oocytes from 4- to 8-wk-old females injected with 5 IU eCG followed by 5 IU hCG 48 h later. Mature oocytes were collected from oviducts at approximately 15 h after the hCG injection. They were freed from the cumulus cells by 5-min treatment with 0.1% bovine testicular hyaluronidase in Hepes-CZB. The oocytes were rinsed and kept in CZB medium at 37°C under 5% CO2 in air for less than 2 h before further treatments. The oocytes were transferred to a 20-µl droplet of Hepes-CZB containing 5 µg/ml cytochalasin B (CB; 100x stock in dimethylsulfoxide), which had previously been placed in the operation chamber on the microscope stage. They were kept there for 5–10 min before enucleation. The metaphase II chromosome-spindle complex was aspirated into a pipette (8–10 µm in the inner diameter) with a minimal volume of oocyte cytoplasm [20], and the resulting oocyte cytoplasts were then cultured in cytochalasin B-free CZB for up to 2 h at 37°C before sperm injection. Haploid androgenetic embryos were made by injection of a single spermatozoon into oocyte cytoplasts according to Kimura and Yanagimachi [19], except that only the sperm head was injected and the operation was performed at room temperature. The sperm head was separated from the tail by applying a few piezo-pulses to the head-tail junction. Because the sperm heads contribute oocyte-activating factors [21], no additional treatment was needed for activation. After further culture in CZB, embryos with 1 male pronucleus were considered haploid androgenones. Diploid androgenones were prepared in the same way as haploid androgenones, but 2 isolated sperm heads, instead of 1, were injected into each oocyte cytoplast. Embryos with 2 male pronuclei were considered diploid androgenones.

Haploid and diploid parthenogenones were prepared from metaphase II oocytes by activating for 6 h in Ca2+-free CZB containing 10 mM Sr2+ at 37°C in a humidified atmosphere of 5% CO2 in air [22], with or without 5 µg/ml CB to suppress second polar body extrusion. Eggs that formed single female pronuclei and second polar bodies were considered haploid parthenogenones and cultured in CZB. Second polar bodies had sharply defined membranes and round centrally located nuclei and thus could be distinguished from first polar bodies, which had granular cytoplasm and scattered chromosomes [23]. For oocytes not treated with CB, eggs with 2 distinct pronuclei with or without a second polar body were discarded. For eggs treated with CB, those with 2 distinct pronuclei were regarded as diploid parthenogenones and cultured in CZB.

Chromosome Analysis

Seven to 8 h after the start of incubation in oocyte-activating Sr2+ medium or sperm injection, a group of pronuclear eggs was transferred into another droplet (0.2 ml) of CZB containing 0.006 µg/ml vinblastine, a microtubule-disrupting agent. Between 19 and 21 h after sperm injection, eggs that had been arrested at the first mitotic metaphase were treated with 0.25% (w/v) pronase (Kaken Pharmaceuticals, Tokyo, Japan) for 5 min to remove zonae pellucida, then exposed to a hypotonic solution (1:1 mixture of 1% Na-citrate and 30% fetal bovine serum) for 10 min at room temperature. Fixation of eggs and spreading of chromosomes were performed as described [24]. The chromosomes on slides were stained with 2% Geimsa solution for 8 min. After conventional chromosome analysis, they were C-banded [25] to detect acentric and dicentric chromosomes [24]. Another group of pronuclear stage eggs was cultured in CZB for 36 h to allow them to develop into 2-cell embryos. They were transferred into CZB containing 0.006 µg/ml vinblastine for 10–14 h to arrest them at the metaphase of the second cleavage. They were processed for chromosome examination as already described.

In Situ Hybridization

Haploid and diploid androgenones (blastocysts, Day 5 of culture) were treated with 0.25% (w/v) pronase as above, then exposed to a hypotonic solution (1:4 mixture of 1.2% Na-citrate and 40:fetal bovine serum) for 10 min at room temperature. Embryos were fixed as described [24]. The in situ hybridization protocol followed the procedure of the supplier of biotin-labeled probes for mouse chromosome X and specific subcentromere and whole chromosome Y probes (Applied Genetics Laboratories Inc., Melbourne, FL). Fluorescence microscopy was performed using a Nikon Microscope ECLIPSE E600 with filter module B2A (Nikon Inc., Melville, NY). Blastomeres having an X chromosome show a small dot of fluorescence, whereas a Y chromosome produces a much larger fluorescence signal. For diploid embryos, XX embryos have 2 small X chromosome signals, whereas XY embryos have the small X chromosome signal and the larger Y chromosome signal. Haploid embryos obviously have either a small X chromosome signal or a small Y chromosome signal. All embryos analyzed fit into these categories. Because diploid blastocysts contain many cells, which in some cases had overlapping nuclei, analyses were limited to those nuclei that had no such overlap and could be unambiguously characterized; between 5 and 10 cells were analyzed for diploid androgenetic blastocysts to ensure accurate genotyping. For haploid androgenetic blastocysts, which contained fewer cells with fewer overlapping nuclei, nearly all cells could be analyzed.

Quantitative RT-PCR Analysis

The abundances of specific mRNAs were measured using the quantitative amplification and dot blotting (QADB) method for quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis [26, 27]. Briefly, embryos were lysed in guanidine thiocyanate buffer and stored at -70°C, and then nucleic acids prepared by ethanol precipitation as described [17]. After heat denaturation and annealing with oligo(dT), reverse transcription and polymerase chain reaction were performed as described [17, 26, 27] to amplify quantitatively the 3' terminal portions of the entire mRNA population. Quantitation of specific mRNAs was achieved by dot blotting and cDNA hybridization as described [26, 27]. The sensitivity and reliability of the QADB assay have been extensively documented [28]. The QADB method is applicable to small amounts of material, even single embryos; provides the ability to quantify expression of a large number of mRNAs; and can provide estimates of actual mRNA abundance [26, 27]. These properties make the QADB method ideal for examining mRNA abundance in nuclear transplant embryos. The QADB method is fully quantitative and produces hybridization signals that are linear over at least 3 orders of magnitude (R2 = 0.992), extending to a very low mRNA abundance, and with excellent reproducibility [28]. The QADB method also exhibits excellent reproducibility between experiments with respect to qualitative patterns of mRNA expression and quantitative estimates of mRNA abundance (e.g., [27, 29]).

Statistical Analysis

Developmental and chromosomal data for haploid and diploid embryos were evaluated using the chi-square test with Yates correction for continuity. For mRNA expression data, the statistical significance of differences in means was evaluated using the Student t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Developmental Potential of Haploid and Diploid Embryos

The methods employed here to produce haploid embryos differed somewhat from those used in earlier studies, most notably in the use of Sr2+ for egg activation and the use of sperm head injection to produce androgenones. The use of Sr2+ for egg activation has advantages over ethanol activation employed in many earlier studies. Sr2+, like spermatozoa, induces repetitive oscillations in intracellular free Ca2+ concentration, whereas other agents like ethanol and calcium ionophores induce a single Ca2+ increase [30]. Thus Sr2+ treatment more closely approximates physiological oocyte activation and leads to enhanced development [30]. In addition, the culture medium employed in this study (CZB) [18] supports enhanced development as compared with media employed in earlier studies. Given these differences, it was necessary to re-evaluate the developmental potential of haploid and diploid androgenones and parthenogenones produced using the methods described above. As seen in earlier studies, preimplantation development was significantly more efficient in diploid parthenogenones than haploid parthenogenones (P < 10-6; Table 1). Haploid androgenones were likewise significantly less efficient in blastocyst formation than diploid androgenones (P < 10-6). As expected, both types of androgenones developed significantly more poorly than their parthenogenetic counterparts. The efficiency of blastocyst formation for diploid androgenones (49%) was as expected and consistent with earlier studies, which revealed the demise of those of the YY genotype (see below) combined with developmental arrest among those of other genotypes due to other, unidentified factors [17]. The blastomeres of haploid androgenones were of irregular size starting from the 4-cell stage. We also observed that the second cleavage division was either delayed or exhibited greater asynchrony between blastomeres than is seen with haploid parthenogenones. This was reflected in a significantly larger fraction of haploid androgenones persisting at the 3-cell stage at 48–50 h of development, as compared to haploid parthenogenetic embryos (Table 2). Differences in developmental rates became more apparent for haploid embryos with continued incubation so that haploid embryos required approximately 24 h longer than their diploid counterparts to attain the blastocyst stage. Haploid androgenones were the least efficient in forming blastocysts, and those few that progressed to the blastocyst stage displayed small blastocoele cavities and poor morphologies (Fig. 1). Thus the results obtained here, using these newer methods of haploid embryo construction, confirm the limited developmental potential of haploid embryos relative to their diploid counterparts.


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  		TABLE 1. In vitro development of mouse haploid and diploid parthenogenetic and androgenetic eggs


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  		TABLE 2. Appearance of mouse haploid androgenetic and parthenogenetic eggs after 48–50 h of development in vitro



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  		FIG. 1. Haploid androgenetic blastocysts. Embryos were photographed after 4 days of culture

Chromosome Analysis of Haploid and Diploid Embryos

To test the possibility that haploid embryos are compromised as a consequence of chromosomal abnormalities, we performed chromosome analyses of embryos on metaphase spreads obtained at the first and second mitotic divisions (Table 3). These analyses revealed that chromosomal abnormalities affected only a small fraction (2%–7%) of haploid androgenones or parthenogenones. This did not differ significantly from the rate of chromosomal abnormalities seen in control biparental embryos made by intracytoplasmic sperm injection (ICSI). Because such a small fraction of haploid embryos displayed chromosomal abnormalities, such abnormalities cannot account for the reduced developmental potential.


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  		TABLE 3. Chromosome analyses of the first and second cleavage metaphase in haploid androgenetic and parthogenetic ova

To evaluate whether sex chromosome composition affected development, we examined this feature of haploid and diploid androgenones at the blastocyst stage (Table 4). As expected based on earlier studies [17], diploid androgenetic blastocysts did not include any YY embryos. There appeared to be a slightly larger than expected number of XY androgenetic blastocysts (2:1 XY:XX ratio predicted), but this difference was not statistically significant. This confirms the earlier observation that XX androgenones are not significantly impaired relative to XY androgenones [17], in contrast to another study reporting such a difference [31]. Not surprisingly, haploid Y chromosome-bearing androgenones also perished before the blastocyst stage. This loss of Y chromosome-bearing haploid androgenones should prevent 50% of these embryos from forming blastocysts. The observed rate (12%), however, is far below 50%, indicating an effect of factors other than just an absence of an X chromosome.


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  		TABLE 4. FISH analysis for haploid and diploid androgenetic blastocysta embryos

Gene Expression Analysis of Haploid and Diploid Embryos

Gene expression patterns have never been compared between haploid and diploid embryos. In order to evaluate the degree to which haploid and diploid androgenones and parthenogenones might differ at the molecular level, we compared these 4 types of uniparental embryos to fertilized control embryos at the morula and blastocyst stages for relative abundance of 11 different mRNAs. Five of these mRNAs were autosomally encoded transcripts, comprising a collection of 4 housekeeping mRNAs that are expected to be constitutively expressed (actin, translation elongation factor EF1{alpha}, ribosomal protein L23, and transcription factors Sp1 and TATA box binding protein TBP), and 1 mRNA encoding another transcription factor (mTEAD2) that is developmentally up-regulated at the blastocyst stage and thus may serve as a molecular marker of developmental progression. Another 6 mRNAs analyzed encompassed X-linked transcripts that provided the ability to evaluate X chromosome function in these embryos. The temporal patterns of expression of these genes have been published elsewhere [26, 28, 29, 32, 33].

Among the housekeeping mRNAs, only a few statistically significant differences were observed among the 4 types of uniparental embryos relative to fertilized embryos, and these were observed only in blastocyst stage embryos (Fig. 2). Diploid parthenogenones displayed an approximately 3-fold reduced mean expression of the L23 mRNA compared with fertilized controls. Haploid and diploid parthenogenones displayed a reduced mean expression (34%–45%) of the EF1{alpha} mRNA as compared with fertilized controls (P < 0.05). EF1{alpha} mRNA mean expression also appeared reduced in haploid androgenones, but this difference was just below the level of statistical significance (P = 0.055). The Sp1 mRNA also appeared reduced in both types of parthenogenones, but the difference in means was not statistically significant (P = 0.088 and 0.104). Actin mRNA expression was variably elevated in haploid androgenones, but the difference in means did not reach the level of statistical significance (P = 0.14).



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  		FIG. 2. Comparison of mRNA abundance between haploid and diploid embryos. The expression of 5 constitutively expressed housekeeping mRNAs is shown in the left-hand set of graphs, and the expression of 6 X-linked transcripts is shown in the right-hand set. The vertical line demarcates samples from morulae and samples from blastocyst stage embryos. Data are expressed in units of mean cpm bound (±SEM) of 5–6 samples (7–10 embryos each). Asterisks denote means that were significantly different from the fertilized controls. A, Androgenones; P, parthenogenones; 1N, haploid; 2N diploid; Fert, fertilized embryos. Expression data were normalized to amount of DNA bound to each dot. Diploid morulae and blastocysts were obtained after 76–80 and 96–100 h of development, respectively. Haploid morulae and blastocysts were obtained after 96–100 and 120–126 h of development, respectively

Among the X-linked transcripts analyzed, a number of significant differences were observed (Fig. 2). Haploid androgenetic blastocysts exhibited a significant 7-fold reduction in mean Hprt mRNA abundance relative to fertilized controls (P < 0.02). Expression of the Pdha1 mRNA was reduced approximately 2-fold (P < 0.03) in diploid androgenetic morulae. The mean Pgk1 mRNA abundance was significantly reduced in diploid androgenones at both the morula and blastocyst stages (P < 0.005 and 0.008), consistent with earlier observations [17, 28]. The mean value for Pgk1 mRNA expression in haploid androgenetic blastocysts was 2-fold lower than for fertilized controls. The mean value for haploid androgenones was not significantly different from the mean for fertilized controls, but neither was it significantly different from the mean for diploid androgenones. Thus it appears that Pgk1 expression in haploid androgenones is variably reduced. Expression of the Prps1 mRNA was significantly reduced in haploid and diploid parthenogenones (P < 0.03). The Bex1 mRNA displayed significantly increased mean expression values in all 4 types of uniparental embryos at the morula stage relative to fertilized control embryos, but only in haploid parthenogenones at the blastocyst stage (P < 0.03–0.01). The expression of Xist RNA appeared to be elevated in haploid androgenetic blastocysts relative to diploid androgenetic blastocysts and fertilized controls, but expression was quite variable and the difference in means was not statistically significant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented here confirm earlier reports that haploid mammalian embryos have severely limited developmental potential, even when newer culture media, newer methods of oocyte activation (parthenogenones), and alternative methods of embryo construction (androgenones) are employed. It could be suggested that physical damage related to sperm injection reduced the developmental potential of androgenones. However, diploid androgenone development was comparable to what is observed using the karyoplast fusion technique [15, 16], and haploid androgenone development was approximately equal to that of 1-pronucleate (presumably haploid) androgenones obtained by insemination under conditions of limiting sperm concentration [34]. Moreover, the production of embryos by intracytoplasmic sperm injection of ovulated oocytes results in up to 93% blastocyst formation [35], thus revealing no apparent negative effect of the injection procedure alone. The data also exclude chromosomal abnormalities as a plausible explanation for the limited development of haploid embryos. Thus it seems most likely that the reduced developmental potential of haploid embryos is related to defects in gene expression.

For some genes, haploidy could inhibit accumulation of transcripts to the normal cellular abundance, whereas for other genes aspects of mRNA stability and the modulation of transcription rate may permit accumulation to normal transcript abundance. Because the QADB method employed here measures mRNA abundance as a fraction of the whole mRNA population, alterations in proportional mRNA abundance through such effects should be detectable. Our data failed to reveal for the genes analyzed here simple 2-fold reductions in mRNA abundance that might be expected to arise through such an effect. Moreover, reduction of cytoplasmic volume fails to improve the developmental potential of haploid embryos [2, 8], arguing against a simple explanation related to a DNA:cytoplasm ratio. Our data reveal, however, subtle effects on X-linked gene expression, which indicate that X chromosome imprinting and inactivation may contribute to developmental arrest in haploid embryos. Hprt mRNA expression was significantly reduced in the haploid androgenones at the blastocyst stage. This could reflect either a developmental delay in these embryos, with a resultant delay in accumulation of this mRNA, or a possible imprinting-related paternal X chromosome inactivation in these embryos. Xist RNA expression tended to be elevated in haploid androgenones, which would be consistent with the latter possibility. Although Xist RNA expression was variable and the difference in means was not statistically significant, a similar degree of variability was observed previously [29], even among single embryos (androgenetic and fertilized) genetically determined to be of the XX genotype [17]. Diploid androgenones were previously found to display a consistently reduced abundance of the Pgk1 mRNA regardless of whether they are XX or XY [17, 29]. Diploid androgenones displayed that same feature in this study. Pgk1 mRNA expression was variable in haploid androgenones. Although one explanation for this is that haploid androgenones are deficient in X chromosome inactivation, another possibility is that these embryos initiate the X chromosome inactivation process, but then begin to die soon thereafter. Those haploid androgenones with the greatest degree of Xist RNA expression, Pgk1 gene repression, and repression of other X-linked genes may die within a narrow period of time just before the blastocyst stage, which would be consistent with the fact that the majority of haploid androgenones fail to progress to the blastocyst stage. This would create the variability observed in Pgk1 mRNA and Xist RNA expression and minimize the apparent difference relative to fertilized control embryos among the surviving haploid androgenones. Diploid androgenones may be able to accumulate larger amounts of X-linked transcripts before repression begins (particularly Pgk1 mRNA), which may allow them to progress to the blastocyst stage.

Haploid parthenogenones overexpressed the Bex1 mRNA relative to fertilized control embryos at both the morula and blastocyst stages. The other 3 classes of uniparental embryos overexpressed this mRNA at the morula stage, but not at the blastocyst stage. The initial overexpression of the Bex1 mRNA by all 4 classes of embryos may reflect a developmental delay relative to fertilized control embryos, because the Bex1 mRNA displays a transient peak in expression at the 8-cell stage, with a second increase in expression at the blastocyst stage when its expression becomes trophectoderm-specific [33]. The modest reduction in expression of the EF1{alpha} mRNA in parthenogenones may also reflect such a delay, as this mRNA increases steadily in abundance over the course of preimplantation development [28]. The continued overexpression of the Bex1 mRNA in haploid parthenogenones, however, distinguishes them from diploid parthenogenones, raising the possibility that this mRNA may be overexpressed because of a lack of dosage compensation in the haploid parthenogenones. This effect may be more readily apparent for the Bex1 gene than for some other genes, because the Bex1 mRNA undergoes a strong increase in abundance at the blastocyst stage, in contrast to other X-linked transcripts (e.g., Hprt, Prps1, Pdha1) that actually decrease in relative abundance between morula and blastocyst stages [29]. Bex1 is preferentially expressed in the trophectoderm and may play a regulatory role in commitment of cells to that lineage. The increased expression of Bex1 in haploid parthenogenetic embryos, therefore, may negatively affect development of the inner cell mass in these embryos, which could in turn inhibit development via an insufficiency of trophic factors for the trophectoderm.

Our observations reveal that expression of X-linked genes is altered in haploid embryos. These alterations appear subtle among the surviving embryos that can be collected for analysis, but may be more severe among those embryos that arrest development and degenerate. It is not clear whether such effects at the X chromosome are sufficient by themselves to account for the limited developmental potential in these embryos or merely contribute to their demise. The difference between haploid androgenones and haploid parthenogenones suggests a possible role for autosomal imprinted genes. The striking differences between haploid and diploid androgenones, and between haploid and diploid parthenogenones, however, indicate that any other imprinting effects must be superimposed on other effects of haploidy. Some genes (imprinted or nonimprinted) may be subject to stochastic inactivating events, such as heterochromatization like that observed for position effect variegation [36]. For such loci, the effects of gene silencing would be expected to be more evident in haploid uniparental embryos, as they would be 100% penetrant, whereas a much smaller fraction of diploid uniparental embryos would be affected, as this would require silencing of both gene copies. It would be informative to devise a strategy for producing haploid embryos that possess a combination of both maternal and paternal chromosomes. This would permit a definitive test of to what degree uniparental chromosomal composition contributes to haploid embryo developmental arrest.


   FOOTNOTES
 
First decision: 3 December 2001.

1 Supported in parts by grants from the NIH (RR15253), the Harold K. Castle Foundation, and the Victoria and Bradley Geist Foundation. Back

2 Correspondence: Keith E. Latham, The Fels Institute for Cancer Research, Temple University School of Medicine, 3307 North Broad St., Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}unix.temple.edu Back

Accepted: February 22, 2002.

Received: November 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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S. Hayashi, J. Yang, L. Christenson, R. Yanagimachi, and N. B. Hecht
Mouse Preimplantation Embryos Developed from Oocytes Injected with Round Spermatids or Spermatozoa Have Similar but Distinct Patterns of Early Messenger RNA Expression
Biol Reprod, October 1, 2003; 69(4): 1170 - 1176.
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