Molecular Human Reproduction, Vol. 8, No. 10, 923-929,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Embryology |
Sex-chromosome linked gene expression in in-vitro produced bovine embryos
1 MTT, Agrifood Research Finland, Animal Production Research, Animal Breeding, FIN-31600 Jokioinen, Finland, 2 Department of Biomedical Sciences, University of Guelph, Guelph, ON, N1G 2W1 Canada and 3 Finnish Game and Fisheries Research Institute, PO Box 6, FIN-00721 Helsinki, Finland
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Abstract |
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The expression of XIST, G6PD, HPRT, ZFX and ZFY were investigated in in-vitro produced bovine embryos. Transcripts of these genes were assayed by RT–PCR in pools of pre-compaction stage embryos and sexed pools of morulae and blastocysts. The expression of XIST, G6PD, HPRT and ZFX in female and male morulae and blastocysts were compared using a semi-quantitative RT–PCR. G6PD, HPRT and ZFX transcripts were noted in all pre-compaction stage embryos and in female and male blastocysts. ZFY transcripts were detected in unsexed pools of 8–16-cell stage embryos and in male blastocysts. XIST transcripts were detected in unsexed pools at the 8–16 cell stage, in male and female morulae, and in female blastocysts. The level of XIST RNA was significantly higher in female morulae than in males. Levels of G6PD and HPRT RNA were also higher in female morulae and blastocysts than in males, but only G6PD levels were significantly different between the sexes. The expression of ZFX was also significantly higher in female than in male blastocysts. These results show sexually dimorphic expression of sex chromosome linked genes prior to the blastocyst stage in in-vitro produced bovine embryos.
G6PD/HPRT/sex-related growth rate/XIST/ZFX/Y
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In humans and cattle, male in-vitro produced embryos appear to develop more rapidly than females (Avery et al., 1991
; Xu et al., 1992; Yadav et al., 1993; Pergament et al., 1994; Ray et al., 1995; Bredbacka and Bredbacka, 1996; Carvalho et al., 1996; Gutiérrez-Adán et al., 1996, 1998; Pegoraro et al., 1998; Peippo et al., 2001). In addition, the survival rates for in-vitro produced human and cattle embryos are low (Hardy et al., 1989; Lonergan, 1994; Holm and Callesen, 1998). It has been suggested that embryo survival may be related to the embryo’s ability to maintain cellular homeostasis and respond to its environment (Betts and King, 2001; Edwards et al., 2001). In humans and cattle G6PD and HPRT are X chromosome linked enzymes (Shimizu et al., 1981) that are important for metabolism and contribute to the detoxification of reactive oxygen species (ROS) through their participation in the pentose-phosphate pathway (PPP) (Rieger, 1992; Iwata et al., 1998, 1999; Nicol et al., 2000). The PPP is important for embryo metabolism in generating reduced nicotinamide adenine dinucleotide (NADP) for the synthesis of lipids and other complex molecules, including ribose-5-phosphate, which itself is the precursor of all the nucleotides (Rieger, 1992). G6PD is the rate-limiting enzyme for PPP activity, which has been reported to be four times greater in female than male bovine blastocysts (Tiffin et al., 1991). It has been suggested that glucose concentration in embryo culture medium associated with ROS production is one of the factors that could influence embryo development in vitro (Rieger, 1992; Bredbacka and Bredbacka, 1996; Gutiérrez-Adán et al., 1998; Peippo et al., 2001).
Female and male mammals, by virtue of their sex chromosome make-up, differ in the complement and number of copies of genes on the sex chromosomes. To compensate for the unequal X-linked gene dosage, females undergo the process of X inactivation early in embryo development, thereby rendering one of the two X chromosomes inactive. The inactive X chromosome becomes heterochromatic and hypoacetylated (Jeppesen and Turner, 1993), replicates late in the S-phase of the cell cycle (Takagi and Oshimura, 1973) and, with the exception of a few genes that escape inactivation, it is transcriptionally quiescent (Graves and Gartler, 1986). The X inactivation is a multi-step process that involves counting of X chromosomes, selection of an X chromosome to remain active and initiation of inactivation of the X chromosome (or X chromosomes in the case of individuals with more than two X chromosomes) to be silenced (Lyon, 1991). In humans, the process of inactivation is thought to be controlled by the X inactivation centre (XIC) and the resident gene X-inactive specific transcript, referred to as XIST (Brown et al., 1991a). XIST RNA lacks an open reading frame and remains as a non-coding nuclear RNA, and X inactivation spreads from its site of transcription to cover nearly the entire length of the X chromosome chosen to become inactive (Brockdorff et al., 1992; Clemson et al., 1996). In cattle, which have been shown to be a good model species for human embryo development (Ménézo et al., 2000, Neuber and Powers, 2000), XIST has been detected at very low levels using nested PCR at the 2-cell stage with increasing amounts detectable by single round PCR at the 8-cell stage on day 3 of in-vitro development of embryos (De La Fuente et al., 1999) at the time of activation of the embryonic genome (Telford et al., 1990). However, late replication of the inactive X chromosome was detected first at the early blastocyst stage on day 7 of in-vitro development (De La Fuente et al., 1999). The percentage of female embryos displaying a late replicating X chromosome and the number of cells per embryo that show this feature increase as the stage of development advances (De La Fuente et al., 1999) suggesting that there is embryo to embryo variation as well as cell to cell variation in the progression and status of X inactivation.
Since X inactivation is thought to be initiated and spread from the XIC (Lyon, 1991), the levels of expression of genes located on the X chromosome at different distances from the XIC could provide valuable insight into the X-inactivation-mediated dosage compensation in embryos. HPRT is known to be closer to the XIC than G6PD on the human X chromosome (Brown et al., 1991b; http://bos.cvm.tamu.edu/htmls/HSA-X.html). The XIST gene maps to the region of the XIC on the long arm of the human X chromosomes (Brockdorff et al., 1991; Brown et al., 1991b) and the locus for the zinc finger protein (ZFX) is mapped to the distal end of the long arm of the bovine X chromosome (Xiao et al., 1998). It has been suggested that timing of methylation, a mechanism involved in X inactivation, may be a function of the distance from XIC and thus of the ‘spreading’ of X chromosome inactivation (Grant et al., 1993).
The aim of the present study was to examine the expression of XIST in early bovine embryos in relation to that of other X-linked loci. Two of the X-linked loci selected, G6PD and HPRT, are known in humans to be subject to inactivation, while another, the ZFX locus, escapes inactivation. The levels of expression of XIST, G6PD, HPRT and ZFX are compared by semi-quantitative RT–PCR in order to determine whether their expression differs in female and male bovine morulae and blastocysts.
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Materials and methods |
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Embryo production
Embryos were produced by a standard IVP protocol as previously described (De La Fuente et al., 1999
) with minor modifications. In brief, 100–125 cumulus-oocyte complexes (COCs) recovered from the ovaries of slaughtered cows were incubated for maturation in 750 µl of maturation medium. After 24 h of maturation, the COCs were washed three times in Talp-hepes and once in IVF-talp before 100–125 COCs were transferred into 500 µl drops of IVF medium supplemented with 20 µg/ml of heparin. Spermatozoa from one straw of a Holstein bull of proven in-vitro fertility was thawed and washed twice with 4.0 ml of sperm-talp at room temperature. After counting, spermatozoa at a final concentration of 1x106 were added to IVF drops and co-incubated for 20 h. Cumulus cells were then removed by vortexing for 90 s in warm Talp-hepes or by vigorous pipetting in the IVF drop. After three Talp-hepes and one IVC-medium wash, 20–25 presumptive zygotes were transferred into 50 µl IVC preconditioned drops containing bovine oviduct epithelial cells (BOEC). Fresh culture medium (25 µl) was added to each IVC drop on day 4 of culture. In-vitro maturation, fertilization and culture were performed at 39°C in 5% CO2 in air.
Embryo samples for gene expression profiles
In the first part of this study, the presence of XIST, G6PD, HPRT, ZFX and ZFY mRNAs during preimplantation stages was assessed using RT–PCR. ß-actin, a gene located on an autosome, was used as a control for the quality of the cDNA sample. Embryos for RT–PCR were collected at 32, 44 and 96 h after exposure to sperm (hpi) and on day 8 of embryo culture, when the development had reached the 2-cell, 4–8-cell, 8–16-cell and the expanded-hatched blastocysts stages respectively. Immature and mature oocytes were also collected and pooled for analysis (Table I). Somatic cells still attached to the zona pellucidae were removed from the oocyte and embryo pools by vortexing or vigorous pipetting in Ca2+ and Mg2+ free phosphate buffered saline (PBS), supplemented with polyvinyl pyrrolidone (PVP) or by removing the zona pellucidae with acid Tyrodes’ solution (pH 1.8) followed by five washes in PBS+PVP. The removal of cumulus cells from immature and matured oocytes was facilitated by the use of 0.1% hyaluronidase (in Ham’s F-10 medium) during prolonged (5 min) vortexing. All embryos were examined for the presence of cumulus cells under a stereomicroscope before transferring them into 1.5 ml eppendorf tubes for freezing in liquid nitrogen and storage at –70°C. Unsexed, intact embryos were pooled and frozen while biopsied embryos were frozen individually. During RNA extraction the sexed day-8 blastocysts were pooled according to sex to increase the amount of RNA obtained.
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RNA quantification
In the second part of this study, day 5–6 morulae and day 8–9 blastocysts were biopsied using a microblade to remove approximately one third of the embryo diameter. Day 5–6 morulae were incubated before biopsy in Ca2+ and Mg2+-free PBS (supplemented with PVP) in order to loosen junctions between blastomeres to minimise cell membrane damage during manipulation. Biopsies were processed for sex determination by RFLP analysis of ZFX/ZFY as previously described (Aasen and Medrano, 1990
; Bredbacka and Peippo, 1992). Embryos were frozen individually and 20 morula stage embryos of each sex were pooled for RNA extractions, whereas three blastocysts of each sex were pooled into one RNA sample. Total RNA was extracted using the RNeasy mini kit (Qiagen) according to manufacturer’s instructions. All samples were treated with DNase (Gibco) for 20 min at room temperature to eliminate any remaining DNA within the samples. The extracted RNA was used for cDNA synthesis using equal amounts of random and oligo-dT primers (500 ng) at 43°C for 2 h with (RT+ve) or without (RT–ve) reverse transcriptase (Superscript, Gibco). After superscript inactivation at 94°C for 5 min, cDNA samples (20 µl in total) were stored at –20°C.
From each RT+ve and RT–ve pool 2.5 µl was used for a 50 µl PCR-reaction (Table II). The resulting PCR products were subjected to electrophoresis using a 2% agarose gel in TAE-buffer containing 0.5 µg/ml ethidium bromide. Bands were visualized and photographed under UV light illumination.
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The levels of mRNA in female and male embryo samples were compared by a semi-quantitative approach using a Quantum RNATM kit (Ambion). Briefly, the linear range for PCR amplification of each specific cDNA was determined by analysing the PCR product after every second amplification cycle between cycles 20–40. The gels were stained with SYBR Green I and the optical density (OD) of each band was estimated using the FMBIO II Image Analysis system (Hitachi Software Engineering Co. Ltd.). The OD value of each sample was plotted against the corresponding cycle number. By using the optimal cycle number (mid-point in the linear range), the optimal 18S rRNA primer:competimer ratios (1:9, 2:8, 3:7 or 4:6 by volume) were determined by separate co-amplification of each 18S:competimer ratio with each specific primer pair. Since 18S rRNA is abundant in embryonic cells, competimers were included into the reaction to adjust the amplification of 18S to the same level as the gene of interest. The 18S to the competimer ratio, at which the intensity of the gene specific bands was closer to that of 18S was selected for analysis of each gene. The RT+ve and RT–ve samples of female and male day 5–6 morulae and day 8–9 blastocysts were analysed using the optimal cycle number and the 18S primer:competimer ratio with each specific primer pair. The amount of cDNA per PCR reaction was 2.5 µl for blastocysts, but was increased to 3.75 µl for morulae. The relative OD value was calculated as the ratio of the OD for gene specific amplicon and OD for 18S co-amplified within each PCR reaction.
Statistical analyses
The relative OD values from semi-quantitative RT–PCR were analysed using analysis of variance. A total of three female and three male pools (referred to as an experimental replicate) were used in the morulae study. The PCR amplification was repeated twice and the OD acquisition was conducted once for each experimental replicate. This design forms a hierarchical design with the experimental replicate and PCR amplification (twice) as random factors and the gene and sex as fixed factors. The main interest was in the differences between sexes within genes of interest.
In total nine female and nine male pools (experimental replicates as above) were used in the blastocyst study. The PCR amplification was repeated twice and within both studies the OD measurements were conducted twice for each experimental replicate. This design also forms a hierarchical design where the experimental replicate, PCR amplification (twice) and the OD acquisition (twice) are random factors and the main interest is focused on the differences between sexes within the relative expression of the genes of interest.
Both studies were analysed with PROC MIXED in SAS (Little et al., 1996; SAS, 2000). The variance–covariance structure was estimated with a ‘variance component’ structure at the experimental replicate, PCR amplification and the OD acquisition levels. In both of the analyses, Kenward–Roger’s approximation was used for determining the correct standard error estimate and degrees of freedom (SAS, 2000). No adjustments were carried out in the pair-wise comparisons after analysis of variance between sexes within genes due to predetermined hypothesis.
A one-tailed null hypothesis was applied, because females with two copies of each gene were expected to express more than males and consequently the P-values were divided by two. A P-value <0.05 was considered significant.
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Results |
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Detection of transcripts
Transcripts of ß-actin, G6PD, HPRT and ZFX were detected in immature and mature oocytes and at all embryo stages (2-cell, 4–8-cell, 8–16-cell, morulae and blastocysts) tested—while no amplification was detected in the corresponding RT–ve samples. ZFY and XIST transcripts were detected in pools of 8–16-cell stage embryos but not in 2-cell and 4–8-cell stage pools. The results are summarized in Table III
. Among the sexed embryos at the blastocyst stage, ZFY expression was detected exclusively in males and XIST exclusively in females. However, in embryos at the morulae stage, XIST RNA was detected in females and males. Amplified fragments of ZFX were sequenced from the 2-cell stage embryos and pooled female blastocysts. Sequence analysis of the PCR products for ZFX using the BLAST program showed 99% homology to the bovine ZFX sequence (Xiao et al., 1998).
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Quantification of transcripts
In both morulae and blastocysts no significant differences were found between repeated PCR amplifications and between repeated OD measurements, and therefore these random factors were left out of the final analysis. Also, the OD of the internal standard 18S rRNA was not affected by the sex of the sample, i.e. the sexxOD of the internal standard-interactions were not found to be statistically significant with any of the genes and therefore this term was also left out of the final analysis.
Three pools each of female and male morulae were analysed for the levels of RNA transcribed by XIST, G6PD and HPRT loci. For each specific primer pair the optimal 18S:competimer ratio was 4:6. One pool of male morulae consisted of only 13 biopsied embryos instead of 20, due to the low numbers of male morulae available for RNA extraction. For semi-quantitative PCR from this sample, 6.0 µl of cDNA was used to equalise the amount of RNA in the embryo samples tested. Two replicates from each female and male embryo sample were analysed for G6PD and HPRT, but only one from the male sample with the reduced number of embryos and the resultant low amount of cDNA available. For the same reason, only one PCR run was performed for comparison of expression of XIST between the sexes. Observed levels of XIST, G6PD and HPRT relative to 18S in female and male morulae replicates are illustrated in Figure 1. The estimated mean values for G6PD and HPRT transcripts were 13% (P = 0.0147) and 6% (P = 0.1096) higher, respectively, in females than in males. XIST expression (based on one semi-quantitative RT–PCR) was 62% (P = 0.0317) higher in female samples compared with male samples of morula stage embryos (Figures 1, 2a and 3a).
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Nine pools (three blastocysts/pool) of female and male embryos were each analysed for the expression of G6PD, HPRT and ZFX, and six were each analysed for the expression of XIST at the blastocyst stage. Each quantification step, starting from the PCR, was repeated twice in order to evaluate the repeatability of the method. Observed levels of G6PD, HPRT and ZFX relative to 18S in female and male blastocysts are illustrated in Figure 1
. At the blastocyst stage, XIST was expressed in female samples only (Figures 1, 2b and 3b). Estimated relative OD of G6PD and HPRT transcripts were 24% (P = 0.0263) and 8% (P = 0.1109) higher, respectively, in female blastocysts compared with male blastocysts (Figure 3b). The relative OD of ZFX was 36% (P = 0.0331) higher in female blastocysts compared with their male counterparts. No amplification was observed in RT–ve samples. |
Discussion |
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In this study, G6PD, HPRT and ZFX transcripts were detected in bovine embryos at all stages of development tested. Since the RT–PCR method used cannot distinguish between maternal and embryo-derived transcripts it could not be determined when transcription of G6PD, HPRT or ZFX from the embryonic genome first occurs. In order to distinguish maternal (i.e. oocyte-derived) and embryo-derived transcripts, coding sequence polymorphism recognised, for example, with restriction endonucleases is required (Taylor et al., 1997
). Unfortunately, such polymorphisms for the genes investigated here have yet to be identified in cattle. The level of G6PD and ZFX was significantly higher in female than in male embryos, but HPRT levels were similar in both sexes. The embryo-derived expression of ZFY and XIST, on the other hand, was detected in pools of 8–16-cell stage embryos and at the morula and blastocyst stages, but not in pools of 2- and 4–8-cell stage embryos. At the blastocyst stage the expression of ZFY was noted exclusively in male embryos, whereas the expression of XIST was confined to the female embryos. The level of ribosomal 18S RNA, the internal standard, did not differ between female and male embryos at the morula or blastocyst stages.
XIST expression in female embryos is expected to parallel the initiation of dosage compensation. As such, the level of expression of G6PD and HPRT, which are subject to dosage compensation, should not be significantly different between the sexes when XIST expression is detected in female embryos. However, since X inactivation is spread over several cell cycles (De La Fuente et al., 1999) and occurs at different times in different cell lines (Monk and Harper, 1979), the equalization of transcript levels is expected to occur gradually. In addition, this equalization between the sexes may be slowed down by the overall increase in cell number from morula to late blastocyst stage, which is reported to be greater in male embryos compared with female embryos in vitro (Yadav et al., 1993; Ray et al., 1995). Nonetheless, it is expected that the differences in expression levels between males and females should become less as development progresses. On the other hand, there should be no sign of dosage compensation evident in the expression of ZFX, which is reported to escape X inactivation in cattle (Farazmand, 2000) and as such ZFX levels should remain higher in females.
However, the results of this study revealed differences from those expected in the expression of these genes at the morula and blastocyst stages. For example, similar levels of HPRT transcripts were detected in pools of male and female morulae and blastocysts 2 or 3 days after the expression of XIST was detected and 1 or 2 days earlier than the time at which the late replication of the inactive X chromosome has been previously detected (De La Fuente et al., 1999). In contrast, the levels of transcripts for G6PD were significantly higher in females than in males in pools of morulae and blastocysts. The difference between males and females was greater at the blastocyst stage than at the morula stage. Furthermore, contrary to expectation, XIST transcripts was detected in male embryos, although a significantly higher level of expression was noted in female embryos of corresponding age (P = 0.0317).
In the present study the level of G6PD was significantly higher in pools of female bovine in-vitro produced embryos than males at both the morula and blastocyst stage. Although the levels of HPRT were higher in female morulae and blastocysts than in males, in contrast to the levels of G6PD, they did not differ significantly. The level of G6PD mRNA has been shown to be similar between male and female 2-cell bovine embryos before activation of the embryonic genome (Lonergan et al., 2000), but higher in females than males at the blastocyst stage (Gutiérrez-Adán et al., 2000; Lonergan et al., 2000). The level of HPRT mRNA has also been shown to be similar in male and female in-vitro produced bovine 2-cell embryos (Lonergan et al., 2000), but in contract with the present study, it has been shown to be significantly higher in female and male blastocysts (Gutiérrez-Adán et al., 2000; Lonergan et al., 2000). These observations might suggest that the processes of dosage compensation for HPRT have occurred by the morula stage, but for G6PD the process was incomplete. The presence of XIST would suggest that inactivation of the X chromosome had begun. Since the inactivation begins at the XIC and spreads along the X chromosome chosen for inactivation (Lyon, 1991) those loci such as HPRT that are close to the XIC would show signs of inactivation and dosage compensation earlier that those further away, such as G6PD. The methylation pattern of mouse X chromosome supports this hypothesis of linear spreading of X inactivation from the XIC (Goto and Monk, 1998).
However, the increase rather than decrease in the difference between male and female levels of mRNA for HPRT and G6PD as the embryos developed from morula to blastocyst noted here, and the effects of embryo production regimens of HPRT and G6PD expression reported by others (Gutiérrez-Adán et al., 2000; Lonergan et al., 2000; Lucas-Hahn et al., 2001) might suggest that factors other than X inactivation may be involved in the equalization of expression of HPRT between females and males. The stability and turnover rate for HPRT mRNA in bovine embryos is not known. It has, however, been shown that the messenger stability associated with the polyadenylation of the transcript is related to developmental potential in bovine oocytes (Brevini-Gandolfi et al., 1999) and that levels of HPRT transcripts are higher in embryos of known high developmental capacity in some in-vitro production systems (Lonergan et al., 2000). Further, in the mouse, it has been shown that the expression of HPRT is more intense from the maternal than the paternal X chromosome (Moore and Whittingham, 1992). Hence the similar levels of HPRT transcripts in morulae and blastocysts could reflect message stability, low metabolism of HPRT and low transcription levels of transcription from the paternal X chromosome in female embryos rather than dosage compensation due to X chromosome inactivation. In the case of G6PD, Luca-Hahn and co-workers (2001) reported that while the levels of G6PD were higher in female than male in-vitro produced bovine blastocysts, the levels were similar in males and females produced in vivo (Lucas-Hahn et al., 2001). This would suggest that either the timing of X inactivation or the synthesis and accumulation of transcripts differ between in-vivo and in-vitro produced embryos and hence are subject to environmental influences.
The ZFX locus is also mapped to the X chromosome in mammals, including cattle, (Page et al., 1987; Xiao et al., 1998) and its male homologue to the sub-pseudoautosomal region of the Y chromosome. The functions of these two genes are not fully understood as yet, although their presence has been shown to be essential for embryo survival and the maintenance and function of germ cells (Luoh et al., 1997). The expression of ZFY was detected in bovine embryos at the 8–16 cell stage of development, which coincides with the beginning of major genomic activation (Telford et al., 1990). The ZFX is one of the many genes on the X chromosome that escape X inactivation in humans (Schneider-Gädicke et al., 1989) and cattle (Farazmand, 2000). It was, therefore, anticipated that the levels of ZFX RNA would differ significantly between males and females and that this transcript would serve to confirm the validity of the semi-quantitative approach used in this study. Our results indeed show that the level of ZFX expression is higher in female than in male blastocysts (P = 0.0331).
Our observations on the expression of XIST in female embryos are in agreement with its reported exclusive expression from the inactive X chromosome and its role in the X inactivation process (Brown et al., 1991a). Expression of XIST is reported to precede the cytological sign of inactivation observed at the late blastocyst stage (De La Fuente et al., 1999). However, the detection of XIST in pools of male embryos is in contrast to this and, viewed in the light of dimorphic levels of X-linked transcripts that persist at the blastocysts, places the role of XIST as a forerunner of X chromosome inactivation in developing embryos in some doubt. It is, however, conceivable that in our study some female embryos were unwittingly included among the pooled male embryos due to the limitation of the sex detection method used in this study. Alternatively, male pools could have included male embryos with an extra X chromosome, which would be subject to X inactivation. Chromosomal abnormalities occur in one or more cells in up to 75% of bovine embryos produced in vitro (Kawarsky et al., 1996; Lechniak, 1996; Viuff et al., 2000). Another possibility is that the male embryos were contaminated by undetected cumulus cells. However, this is unlikely, since the chance of cumulus cells being attached to the zona pellucida for 5 or 6 days after fertilization and remaining undetected in replicate runs is remote. A more plausible explanation for our observation is that the RT–PCR product was transcribed from normal male bovine embryos. XIST RNA has been detected in human male zygotes (Daniels et al., 1997), cleavage stage embryos (Daniels et al., 1997) and blastocysts of both the human (Ray et al., 1997) and the mouse (Lee et al., 1999). It was suggested that since XIST was detected in both male and female embryos, it may not play a role in the choice of X chromosome to be inactivated (Daniels et al., 1997). More recently, it has been suggested that the XIST transcribed from the maternal X chromosome in the male mouse blastocyst in fact includes its antisense form, which is not distinguishable from XIST by conventional PCR (Lee et al., 1999), and plays a role in the choice of active and inactive X chromosomes (Lee, 2000). While it is beyond the scope of this study to resolve whether or not XIST plays a role in inactivation of one of the X chromosomes or in the choice of the X chromosome to remain active, it is clear that in the cow, as in humans and mice, XIST is present in both male and female pre-hatching embryos.
In summary, this study demonstrates a dimorphic expression of some but not all genes located on the X chromosomes during development of in-vitro produced bovine morulae and blastocysts. It is suggested that the equalization of levels of mRNA detected in males and females may be influenced by factors not necessarily related to inactivation of one of the X chromosomes in females.
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Acknowledgements |
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We extend our gratitude to Ed R.Reyes, Elizabeth St John, Dorota Lechniak, Dean Betts and Harpreet Kochhar for their assistance and to Brad Cooney for production of primers and for sequencing of PCR products. Angela Hollis is acknowledged for helping with the OD measurements. Funding was provided by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. Jaana Peippo was also a recipient of a scholarship from The Finnish Indigenous Cattle Breeding Society.
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Notes |
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4 Present address: Leiras OY, Research & Development, Clinical Research, PO Box 415, FIN-20101 Turku, Finland
5 To whom correspondence should be addressed. E-mail: jaana.peippo{at}mtt.fi
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References |
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Submitted on June 25, 2001; resubmitted on March 1, 2002; accepted on July 23, 2002.
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