BIOLOGY OF REPRODUCTION 60, 769–775 (1999)
X Inactive-Specific Transcript (Xist) Expression and X Chromosome Inactivation in the Preattachment Bovine Embryo 1 Rabindranath De La Fuente, Ann Hahnel, Parvathi K. Basrur, and W. Allan King2 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1 ABSTRACT
The inactive X chromosome displays several characteristics that are useful for its identification: it is heterochromatic and hypoacetylated [9–11]; it replicates later in the cell cycle than other chromosomes [10, 12]; and except for genes that escape inactivation, loci on the inactive X chromosome are transcriptionally silent [8]. Cytogenetic and biochemical analyses of the pattern of X chromosome inactivation in the mouse embryo [1, 13] have shown that at the blastocyst stage (Days 3.5–4 of development), the paternal X chromosome in the trophectoderm becomes late replicating and transcriptionally inactive [14, 15], while in the embryonic ectoderm, X inactivation occurs later (on Day 6.5 of development) and involves either the paternal or maternal X chromosome [16]. Molecular events leading to the inactivation of the X chromosome are not fully understood. However, cytogenetic and molecular evidence suggests the presence of an X inactivation center (XIC) that participates in the initiation (and spreading) of inactivation in one of the X chromosomes [17–20]. The human X inactive-specific transcript (Xist), and its murine homologue (Xist) exclusively transcribed from the inactive X chromosome, map to XIC, suggesting its possible role in the process of X chromosome inactivation [18, 19, 21]. Studies showing that Xist expression is a prerequisite for dosage compensation in vitro and during early mouse embryo development are consistent with a role for Xist in the initiation of X chromosome inactivation [22–24]. Comparison of Xist sequence in a range of mammals revealed a highly conserved region at the 59 end [25], providing a molecular tool for further characterization of this phenomenon in species other than the human and mouse. However, despite knowledge of the XIC and the considerable agricultural interest in embryo sex identification based on X-linked gene activity, information on X chromosome inactivation during embryogenesis in domestic animals is scanty. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) was used to test for Xist expression in bovine somatic tissues and preattachment embryos and to determine the temporal relationship of Xist expression to bovine X chromosome inactivation.
Expression of the X inactive-specific transcript (Xist) is thought to be essential for the initiation of X chromosome inactivation and dosage compensation during female embryo development. In the present study, we analyzed the patterns of Xist transcription and the onset of X chromosome inactivation in bovine preattachment embryos. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of Xist transcripts in all adult female somatic tissues evaluated. In contrast, among the male tissues examined, Xist expression was detected only in testis. No evidence for Xist transcription was observed after a single round of RT-PCR from pools of in vitroderived embryos at the 2- to 4-cell stage. Xist transcripts were detected as a faint amplicon at the 8-cell stage initially, and consistently thereafter in all stages examined up to and including the expanded blastocyst stage. Xist transcripts, however, were subsequently detected from the 2-cell stage onward after nested RT-PCR. Preferential [3H]thymidine labeling indicative of late replication of one of the X chromosomes was noted in female embryos of different developmental ages as follows: 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11 (72.1%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts. These results suggest that Xist expression precedes the onset of late replication in the bovine embryo, in a pattern compatible with a possible role of bovine Xist in the initiation of X chromosome inactivation.
INTRODUCTION
The sex chromosome complement of the mammalian conceptus is determined at fertilization, when an oocyte carrying the X chromosome is fertilized with either an Xor a Y-bearing sperm. Female embryos carrying two X chromosomes could potentially produce twice the amount of X-linked enzymes relative to the male embryos with only one X chromosome [1]. However, during early embryonic development, dosage compensation for X-linked enzymes takes place through the inactivation of one of the two X chromosomes of the female conceptus [2]. This process, thought to have evolved approximately 150 million years ago, is highly conserved among mammals [3] and is essential for embryogenesis [4–6]. Unfortunately, the lack of data and specialized reagents for species other than humans and mice has limited our understanding of the extent to which this process has been conserved among mammalian species [7]. Indeed, differences between the human and murine X chromosome have been observed with regard to the number and type of genes that escape inactivation [8], emphasizing the need for studies in other mammals.
MATERIALS AND METHODS
In Vivo Bovine Embryo Collection
Holstein-Freisian cows were superovulated with 3.0, 2.5, 2.0, and 1.5 mg Folltropin (Vetrepharm, Willowdale, ON, Canada) administered twice daily as i.m. injections starting on Day 9 of the estrous cycle (day of estrous 5 Day 0), followed by 2 injections (i.m.; 12 h apart) of Cloprostenol (Estrumate; Schering Canada, Inc., Pointe-Claire, PQ) on the third day of treatment. Artificial insemination with frozen-thawed semen was performed twice, at a 12-h interval, after estrus detection. Embryos were recovered on Days 14 and 15 of development by a transcervical uterine flush as
Accepted October 27, 1998. Received July 22, 1998. 1 This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada, Cattle Breeding Research Council, and the Ontario Ministry of Agriculture, Food and Rural Affairs. R.D. was a recipient of a Government of Canada Award. 2 Correspondence. FAX: 519 767 1450; e-mail:
[email protected]
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TABLE 1. Number of embryos at each stage of in vitro development used for RT-PCR amplification of bovine Xist.* 2–4 Cell 65 68
8 Cell
8–16 Cell
Morula
Blastocyst
Expanded blastocyst
75 65
65 40
38 65
57 25
48 25
* From two independent replicates.
TABLE 2. Nucleotide sequence of bovine Xist primers. Gene Bovine Xist Nested bovine Xist*
Primer sequence P133: P596: P190: P459:
59-AGCATTGCTTAGCATGGCTC-39 59-TGGCTGTGACCGATTCTACC-39 59-TTGCCGCAGTGTTCCAATGG-39 59-CCGCCAAGAATGTAACGGTC-39
* Primers used for nucleotide sequencing of amplified Xist PCR products and second-round PCR.
described by Betteridge [26]. Elongated blastocysts were transferred into holding medium consisting of PBS supplemented with 1.0 mg/ml glucose, 0.036 mg/ml sodium pyruvate, and 10 mg penicillin-streptomycin at a final concentration of 10 000 U/ml penicillin G, and 10 000 mg/ml streptomycin (Canadian Life Technologies, Burlington, ON, Canada), 4 mg/ml BSA fraction V (Sigma Chemical Co., St. Louis, MO), and 200 ml/L fetal calf serum for classification and measurement. A biopsy of approximately 2 mm in length was taken from the tip of each trophoblast and transferred to 1.0 ml of in vitro culture medium (IVC) consisting of tissue culture medium (TCM)-199 (Canadian Life Technologies) supplemented with 10% steer serum (SS), 0.35% BSA, 0.2 M sodium pyruvate (Sigma), 0.5% penicillin-streptomycin, 50 mg/ml gentamycin, and 0.2 M Lglutamine (Sigma) for incubation in an atmosphere of 5% CO2 in air at 398C.
of granulosa cells were removed by vigorous pipetting before transferral of pools of 40–75 embryos in 5 ml of PBS 1 0.1% PVP medium into microcentrifuge tubes. Tubes were plunged directly into liquid nitrogen and stored at2708C until RNA extraction. The number of embryos used for RNA extraction at each stage of development is shown in Table 1. RNA from groups of embryos was extracted as described by Hahnel et al. [28]. DNase treatment was performed as described by Gaudette et al. [29] in 50 ml of DNase buffer (2 M NaCl, 1 M Tris, pH 8.0, 1 M MgCl2, 0.1 M CaCl2). Total RNA from adult somatic tissues and embryos was digested for 15–20 min with 5 units of RQ1 DNase (Promega Corp., Madison, WI) at 378C, followed by a Tris-saturated phenol:chloroform extraction and ethanol precipitation.
In Vitro Bovine Embryo Production
RT-PCR
Bovine embryos were produced by in vitro oocyte maturation, fertilization, and culture as previously described [27]. Cumulus-oocyte complexes were obtained by follicular aspiration and collected into Ham’s F-10 medium (Canadian Life Technologies) supplemented with 2.0% SS, 1.0% Hepes buffer (Canadian Life Technologies), 1.0% NaHCO3 (Fisher Scientific, Nepean, ON, Canada), 2 IU/ml heparin (Organon Teknica, Toronto, ON, Canada), and 1.0% penicillin-streptomycin maintained at 378C. In vitro maturation was carried out for 22–24 h, at 398C in a humidified atmosphere of 5% CO2 in air, in Hepes-buffered TCM-199 supplemented with 0.2 M sodium pyruvate, 0.2 M L-glutamine (Sigma), 0.5% penicillin-streptomycin, and 10% SS, under silicone oil (Fisher). Cumulus cells were removed from cumulus-oocyte complexes by vigorous pipetting in 3 ml Hepes-buffered Tyrode’s albumin lactate pyruvate (TALP) medium and rinsed in TALP supplemented with 20 mg/ml heparin (IVF-TALP). Twenty oocytes were transferred into a 95-ml droplet of IVF-TALP containing 5 ml of bovine oviductal epithelial cell (BOEC) suspension under silicone oil. Approximately 1 3 106 sperm/ml were added to the 95-ml droplets containing oocytes. At 18 h postinsemination, presumptive zygotes were washed twice in 1 ml IVC medium and cocultured with BOEC (5-ml suspension) in 50 ml IVC medium for 8 days in a humidified atmosphere of 5% CO2 in air at 398C. RNA Extraction from Adult Tissues and Preattachment Embryos
Total RNA was extracted from male and female adult tissues and from the chorioallantois of a Day 90 female fetus with a combination of phenol and guanidine isothiocyanate procedure (Trizol) according to specifications of the manufacturer (Canadian Life Technologies). Preattachment embryos at various stages (from the 2-cell to the hatched blastocyst stage) were washed four times with PBS supplemented with 0.1% polyvinylpyrrolidone (Sigma). Remnants
RNA samples were divided into two aliquots for RT; one was used in the presence of reverse transcriptase and the other in the absence of reverse transcriptase as a control for genomic DNA contamination. Complementary DNA synthesized from 1 mg of total RNA extracted from tissues, and all embryo RNA extracts, were primed with 25 mg/ml oligo-dT (New England Biolabs, Boston, MA) in 4 ml (5strength) first strand buffer (Canadian Life Technologies), 10 mM dithiotreitol (Canadian Life Technologies), 0.2 mM dNTPs, 0.5 U/ml RNasin (Promega Corp.), and 200 U Maloney murine leukemia virus reverse transcriptase (Superscript II; Canadian Life Technologies). RT was carried out at 458C for 1 h. Total cDNA was diluted with 30 ml doubledistilled H2O and stored at2208C. Primers were designed to amplify a 463-base pair (bp) PCR product from the 59 region of bovine Xist (a 650-bp nucleotide sequence) described by Hendrich et al. [25]. Primer sequences (59–39) for upstream primer P133 and downstream primer P596 are illustrated in Table 2. Primers that cross-react with bovine b- and g-actin forms [30] were used as positive controls for the presence of cDNA. In all, 1 ml cDNA from adult tissues or 5 ml of embryo cDNA per 50-ml reaction mixture was used for amplification of actin transcripts. For the analysis of Xist gene expression, 1 ml cDNA from adult tissues or 30 ml of embryo cDNA per 50-ml PCR reaction mixture was used. Sequence amplification from cDNA samples was performed according to Kay et al. [22]. In brief, a 50-ml PCR reaction mixture consisting of 0.2 mM dNTPs, 0.50 mM primers, 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 0.1% Triton X, and 1.25 U Taq DNA polymerase (Promega) was heated to 958C and held for 5 min; this step was followed by 30 cycles of denaturation at 958C for 1 min, annealing at 558C for 1 min, and extension at 728C for 2 min with a final extension step at 728C for 15 min. PCR products were resolved in 2% agarose gels and photographed under ultraviolet light. As a control for contamination, a blank lane consisting of PCR reaction mixture
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with double-distilled water instead of cDNA was included in each gel. For the second-round PCR reaction in embryo samples, nested primers (Table 2) that amplify a 283-bp sequence internal to the first-round primers in a 50-ml PCR reaction mixture, with cycling conditions exactly as described above, were used with the exception that 1 ml firstround PCR product was used as a template. Purification and Sequencing of PCR Products
PCR products obtained after amplification of cDNA samples from somatic tissues and preattachment embryos were gel purified with a Qiaquick DNA purification kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer’s specifications. Sequencing was performed using 5 ml of DNA template with 2 pmol/ml of nested primers (Table 2) by the method of dye terminator labeling in an ABI 377 Prism automated sequencer (Guelph Molecular Centre, Guelph, ON, Canada). Sequence identity was determined with the basic local alignment search tool (BLAST) algorithm [31]. Autoradiography
The late-replicating X chromosome was identified by the preferential deposition of silver grains on the X chromosome of cells after [3H]thymidine incorporation and autoradiography. In vitro-produced morulae, early blastocysts, blastocysts, expanded blastocysts, and hatched blastocysts were incubated in IVC medium supplemented with 2 mCi/ ml [3H]thymidine (Amersham, Oakville, ON, Canada) for a 4-h period, washed twice in freshly prepared culture medium, and incubated in the presence of 0.05 mg/ml Colcemid (Canadian Life Technologies) for an additional 4 h. Embryos were exposed to a hypotonic solution (1.0% sodium citrate) for 4 min and individually fixed on glass slides with methanol:acetic acid [32]. After staining of the slides with 4% buffered Giemsa, a total of 62 embryos identified as females were selected, and metaphases were photographed before destaining and processing of the slides for autoradiography [32]. Radiolabeled slides were dipped into NTB2 Kodak emulsion (Kodak Tetrachem, Rexdale, ON, Canada) maintained in a water bath at 408C and were air dried in a dark room for 3–4 h, stored in light-proof boxes, and maintained at 48C for 24 h. The slides were developed in D-19 (Kodak Tetrachem) for 3 min and stained with 4.0% buffered Giemsa. Silver grain deposition indicative of labeled thymidine incorporation was examined under a Leitz Aristoplan (Leitz Wetzlar GBH, Wetzlar, Germany) light microscope at 3100 objective. Preferential deposition of silver grains on one of the X chromosomes was considered to be indicative of a late-replicating X chromosome. A group of 14 female embryos at the elongated blastocyst stage (nine Day 14 and five Day 15) were cultured in IVC medium supplemented with 2 mCi [3H]thymidine (Amersham) for a period of 4 h followed by a further incubation for 2 h in the presence of Colcemid (0.05 mg/ml; Canadian Life Technologies). After a hypotonic treatment for 10–15 min in 1% sodium citrate, embryos were fixed in 1.0 ml of methanol:acetic acid 3:1 (v:v) for 30 min and transferred to fresh fixative for a minimum of 12 h. The fixative was removed, and 0.5 ml of 50% acetic acid in distilled water was added to the embryos to disperse the cells into a suspension and placed on previously cleaned glass slides [33]. Slides were stained with 4% Giemsa for 4 min, and female embryos showing well-spread meta-
FIG. 1. Ethidium bromide-stained agarose electrophoresis gel of total RNA extracted from female bovine tissue after incubation in the presence (1) or absence (2) of reverse transcriptase followed by PCR amplification with primers for actin (A) or Xist (B). Molecular weight markers, lane 1; liver, lanes 2(1), 3(2); kidney, lanes 4(1), 5(2); chorioallantois, lanes 6(1), 7(2); bovine oviduct epithelium, lanes 8(1), 9(2); blank, lane 10. Note the presence of a 450-bp amplicon of the expected size for actin in lanes 2, 4, 6, and 8 in B and a 463-bp amplicon of the expected size for Xist in lanes 2, 4, 6, and 8 in B.
phases were photographed and destained before processing for autoradiography as described above. RESULTS
Xist Expression in Somatic Tissues
Complementary DNA samples from adult liver, kidney, chorioallantois, and bovine oviductal epithelial cells, subjected to RT-PCR amplification with primers designed from the bovine Xist nucleotide sequence [25], revealed the presence of Xist transcripts in female somatic tissues (Fig. 1). The female samples evaluated as positive controls for this experiment showed a consistent PCR product (Fig. 1A) corresponding to the expected actin amplicon size (450 bp), attesting to the technical reliability of the RT-PCR used in the present study. Samples of RNA in which reverse transcriptase was omitted (the negative control for the RT reaction; Fig. 1, lanes 3, 5, 7, and 9), as well as the blank PCR mixture (lane 10), showed no amplification, thus eliminating the possibility of genomic DNA contamination or artifact. A distinct band representing the amplified Xist sequence was detected only from female samples including the chorioallantois, in which RNA RT appears to have been successful (lanes 2, 4, 6, and 8); however, the amplicon from kidney samples was faint (Fig. 1B). Actin amplicons of the expected size (450 bp) were displayed by all male somatic tissues tested (Fig. 2A). In contrast, Xist expression was not detected in male tissues except in the testis (Fig. 2B, lane 6), in which a faint amplicon of the expected size was con-
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FIG. 2. Ethidium bromide-stained agarose electrophoresis gel of total RNA extracted from male bovine tissues after incubation in the presence (1) or absence (2) of reverse transcriptase following PCR amplification with primers for actin (A) or Xist (B). Molecular weight markers, lane 1; liver, lanes 2(1), 3(2); kidney, lanes 4(1), 5(2); testes, lanes 6(1), 7(2); blank, lane 8. Note the 450-bp amplicon of the expected size for actin in lanes 2, 4, and 6 in A and the 463-bp Xist amplicon in lane 6 in B.
sistently observed. Nucleotide sequence analysis of the PCR products after gel purification revealed a 96% homology with the murine Xist, and the BLAST sequence search comparison [31] proved identity with the previously reported bovine Xist (clone pcow1) from Bison bonasus, [25] from which Bos taurus Xist differs only in T for A substitutions at positions 558–561. Xist Expression in Preattachment Embryos
Expression patterns of Xist and of actin (used as a positive control) during bovine embryonic development are illustrated in Figure 3. Actin transcripts were detected at all developmental stages examined from the 2-cell stage (48 h postinsemination; lane 2) to the expanded blastocyst stage on Day 9 of in vitro development (lane 12), with greater intensity at blastocyst stages (lanes 10 and 12). No Xist expression was evident in embryos at the 2-cell stage (Fig. 3B, lane 2) after a single round (30 cycles) of PCR amplification. The first indication of Xist expression was seen in samples of 8-cell-stage embryos (on Day 3 of in vitro development), in which a distinct amplicon of the size corresponding to bovine Xist (463 bp) was consistently observed. Xist amplicons were faint but consistently detected from the 8-cell stage and at all stages examined up to and including expanded blastocyst stage on Day 8 of in vitro development (lanes 4, 6, 8, 10, and 12). No amplification products were evident in samples representing DNase-treated RNA samples amplified in the absence of reverse tran-
FIG. 3. Ethidium bromide-stained agarose electrophoresis gel of total RNA extracted from pools of bovine embryos after incubation in the presence (1) or absence (2) of reverse transcriptase followed by PCR amplification with primers for actin (A) or Xist (B). Molecular weight markers, lane 1; 2- to 4-cell embryos, lanes 2(1), 3(2); 8-cell embryos, lanes 4(1), 5(2); 8- to 16-cell embryos, lanes 6(1), 7(2); morulae, lanes 8(1), 9(2); blastocysts, lanes 10(1), 11(2); expanded blastocysts, lanes 12(1), 13(2). Note the 450-bp amplicon of the expected size for actin in lanes 2, 4, 6, 8, 10, and 12 in A and the 463-bp amplicon of the expected size for Xist in lanes 4, 6, 8, 10, and 12 in B. In addition, a 700-bp amplicon corresponding to an E. coli rRNA subunit used as a carrier in extraction and a 400-bp amplicon of unknown identity appear in lanes 2, 4, 6, 8, 10, and 12 in B.
scriptase or in blank PCR reaction mixtures in which cDNA was not included, indicating absence of exogenous DNA contamination. Nucleotide sequence analysis of the 463-bp band revealed 97% homology with bovine (Bison bonansus) Xist (clone pcow1). Amplification of 400 and 700 bp was observed in lanes containing RNA samples from preattachment embryos except for expanded blastocysts and not in blank or RT control lanes. Although amplified with the bovine Xist outside primers, neither of the additional bands had other significant homology to Xist. Analysis of the 700bp band revealed identity with a ribosomal subunit of Escherichia coli, corresponding to the ribosomal RNA used as a carrier in our RNA extraction protocol. After BLAST sequence comparison, the 400-bp band showed no homology with known bovine or E. coli sequences and only low homology with other known sequences (less than 60% homology with a genomic sequence from Caenorhabditis elegans) of unknown function. In order to increase the specificity and resolution of Xist transcript detection, a secondround PCR reaction (30 cycles) was undertaken using nested primers (Table 2). With the inside primers, only amplicons of 283 bp, corresponding in size to the Xist transcripts, were consistently detected from the 2-cell stage to the blastocyst stage. No other bands were observed on second-round PCR. The 283-bp amplicons were observed only with samples that had been reverse transcribed and not in PCR blanks, again indicating no DNA contamination (Fig. 4). Nucleotide sequence analysis of PCR product amplified with the nested primers revealed 100% homology with bovine Xist (clone pcow1).
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FIG. 4. Ethidium bromide-stained agarose electrophoresis gel of cDNA previously RT-PCR amplified with primers for a 463-bp sequence of the Xist gene following second-round PCR using internal primers for a 283bp sequence of the gene. In description of lanes below, (1) denotes that the original embryo RNA extract was incubated in the presence of reverse transcriptase and (2) denotes incubation in the absence of reverse transcriptase. Molecular weight markers, lane 1; 2-cell embryos, lanes 2(1), 3(2); 8-cell, lanes 4(1), 5(2); morulae, lanes 6(1), 7(2); blastocyst, lanes 8(1), 9(2). Lanes 10 and 11 represent bovine oviduct epithelium cell controls. Note the 283-bp amplicon of the expected size for the secondround Xist primers in lanes 2, 4, 6, 8, and 10.
X Chromosome Inactivation
Metaphase plates from cultured bovine blastocysts displaying a normal female chromosome complement, including the two submetacentric X chromosomes, are presented in Figure 5. A late-replicating X chromosome, as evidenced by preferential deposition of silver grains on one of the X chromosomes in at least one metaphase per embryo, was observed in 30 of 48 (62.5%) female blastocysts of different developmental stages and none of 13 female morulae produced in vitro. Among the 48 blastocysts, 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11 (72.7%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts revealed a late-replicating X chromosome. All Day 14 (n 5 9) and Day 15 (n 5 5) female elongated blastocysts revealed metaphase spreads with a late-replicating X chromosome. Of the 109 labeled metaphases from Day 14 embryos and the 150 from Day 15 embryos, 17 (16.0%) and 82 (55.0%), respectively, revealed a late-replicating X chromosome. Figure 5, A and B, presents the two X chromosomes of an early blastocyst before autoradiography and after autoradiography, respectively, the latter showing preferential deposition of silver grains on one of the X chromosomes representing the latereplicating X chromosome. Metaphase plates from a Day 14 and a Day 15 female embryo before and after autoradiography are presented in Figure 5, C–F; preferential deposition of silver grains is seen on one of the X chromosomes after autoradiography in Figure 5, D and F. Latereplicating regions were also observed near the centromere on a few autosomes. DISCUSSION
Our molecular and cytogenetic analyses of X chromosome inactivation in female bovine preattachment embryos show that Xist expression is evident in preattachment embryos and that it precedes the onset of the late replication of one of the X chromosomes. Our studies also indicate that Xist expression is detectable after a single round of PCR amplification as a faint amplicon at the 8-cell stage (on Day 3 of development) and consistently thereafter as a stronger band (at the morula and blastocyst stages). Xist transcripts, however, could be detected as early as the 2cell stage with the use of nested RT-PCR amplification. X chromosome inactivation, indicated by late replication, was
FIG. 5. Chromosome spreads from female bovine embryos incubated in the presence of [3H]thymidine displaying late-replicating (thick arrows) and isocyclic (thin arrows) X chromosomes. A and B) Early blastocyst stage metaphase stained with Giemsa before (A) and after autoradiography (B). C and D) Day 14 elongated blastocyst stage metaphase spread stained with Giemsa before (C) and after autoradiography (D). E and F) Day 15 elongated blastocysts stained with Giemsa before (E) and after autoradiography (F). Note the more abundant silver grain deposition over one of the X chromosomes in B, D, and F, indicating later replication relative to the other chromosomes.
first evident at the early blastocyst stage (on Day 8) and even at that stage only in some embryos, although by the elongated blastocyst stage (Days 14 and 15 of development) it was unequivocally detectable in all female embryos. Our observations, therefore, are in accord with the suggestion that Xist is involved at least in the initiation of X chromosome inactivation in human embryos [19, 21,24, 34] and with the findings that Xist expression in mouse embryos precedes the initiation of late replication [6, 24] and dosage compensation [1]. The patterns of Xist expression in human preimplantation embryos appear to vary greatly. Xist transcripts were detected using nested-primer PCR at the 1-cell stage in some embryos [35], while Ray et al. [36] reported Xist expression consistently from the 5- to 10-cell stage onward. The reason(s) for the difference in the timing of Xist expression in the human embryos is not clear. However, Daniels et al. [35] suggest that Xist expression at the 1-cell stage may be due to a genome-wide demethylation reported to be taking place during early cleavage [37]. In contrast, Xist expression in the mouse embryo was detected only from the 4-cell stage onward using a nested-primer approach [22, 23]. Furthermore, human embryos display Xist expression from both the paternal and the maternal X chromo-
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somes during early cleavage stages [35, 36]. This pattern is substantiated by in situ hybridization data that confirmed a low level of Xist expression from both the active and inactive X chromosomes in murine embryos [38, 39]. In our study, Xist transcripts were initially detected in pools of embryos at the 8-cell stage after a single round of PCR amplification. Consistent detection in bovine embryos from the 8-cell stage onward may reflect a substantial increase in embryonic Xist transcription. It is possible that the transcripts detected at the 2-cell stage correspond to the low level, biallelic Xist expression reported by in situ hybridization in mouse embryos at the 8-cell stage [38, 39]. Nonspecific amplification of a 700-bp and a 400-bp sequence was noted in embryo samples that, due to small amounts of total RNA, required addition of E. coli carrier RNA to facilitate extraction. The 700-bp sequence had identity with E. coli RNA, while the 400-bp sequence remains unknown. Since neither sequence was present in reverse transcriptase-negative or blank controls, the possibility of exogenous DNA contamination as a source of the sequences was ruled out. The presence of both sequences at the 2- to 4-cell stage when Xist amplicons were not visible suggests that Xist expression is independent of these two sequences. Also, neither sequence was amplified in the second-round PCR, indicating lack of shared homology with the internal primers. Although a low level of transcription is evident in 2cell-stage bovine embryos [40], a major burst of embryonic transcription in this species occurs at the 8-cell stage [41, 40]. This pattern of transcription is consistent with the rapid translation of maternal mRNA during the first cleavage divisions [42], followed by the transition from maternal to embryonic control of gene expression. Our observations on Xist expression suggest that even after the transition from maternal to embryonic control [41], the levels of de novo mRNA synthesis may not be adequate to promote X inactivation in the relatively large number of cells generated in early embryos prior to the blastocyst stage. Alternatively, it is possible that the Xist transcripts are rapidly degraded. The relatively faint Xist amplicons detected in preattachment embryos as compared to adult female cells may be a reflection of the smaller number of cells undergoing inactivation at this stage. Our cytogenetic data revealed that the number of metaphases showing an inactive X chromosome increases during subsequent divisions as more blastomeres ‘‘commit’’ to X chromosome inactivation. Similarly, the higher levels of Xist expression evident in adult female somatic tissues relative to embryonic Xist expression in the present study may be the result of accumulated transcripts in the former, since quantitative RT-PCR assays have shown that the levels of Xist expression during embryonic development and in embryonic stem cell lines are considerably lower than those observed in adult female somatic tissues [43]. Xist expression was not detected in any adult male tissue examined with the exception of testis, confirming its predominantly female expression. Testicular cDNA displayed a single 463-bp PCR product of somewhat lower intensity compared to that obtained from female tissues as has been reported in human and mouse testes [22]. The role of Xist during spermatogenesis is not clear, although X inactivation has been considered to be essential for germ cell survival in normal males [44, 45], possibly through protecting the unpaired regions of the X and Y chromosomes from nuclease digestion or from incorrect (nonhomologous) pairing [46].
Late replication was first observed in a few cells of a small percentage of bovine blastocysts on Day 8 of development. At this stage, embryos contain fewer than 80 blastomeres and a small blastocoele. The percentage of embryos showing an inactive X chromosome was strikingly higher at the expanded blastocyst stage (on Day 8 after fertilization), when conceptuses display more than 100 cells and a fully differentiated inner cell mass and trophectoderm. Elongated blastocysts (Days 14 and 15 of development) showed evidence of late replication in all female embryos evaluated, indicating that cells exhibiting X inactivation increase progressively as embryos reach more advanced developmental stages. The higher percentage of cells displaying a late-replicating X chromosome noted in Day 15 embryos could well be a reflection of the larger cell number in these embryos and the higher number of cells undergoing X inactivation in the trophectoderm. In conclusion, Xist expression in the bovine embryo appears to be initiated as early as the 2-cell stage, while a late-replicating (inactive) X chromosome is not readily evident until the early blastocyst stage (on day 8 after in vitro fertilization). Cells committed to this process increase progressively during embryo development, and it is strikingly evident as the blastocyst elongates. Although the process of X inactivation is a highly conserved characteristic among female mammals [3], the pattern of dosage compensation for human genes differs from that in their mouse homologues [8], suggesting evolutionary modifications in some cases. Further analysis of the activity status of different X-linked genes could increase our understanding of X chromosome inactivation in mammals and identify the mechanisms adopted by different species for gene control. ACKNOWLEDGMENTS The authors are grateful to Dr. M. Viveiros for critical reading of the manuscript, to Dr. K.J. Betteridge and the group at the Animal Biotechnology Embryo Laboratory for providing elongated blastocysts and ovaries, to Angella Hollis for assistance with nucleotide sequence analysis, to Liz St. John and Ed R. Reyes for assistance with embryo production, and to S. Kawarsky for assistance with the preparation of figures.
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