| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Embryo |
a Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
developmental regulation, embryo, gametogenesis, gene regulation, oocyte development
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
The molecular basis for genome activation is now better understood. For example, genome activation, which is accompanied by a change in promoter utilization such that TATA-less promoters are more efficiently used [5, 6], appears to be relatively promiscuous (i.e., genes whose promoters are accessible to the transcription machinery are expressed) [7]. Superimposed on genome activation is the development of a chromatin-mediated, transcriptionally repressive state [1] that would reduce the expression of inappropriate genes but permit the continued expression of genes that are regulated by strong promoters/enhancers. The expression of these genes would, therefore, be critical for continued development.
The identity of embryo-specific genes (i.e., genes that are not expressed in the oocyte or whose expression is markedly up-regulated compared to the oocyte) is very poorly defined. Although efforts have been made to identify these genes, the methods employed to date have major drawbacks. For example, mRNA differential display is labor intensive, has a high degree of false positives, and is biased toward detecting more abundant transcripts [7]. Likewise, analysis of cDNA libraries derived from oocytes and preimplantation embryos is only as robust as the quality of the libraries, in which rare transcripts may be underrepresented [8]. Last, subtraction methods employed to date are also biased toward detecting abundant transcripts [9].
Suppression subtractive hybridization (SSH) [10] provides an attractive solution to identify embryo-specific (and oocyte-specific) genes and, in particular, rare transcripts that may encode regulatory proteins; see Diatchenko et al. [10] for an excellent discussion of SSH. The advantage of this method stems from its ability to normalize the mRNA population so that abundant mRNAs are reduced while rare transcripts are enriched. The disadvantage is that the amounts of starting mRNA required for the hybridization are not readily obtained from mouse oocytes/embryos. For example, SSH requires approximately 2 µg of mRNA, and this amount would require 25 000 oocytes (80 pg mRNA/oocyte) [11].
We describe here a method in which small amounts of mRNA from mouse oocytes and preimplantation embryos are amplified linearly to generate sufficient amounts of material to conduct SSH. Using both forward and reverse subtraction protocols, oocyte- and 8-cell-specific transcripts were identified.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
The mRNA samples were prepared from a minimum of 100 fully grown, germinal vesicle-intact oocytes as well as 2-cell and 8-cell preimplantation embryos as previously described [7] using the Micro-FastTrack 2.0 mRNA extraction kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Oocytes were obtained from CF-1 female mice, and embryos were obtained from CF-1 females mated to B6D2F1/J males. The mRNA was also isolated from adult heart.
First-strand cDNA synthesis was carried out by incubating 3.5 µl of mRNA (0.5–14 ng) with 0.5 µl (10 µM) of cDNA synthesis primer (Table 1) at 70°C for 2 min; 0.5 ng of mRNA corresponds to approximately six oocytes. A reverse transcription (RT) reaction (total volume, 10 µl) was then prepared with the above mRNA/primer mix in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 6 mM MgCl2, 2 mM dithiothreitol (DTT), 1 mM of each dNTPs, 20 U of RNase inhibitor, and 0.5 µM T7-GGG primer. Two hundred units of Superscript II (Invitrogen) were added, and the samples were incubated at 42°C for 1 h. This RT reaction also results in dC tailing.
|
Double-stranded cDNA was generated by combining the 10 µl of first-strand cDNA sample with 10 µl of 10x Advantage 2 polymerase chain reaction (PCR) buffer (Clontech, Palo Alto, CA), 0.2 mM of each dNTPs, 2 U of RNase H (Promega, Madison, WI), and 2 µl of 50x Advantage 2 polymerase mix (Clontech) in a total reaction volume of 100 µl. Following an incubation at 37°C for 3 min to degrade the mRNA template, second-strand synthesis was then performed (95°C for 2 min and then 72°C for 20 min), followed by two cycles of PCR with the following conditions: 95°C for 30 sec and then annealing/extension at 72°C for 15 min.
Before sense RNA (sRNA) amplification, the successful synthesis of double-stranded cDNA was first established by PCR analysis using 1 µl of the resulting double-stranded cDNA and the flanking primer 5' T7 PCR primer (Fig. 1, black rectangle) and 3' PCR primer (Fig. 1, open rectangle). The size distribution of the resulting material was then assessed by electrophoresis in 1.2% (w/v) agarose gel containing ethidium bromide. In addition, 1 µl of the double-stand cDNA was used to detect specific transcript using gene-specific primers (e.g., for glyceraldehyde-3-phosphate dehydrogenase [G3PDH]). Preparations that displayed a broad size range and contained specific transcripts were further processed as follows: The remainder of the double-stranded cDNA was phenol/chloroform extracted, ethanol precipitated, and resuspended in 20 µl of diethyl pyrocarbonate (DEPC)-treated water. Ten microliters of the sample were drop-dialyzed on a 0.25-µm Millipore filter (Millipore Corporation, Bedford, MA) [12] against 50 ml of DEPC-treated water for a duration of 4 h to overnight to remove unincorporated nucleotides and excess salts.
|
For sRNA amplification, the purified double-stranded cDNA template was incubated at 37°C for 4 h in 40 mM Tris-HCl (pH 7.5) containing 7 mM MgCl2, 10 mM NaCl, 8 mM DTT, 20 U of RNasin, 1 mM of each NTP, and 2000 U of T7 RNA polymerase (Epicentre Technologies, Madison, WI) in a final reaction volume of 40 µl. The amount of double-stranded cDNA used was estimated to be 0.5–2.0 ng of the original mRNA assuming 80 pg mRNA/oocyte and 14 pg mRNA/8-cell embryo and an overall recovery rate of 90% of the initially isolated mRNA. To generate radiolabeled sRNA, the same conditions were used as above, except 30–60 µCi of [
-32P]CTP (3000 Ci/mmol; 10 mCi/ml; Amersham, Biosciences, Piscataway, NJ) were added. The size distribution of the radiolabeled products was determined by electrophoresis in a 1.2% denaturing agarose gel and autoradiography.
Nonradiolabeled and radiolabeled sRNA were then purified on Chromospin-100 columns (Clontech). The amount of sRNA generated was determined by RiboGreen staining (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. The amount of radiolabeled sRNA was quantified by trichloroacetic acid (TCA) precipitation.
Sense RNA Characterization
To establish the suitability of the sRNA for conversion to cDNA that would be used for SSH or microarray analysis, RT was performed with purified oocyte sRNA with either an oligo-dT primer (Table 1) or random hexamers. One-fortieth (1/40) of the Chromospin-purified sRNA (see above) was added to 0.5 µl of dT primer (10 µM) or 0.5 µl of random hexamer (10 µM). The sample was incubated for 3 min at 70°C and then combined with first-strand buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, and 3 mM MgCl2); 5 mM DTT; 1 mM each of dATP, dGTP, and dTTP; 12 µM dCTP; 1 µM of [-32P]dCTP; 40 U of RNasin; and 300 U of Superscript II in a total volume of 10 µl. When the random hexamers were used, the sample was initially incubated at room temperature for 10 min. In both cases, RT was conducted at 42°C for 90 min. The RNA strand in the cDNA was then degraded by incubating the sample at 37°C for 30 min with 4 U of RNase H. Following phenol extraction, first-strand radiolabeled cDNA was subjected to electrophoresis in a 1.2% agarose gel and size distribution visualized with a Storm imaging system (Amersham Biosciences).
To assay for the presence of a specific transcript, nonradiolabeled cDNA was prepared as described above, except radiolabeled dCTP was replaced with 1 mM dCTP. One-tenth (1/10) of the nonradiolabeled cDNA primed with dT (cDNAST) or random hexamer (cDNASR) was used for PCR using gene-specific primers.
Suppression Subtractive Hybridization
Suppression subtractive hybridization was conducted with 2 µg of purified 8-cell and oocyte sRNA. The cDNA synthesis and subtraction were performed with the PCR-select cDNA subtraction kit (Clontech) according to manufacturer's instructions, except the mRNA was replaced by amplified sRNA.
Eight-cell transcript-enriched subtracted cDNA and oocyte transcript-enriched subtracted cDNA were generated by forward and reverse SSH. These subtracted cDNAs were cloned into a TA vector (Invitrogen) to generate an 8-cell- and an oocyte-subtracted cDNA library, respectively. Differential screening was carried out using the PCR-select differential screening kit (Clontech) according to the manufacturer's protocol. The PCR products of randomly selected clones were then alkaline denatured and spotted onto a nylon membrane in two replicates that were then hybridized with radiolabeled probes generated from the 8-cell and oocyte cDNA subtracted probes. The relative extent of differential expression of each clone was estimated by calculating the ratio of the signal with the homologous subtracted probe to that with the heterologous subtracted probe. The clones for which the ratio was >5 were sequenced, and these sequences were then used for a BLAST search against GenBank database after RepeatMasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html) and Contig Assembly Program (http://www.infobiogen.fr/sevices/analyseq/cgi-bin/cap_in.pl) were used to identify redundant clones.
PCR and RT-PCR of Selected Genes of Interest
Selected genes identified by SSH were used for PCR and RT-PCR to validate their differential expression. To confirm the presence of the oocyte-specific histone H1 gene (H1oo), PCR was performed using the 8-cell subtracted and oocyte subtracted cDNA with H1oo gene-specific primers (Table 1). The PCR (25 cycles) was performed as follows: 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min.
The differential expression of several other candidate genes revealed from the sequencing analysis was also confirmed by PCR as above using gene-specific primers (Table 1). The PCR conditions were the same as those used for H1oo, except 28 cycles were used for Kim-1 and 35 cycles for all other genes.
To confirm the differential expression of selected transcripts detected after SSH, RT-PCR using total RNA isolated from equal numbers of oocytes and 8-cell embryos was also conducted. The isolated RNA was reversed transcribed using an oligo-dT primer, and five oocyte/embryo equivalents were used for RT-PCR as previously described [13] using 30 cycles and the aforementioned PCR conditions. The radiolabeled PCR products were run on an 8% acrylamide gel that was imaged with a Storm imaging system.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
To obtain the amounts of mRNA required for SSH (2 µg), we developed an amplification scheme to generate sRNA from small numbers of oocytes/embryos (Fig. 1). The method couples the Eberwine linear RNA amplification procedure [12] with Clontech's SMART technology to select for long-length cDNAs. Following RT, dC-tailing, template switching and extension, and second-strand cDNA synthesis, two rounds of PCR amplification were performed before sRNA amplification. The rationale for the PCR step was to provide enough starting material for the linear amplification step, which results in an approximately 2000-fold amplification, to generate sufficient amounts of material for SSH without the need for reamplification. Our concern was that reamplification could distort the representation of the original mRNA population, whereas two rounds of PCR before linear amplification would not.
The size distribution of the resulting radiolabeled material from either oocytes or 8-cell embryos before and after purification ranged from 0.5 to 12 kilobases (kb) (Fig. 2). The amplification was reproducible, and a similar size distribution was also observed for 2-cell embryos and adult heart tissue (data not shown). The procedure was optimized for starting with as little as 0.5 ng of mRNA, which corresponds to approximately six oocytes. The extent of amplification was routinely found to be 4000- to 8000-fold, as determined by either TCA precipitation of the radiolabeled sRNA or by RiboGreen fluorescence, and generated microgram amounts of sRNA from the original mRNA sample that were sufficient for SSH.
|
To establish that the sRNA generated from the linear amplification procedure could serve as a template for cDNA synthesis and contained bona fide sequences present in the original mRNA population, sRNA was first reverse transcribed with either the poly dT primer (cDNAST) or random hexamers (cDNASR). As a result, cDNAST and cDNASR with a size distribution from 0.5 to 12 kb (Fig. 3A) was generated; the size distribution was similar to that of the original sRNA. The presence of known genes in the cDNA population was confirmed by PCR amplification of the cDNAST and cDNASR with gene-specific primers for G3PDH (Fig. 3B). The presence of several other genes (Hdac2, Mos, Plat, and Sin3a) was also readily detected using gene-specific primers (data not shown).
|
SSH to Identify Genes Differentially Expressed in Mouse Oocytes and 8-Cell Embryos
We elected to use 8-cell rather than 2-cell embryos for SSH for the following reasons: First, genome activation during the 2-cell stage appears to be a relatively promiscuous process [7]. Thus, transiently and inappropriately expressed transcripts would be present and detected and, thereby, confound the analysis. In contrast, development of the transcriptionally repressive state is fully operational by the 8-cell stage [1], thus minimizing the likelihood of detecting inappropriately expressed transcripts. Moreover, the degradation of maternal mRNA is not yet complete by the 2-cell stage, whereas most maternal mRNAs are degraded by the 8-cell stage. Thus, it is more appropriate to use 8-cell material for subtraction to identify oocyte-specific transcripts. Equal amounts of amplified sRNA obtained from oocytes and 8-cell embryos were used for SSH. The general strategy for SSH and construction of 8-cell and oocyte subtracted libraries is briefly outlined in Figure 4. The average insert size of randomly picked clones was approximately 600 base pairs (data not shown), which is consistent with the average size generated by the Rsa I step in the SSH protocol.
|
Following SSH, the resulting subtracted 8-cell and oocyte cDNAs were used to construct 8-cell and oocyte subtracted libraries, respectively. These resulting libraries would contain what are termed oocyte- and embryo-specific transcripts, whereas in actuality, most of them contain genes that are differentially expressed (as opposed to an all-or-none expression profile). Accordingly, a differential screening of randomly picked clones was performed (Fig. 5). Candidate genes that were differentially expressed were sequenced and then subjected to a BLAST search in GenBank ( Tables 2 and 3 ). Detection of the oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1oo transcripts [3, 14] in the oocyte subtracted library increased confidence in the validity of the subtraction procedure. In addition, many novel genes (i.e., clones whose sequences are not present in the existing databases) were detected in both the oocyte- and 8-cell-specific cDNA libraries (data not shown).
|
|
|
|
|
To confirm the differential expression pattern in the subtracted population for both the oocyte and embryo subtracted libraries, PCR of the subtracted cDNA was performed with gene-specific primers. As anticipated, the oocyte-specific histone H1oo gene was not present in the subtracted 8-cell library but was readily detected in the subtracted oocyte library (Fig. 6A). Likewise, Oas15, Bpgm, and two other unknown genes (3E4 and 3H3) that were preferentially expressed in the oocyte subtracted library (Table 1) were also detected in the oocyte, but not in the embryo, subtracted library. Reciprocally, the Kim-1 and Zmpste24 genes that were preferentially expressed in the 8-cell embryo (Table 2) were also readily detected in the 8-cell, but not in the oocyte, subtracted library (Fig. 6A). Final validation of the method was achieved by assaying with RT-PCR the relative level of expression of Zmpste24, Bpgm, and two unknown oocyte-specific genes (3E4 and 3E6) in oocytes and 8-cell embryos (Fig. 6B). Results of these experiments documented the preferential expression of Bpgm, 3E4, and 3E6 in the oocyte and of Zmpste24 in the 8-cell embryo.
|
The intrinsic strengths of SSH are, first, that the only underlying assumption is that genes are differentially expressed and, second, that the normalization procedure enhances the ability to detect rare transcripts that potentially encode regulatory proteins. The inherent strength of microarray analysis is that it permits analysis of global patterns of gene expression, but it is limited by the quality of the cDNA library used to generate the array (e.g., rare transcripts may not be arrayed). The method we describe here to amplify linearly and reproducibly mRNA from mouse oocytes and preimplantation embryos generates sufficient amounts of material for SSH (to identify oocyte- and embryo-specific genes), and preliminary results indicate that it is also suitable for microarray analysis of gene expression (unpublished results). Combining these two complementary approaches, coupled with further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries, should furnish a trove of information regarding temporal changes in gene expression during gametogenesis and preimplantation development in the mouse.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Richard M. Schultz, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018. FAX: 215 898 8780; rschultz{at}mail.sas.upenn.edu
Received: 29 May 2002.
First decision: 14 June 2002.
Accepted: 9 July 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  T. Hamatani, M. Yamada, H. Akutsu, N. Kuji, Y. Mochimaru, M. Takano, M. Toyoda, K. Miyado, A. Umezawa, and Y. Yoshimura What can we learn from gene expression profiling of mouse oocytes? Reproduction, May 1, 2008; 135(5): 581 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vallee, K. Aiba, Y. Piao, M.-F. Palin, M. S H Ko, and M.-A. Sirard Comparative analysis of oocyte transcript profiles reveals a high degree of conservation among species Reproduction, April 1, 2008; 135(4): 439 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Susor, Z. Ellederova, L. Jelinkova, P. Halada, D. Kavan, M. Kubelka, and H. Kovarova Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1 Reproduction, October 1, 2007; 134(4): 559 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xing, R. Gosden, P. Lasko, and H. Clarke Murine homologues of the Drosophila gustavus gene are expressed in ovarian granulosa cells. Reproduction, May 1, 2006; 131(5): 905 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-W. Chen, J. J.W. Chen, S.-L. Yu, H.-N. Li, P.-C. Yang, C.-M. Su, H.-K. Au, C.-W. Chang, L.-W. Chien, C.-S. Chen, et al. Transcriptome analysis in blastocyst hatching by cDNA microarray Hum. Reprod., September 1, 2005; 20(9): 2492 - 2501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Serafica, T. Goto, and A. O. Trounson Transcripts from a human primordial follicle cDNA library Hum. Reprod., August 1, 2005; 20(8): 2074 - 2091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Vallee, C. Gravel, M.-F. Palin, H. Reghenas, P. Stothard, D. S. Wishart, and M.-A. Sirard Identification of Novel and Known Oocyte-Specific Genes Using Complementary DNA Subtraction and Microarray Analysis in Three Different Species Biol Reprod, July 1, 2005; 73(1): 63 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Seli, M. D. Lalioti, S. M. Flaherty, D. Sakkas, N. Terzi, and J. A. Steitz An embryonic poly(A)-binding protein (ePAB) is expressed in mouse oocytes and early preimplantation embryos PNAS, January 11, 2005; 102(2): 367 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tanaka, M. Kihara, J. D. Hennebold, J. J. Eppig, M. M. Viveiros, B. R. Emery, D. T. Carrell, N. J. Kirkman, B. Meczekalski, J. Zhou, et al. H1FOO Is Coupled to the Initiation of Oocytic Growth Biol Reprod, January 1, 2005; 72(1): 135 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Ellederova, P. Halada, P. Man, M. Kubelka, J. Motlik, and H. Kovarova Protein Patterns of Pig Oocytes During In Vitro Maturation Biol Reprod, November 1, 2004; 71(5): 1533 - 1539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pennetier, S. Uzbekova, C. Perreau, P. Papillier, P. Mermillod, and R. Dalbies-Tran Spatio-Temporal Expression of the Germ Cell Marker Genes MATER, ZAR1, GDF9, BMP15,andVASA in Adult Bovine Tissues, Oocytes, and Preimplantation Embryos Biol Reprod, October 1, 2004; 71(4): 1359 - 1366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Rinaudo and R. M Schultz Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos Reproduction, September 1, 2004; 128(3): 301 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Hennebold Characterization of the ovarian transcriptome through the use of differential analysis of gene expression methodologies Hum. Reprod. Update, May 1, 2004; 10(3): 227 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Y. Sun, F.-X. Li, J. Li, Y.-F. Tan, Y.-S. Piao, S. Tang, and Y.-L. Wang Determination of Genes Involved in the Early Process of Embryonic Implantation in Rhesus Monkey (Macaca mulatta) by Suppression Subtractive Hybridization Biol Reprod, May 1, 2004; 70(5): 1365 - 1373. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
