Abstract:
Early embryonic development is initially dependent on mRNAs that have been transcribed during oocyte growth (maternal transcripts). Newly formed zygotic transcripts then become required during what is called the maternal-to-zygotic transition. In cattle, this transition initiates at the 8-cell stage and ends before the morula stage. Because of its decisive role in the further development of the embryo we are interested in characterizing the regulative functions of those cattle zygotic transcripts that are differentially expressed at the end of this transition. For that reason a subtracted cDNA library corresponding to the first zygotic transcripts was established at the early compacted morula stage using suppressive subtractive hybridization (SSH; Clontech, LePont de Claix, France). Morula derived cDNAs were used as Tester and 4-cell stage cDNAs as Driver materials. Cattle embryos were obtained from slaughterhouse-derived ovaries using standard in vitro maturation and fertilization techniques. Since, in cattle, early cleaving (2-cell-stage) zygotes are more likely to develop to the blastocyst stage than their later-cleaving counterparts, all embryos used to establish the cDNA library were selected from zygotes that were already at the 2-cell stage 32 h post-in vitro fertilization. Total RNA was extracted from batches of 140 (morula) and 200 (4-cell-stage) embryos and the amount of PolyA+ RNAs was estimated according to Duranthon and Renard (in Biology and Pathology of the Oocyte, Trounson and Gosden eds, Cambridge Univ. Press, 2003, p. 96). Double-stranded cDNAs were synthesized with the SMART cDNA amplification kit (Clontech) before SSH was undertaken. Upon RNA extraction, exogenous transcripts obtained from Arabidopsis thaliana (Stratagene, La Jolla, CA, USA) were added either to the Tester only (at three concentrations: 10?3, 5 _ 10?3, 5 _ 10?2) or to both the Tester and the Driver materials (at two concentrations: 5 _ 10?3, 5 _ 10?2). These transcripts allowed us to report on the efficiency of our subtraction procedure and on the quality of the bacterial library in terms of tester-specific transcript enrichment. We found the library to be enriched in specific transcripts of the Tester (morula stage) by a factor of 300. Normalization of the library, as determined from the proportion of exogenous transcripts after bacterial transformation, was effective for those added initially at low (10?3) or moderate (5 _ 10?3) concentrations but not for abundant ones (5 _ 10?2). These conditions are thus beneficial for the isolation of rare zygotic transcripts present at an initial concentration of only 10?3 of the messengers. Ongoing study using various differential screening of this cattle library with morula- and 4-cell-stage probes will now allow us to identify zygotic transcripts specifically expressed at the onset of genome activation and not present in the pool of maternal transcripts up to the 4-cell stage. This work was supported by an INRA CIRAD grant (BioDiva) to LCB.