Analysis of Gene Expression in the Bovine Blastocyst Produced In Vitro Using Suppression-Subtractive Hybridization1

Abstract

Successful embryonic development is dependent on time and location-specific expression of appropriate genes. Unfortunately, information on stage-specific gene expression during early embryonic development in the bovine is lacking. In the present study, we compared gene expression between in vitro-produced Day 7-8 intact blastocysts (driver) and Day 9-10 hatched blastocysts (tester) using suppression-subtractive hybridization. Pools of 30 embryos for both driver and tester were used in the RNA extraction process. From limited amounts of starting material ( approximately 400 ng of total RNA), a reverse transcription-polymerase chain reaction (PCR) procedure was used to amplify the mRNA and generate sufficient cDNA to conduct suppression-subtractive hybridization. The subtracted cDNA products were cloned, and 126 cDNAs representing expressed mRNAs were isolated, sized, single-pass sequenced, and compared to known sequences in GenBank. Ninety-two clones provided sequence information for further analysis. Among these, 31 exhibited high homology to known genes. Three, 26S proteasomal ATPase (PSMC3), casein kinase 2 alpha subunit (CK2), and phosphoglycerate kinase (PGK) were selected and further characterized using real-time quantitative PCR to assess their differential expression in hatched blastocysts. Overall, a 1.3-, 1.6-, and 1.5-fold increase in expression level was observed in hatched blastocysts compared with intact blastocyst for PSMC3, CK2, and PGK, respectively. These results show that construction of subtracted cDNA libraries from small numbers of embryos is feasible and can provide information on gene expression patterns during preattachment embryogenesis.

Peroxisome proliferator-activated receptors in reproductive tissues: from gametogenesis to parturition

“…3) PPAR is required for the attachment of embryos to the endometrium and the development and function of the placenta (Barak et al 1999). Strong PPAR expression has been detected in the trophectoderm and inner-cell mass of the blastocyst (Mohan et al 2002). PPAR inactivation leads to impaired placental vascularization, resulting in the death of the embryo.…”

Un rôle pour PPARγ dans la reproduction ?

“…Ces résultats sont en accord avec la forte expression de ce récepteur dans le trophectoderme et la masse cellulaire interne du blastocyste [27]. L'inactivation de PPARγ induit plus particulièrement un défaut de vascularisation du placenta, ce qui conduit à la mort de l'embryon en milieu de gestation.…”

Independent Downstream Gene Expression Profiles in the Presence of Estrogen Receptor α or β1

“…The cDNA was cut with RsaI (BD Biosciences Clontech), and two different adaptors [34] were ligated onto individual aliquots of the tester population. Hybridizations were carried out as described by Mohan et al [35] with modifications. The first hybridization was carried out on the tester with either Adaptor 1 or 2R, and these were heat denatured and hybridized in the presence of excess denatured driver.…”

“…A second hybridization was then performed where the two tester populations were combined in the presence of fresh denatured driver. Due to the presence of the adaptors, only those genes unique to the tester population were enriched and amplified in the polymerase chain reaction (PCR) [35]. PCR products from SSH were then compared against unsubtracted products.…”