Changes in the Temporal and Spatial Expression: MATERIALS AND METHODS(1)

4 Jun



Mated female Sprague-Dawley rats were housed on a 14L:10D cycle at the University of Missouri-Kansas City and provided rat chow and water ad libitum. The presence of sperm in the vagina was determined by the vendor (Ta-conic, Germantown, NY) and was counted as Day 1 of pregnancy. Animals were treated in accordance with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals. Protocols for the care and use of animals were approved by the University of Missouri Animal Care and Use Committee. Rats were injected in the lateral tail vein 15 min prior to euthanasia and necropsy with 1% (w/v) Evans blue dye (Sigma Chemical Co., St. Louis, MO) to visualize implantation sites. The endometrium was decidualized in ovariectomized hormone-treated rats by injecting 50 ^l of sesame oil per uterine horn as described by us in detail elsewhere. The contralateral uterine horns from the same animals were used as control uterine tissues for the hormone treatments. Placentas from pregnant (Days 10, 12, and 14) Sprague-Dawley rats were a gift from Michael J. Soares (University of Kansas Medical Center).

RNA Fingerprinting

Isolated implantation sites (Evans blue dye positive) and intersite regions were pooled from the uterus of three rats at Day 5 of pregnancy. Total RNA was isolated by homog-enization of the tissues in guanidine thiocyanate. Complementary DNAs were synthesized from RNA samples using primers and reagents provided in an RNA fingerprinting kit (Delta RNA Fingerprinting Kit K1810-1, Clontech, Palo Alto, CA). Select cDNA populations were amplified by fingerprinting using combinations of one oligo-dT (T) primer and one 5′ (P) primer and 10 ^Ci of [a-35S]dATP Polymerase chain reaction (PCR) conditions were 40 cycles at 94°C for 30 sec, 40°C for 2 min, and 72°C for 5 min. Amplified products were separated by electrophoresis through 6% polyacrylamide sequencing gels at 1700 V. Gels were exposed to x-ray film for 3-6 days. A cDNA unique to the implantation site was excised from the gel and reamplified with PCR using the same primer pair corresponding to that used for the original display. The PCR product was subcloned into Bluescript T vector [26], and the cDNA was sequenced using an automated sequencer (model 377; Perkin Elmer, Foster City, CA) with the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction System with AmpliTaq DNA Polymerase, FS (Perkin Elmer). Sequences were examined in the GenBank and EMBL data bases, using the BLAST program.