Preferential Amplification of the Rat H/358 cDNA from the Implantation Site
Complementary DNAs prepared from total RNA that was isolated from the implantation sites (Evans blue dye positive) and the intersite regions of the uterus at Day 5 of pregnancy were amplified using RNA fingerprinting. One PCR product unique to the implantation site was excised and reamplified by PCR using the same primer pairs (5′ primer, P4; 3′ primer, T2). This amplified product was subcloned into the EcoRV site of a pBluescript SK(+) cloning vector (Stratagene, La Jolla, CA) prepared for direct cloning of PCR products. Examination of the sequence (Fig. 1A) suggested this cDNA to be the rat homolog of the mouse Hp58 gene shown to be essential for embryogenesis, and the yeast pep8 gene, a gene coding for a protein involved in vacuole protein trafficking. Alignment of the predicted amino acid sequences of the rat with reported sequences from yeast, chicken, and mouse sequences showed a high degree of identity and conservative substitutions of amino acid residues among those species (Fig. 1B). The rat Hp58 open reading frame exhibited 17 out of 20 amino acids identity (85%) to the mouse and chicken proteins. Over the region compared, 15 of 20 amino acids were identical (75%) to the yeast vacuolar protein.
Endometrial Expression of H/358 mRNA Does Not Require Decidualization
Previous experiments in the mouse showed H358 transcripts were present in the embryo, fetal placenta, and maternal placenta (decidua). To investigate whether uterine expression of H358 was induced by decidualization of the endometrium, RNA samples pooled from artificially deci-dualized endometria, from the control contralateral nonde-cidualized uterine horns, and from implantation sites at Day 5 of pregnancy were analyzed by Northern blotting (Fig. 2). Results from Northern blot analysis revealed a singlesized H358 transcript of approximately 2.1 kilobases (kb) in the endometrium (Fig. 2). The mRNA was evident in the uterus of hormonally sensitized rats stimulated to undergo decidualization (lane 1), in the contralateral uterine horns exposed to the same hormonal treatment but lacking a decidual stimulus (lane 2), and in the isolated implantation sites from Day 5 pregnant rats (lane 3). Taken together, those results indicated that H358 transcripts were expressed in the endometrium of rats hormonally prepared for deci-dualization, but decidualization and the presence of a blastocyst was not required for endometrial expression.
FIG. 1. Sequence analysis of rat H^58. A) Nucleotide sequence and predicted amino acid sequence of rat H^58 amplified from the site of implantation by RNA fingerprinting. B) Sequence alignment of the predicted amino acid sequence of rat H^58 with the predicted amino acid sequence of the homologous gene in yeast, chicken, and mouse. The numbers shown on the right side of the figure refer to the amino acid sequences in each species. Identical amino acid sequences are boxed.
FIG. 2. Expression of H^58 in the rat uterus does not require the presence of a blastocyst. Total RNA was isolated from ovariectomized, hormone-treated rats (lanes 1 and 2) or from the isolated implantation sites of pregnant rats (lane 3) as described in the text. Samples (20 ^g) were analyzed for H^58 mRNA by Northern blotting. Lane 1, RNA sample pooled from hormonally treated rats in which the endometrium was stimulated to undergo decidualization; lane 2, RNA sample pooled from non-decidualized control uterine horns of hormone-treated rats; lane 3, RNA sample pooled from the implantation sites of Day 5 pregnant rats. The RNA size markers are indicated on the left. Arrow indicates H^58 mRNA. The lower panel shows the RNA samples stained with ethidium bromide in the agarose gel before transfer. Arrows indicate the 28S and 18S rRNA subunits.