Molecular cloning of spermidine/spermine N1-acetyltransferase from the periimplantation porcine uterus by messenger ribonucleic acid differential display: temporal and conceptus-modulated gene expression.
Using messenger RNA (mRNA) differential display, we isolated several putative differentially expressed complementary DNAs (cDNAs) from the periimplantation (days 11-12) endometrium of unilaterally pregnant pigs. Nucleotide sequence analysis revealed that one cDNA clone was 87% homologous to human spermidine/ spermine N1-acetyltransferase (SSAT) over a stretch of 201 bp and represents the porcine homologue of this cDNA. A second differentially expressed cDNA encoded the porcine equivalent of the human fragile X mental retardation gene (FMR1), whereas a third specified an open reading frame with significant homology to the Escherichia coli N-acetylglucosamine transfer protein. Because SSAT is the rate-limiting enzyme in polyamine metabolism and polyamines are required cytosolic components for cell growth and differentiation, we characterized the expression of the porcine SSAT gene as a potential marker for endometrial growth and/or differentiation during early pregnancy. Further, using the consensus sequence from human and mouse cDNAs, PCR primers were designed and used to generate a 568-bp cDNA fragment from gravid endometrium that encompassed the entire open reading frame for porcine SSAT and which was subsequently used for Northern hybridization analysis. Two distinct SSAT transcripts, a major species of 1.3 kilobase pairs (kb) and a minor species of 3.5 kb were detected in endometrium, each with similar temporal patterns of expression. The levels of SSAT mRNA were higher (P = 0.03) in gravid than in nongravid uterine endometrium of unilaterally pregnant pigs on days 11-12. Similarly, SSAT mRNAs were more abundant (P = 0.0004) in day 12 pregnant than in day 12 cyclic, and in days 30, 60, 90, and 105 pregnant pig endometria. Uterine endometrial luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells expressed the SSAT gene, but mRNA abundance varied among cell types (LE > GE > ST). Expression of SSAT gene in ovariectomized gilts treated with estrogen (E2, 100 microg/day), progesterone (P4, 200 mg/day) or E2 + P4 for 11 days was highest (P = 0.03) in the endometria of the P4 group. In contrast, E2 (10 nM), P4 (10 nM) and E2 + P4 had no effect on SSAT mRNA abundance in uterine endometrial explants from day 12 pregnant pigs. However, steady-state SSAT mRNA levels were induced in day 12 pregnant uterine explants by conditioned medium from day 12 filamentous but not spherical conceptuses. These data demonstrate that the temporal induction of the endometrial SSAT gene during periimplantation is modulated by a factor(s) secreted by the periimplantation conceptus and suggest that this enzyme may have an important role in uterine endometrial growth, remodeling and/or differentiation during periimplantation.
Green
M L
ML
Department of Animal Science, University of Florida, Gainesville 32611, USA.
Blaeser
L L
LL
Simmen
F A
FA
Simmen
R C
RC
D000123:Acetyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D010064:Embryo Implantation; D004622:Embryo, Mammalian / Q000502:physiology*; D004717:Endometrium / Q000166:cytology / Q000502:physiology; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D014599:Uterus / Q000201:enzymology*; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D018076:DNA, Complementary; D012333:RNA, Messenger; D000099228:Diamine N-Acetyltransferase