Transient expression of the cytochrome P450 aromatase gene in elongating porcine blastocysts is correlated with uterine insulin-like growth factor levels during peri-implantation development.
The insulin-like growth factors (IGFs-I and -II) are mediators of cellular growth and differentiation. The expression of these growth factor genes is temporally and hormonally regulated in the uterus during pregnancy, suggesting potentially important roles in embryonic development, implantation, and successful progression of pregnancy. A known regulator of uterine IGF-I secretion is estrogen, which is produced by pre-implantation mammalian embryos of several species and whose amounts may be influenced by growth factors via their effects on the transcriptional activities of steroidogenic enzyme genes. We have previously proposed that within the uterine microenvironment, a positive feedback loop may link uterine secretion of IGFs with embryonic production of estrogens to maintain and coordinate the timing of biological signals essential for embryo development. The present study examined the temporal relationships between the levels of conceptus cytochrome P450 aromatase mRNA and protein and concentrations of IGF-I and -II in uterine luminal fluids of pigs. A DNA fragment encoding a highly conserved region among mammalian aromatase P450 proteins was isolated by hybridization screening of a porcine genomic DNA library with a human aromatase P450 cDNA fragment as probe. A synthetic oligopeptide DDVIDGYPVKKGTNI within this highly conserved region was used to generate an antiserum in sheep that recognized a protein of M(r) 49,000 in Western blot analysis of porcine ovarian, placental, endometrial, and conceptus extracts. A radioimmunoassay (RIA) for aromatase P450 was established and validated using this antiserum. RIA demonstrated highest levels of aromatase P450 protein in extracts of days 10, 11, and 12 porcine conceptuses with significantly diminished levels in elongated conceptuses at days 15 and 18. In the conceptus, aromatase P450 was localized to the inner cell layer (hypoblast) of the trophectoderm. A major mRNA transcript of approximately 3 kb in length was demonstrated by Northern blot analysis of conceptus RNA with a porcine aromatase P450 antisense RNA probe. The relative levels of aromatase P450 mRNA were higher in conceptuses at day 12 than at days 15 and 18, in parallel with the levels of aromatase P450 protein. RIA of uterine luminal fluids demonstrated maximal concentrations of IGF-I at day 12, which were significantly decreased by day 15, and increased concentrations of IGF-II by day 12, which were maintained until day 18 of pregnancy. These results demonstrate that the transient expression of conceptus aromatase P450 mRNA and protein in elongating pig blastocysts is coincident with their capacity to secrete estrogens and with the rapidly changing concentrations of IGFs within the uterine microenvironment.(ABSTRACT TRUNCATED AT 400 WORDS)
Ko
Y
Y
Dairy Science Department, University of Florida, Gainesville 32611-0691.
Choi
I
I
Green
M L
ML
Simmen
F A
FA
Simmen
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D001755:Blastocyst / Q000201:enzymology*; D017124:Conserved Sequence; D010064:Embryo Implantation; D005314:Embryonic and Fetal Development; D004717:Endometrium / Q000201:enzymology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014599:Uterus / Q000502:physiology*
D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D001141:Aromatase