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Expression of the pregnancy-specific beta 1-glycoprotein gene in cultured human trophoblasts
1Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Insights
Human pregnancy-specific beta 1-glycoprotein (PS beta G) synthesis and regulation were studied in trophoblast cells. In vitro culture promoted PS beta G expression, with specific transcript variants and polypeptide profiles observed.
Area of Science:
- Reproductive Biology
- Molecular Endocrinology
- Cellular Differentiation
Background:
- Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a major placental glycoprotein.
- PS beta G belongs to the immunoglobulin superfamily and shares sequence similarity with carcinoembryonic antigen.
- Understanding PS beta G's role in placental development requires examining its synthesis and regulation.
Purpose of the Study:
- To investigate the synthesis and regulation of PS beta G in primary trophoblast cell cultures.
- To analyze the expression of different PS beta G transcript variants during trophoblast differentiation in vitro.
- To characterize the polypeptide profile of PS beta G synthesized by primary trophoblasts compared to placental tissue.
Main Methods:
- Primary human cytotrophoblasts were cultured in vitro to assess differentiation.
- Molecular cloning and Northern blot analysis were used to study PS beta G mRNA expression.
- Ribonuclease protection assays identified specific PS beta G transcript variants.
- Western blot analysis or similar techniques were used to examine PS beta G polypeptide synthesis.
Main Results:
- Trophoblast cells cultured in vitro showed increased PS beta G mRNA and protein synthesis, indicating in vitro differentiation.
- Multiple PS beta G mRNA transcripts (2.3, 2.2, and 1.7 kb) were detected, with preferential increase of PSG95-3' containing transcripts in culture.
- Primary trophoblasts synthesized a distinct pattern of PS beta G polypeptides (64K, 72K, 62K, 54K) compared to purified placental PS beta G (72K, 64K, 54K).
- 8-bromo-cAMP slightly reduced PS beta G expression, while sodium butyrate markedly inhibited it.
Conclusions:
- Primary trophoblast cells differentiate in vitro, leading to altered PS beta G gene expression.
- The regulation of PS beta G expression in trophoblasts involves specific transcript variants and is influenced by chemical agents.
- These findings provide insights into the molecular mechanisms governing PS beta G expression during pregnancy and placental development.
Abstract:
Human pregnancy-specific beta 1-glycoprotein (PS beta G), the major placental glycoprotein, shares strong sequence similarity with carcinoembryonic antigen and is a member of the immunoglobulin superfamily. To understand the role of PS beta G in placental ontogeny during pregnancy, we examined its synthesis and regulation in primary cultures of trophoblast cells. Freshly plated (12 h) cytotrophoblasts expressed little PS beta G transcripts; however, within 24 h of culture, PS beta G mRNAs became detectable. PS beta G synthesis and mRNA expression increased with time in culture, and maximal synthesis was achieved at 4 days, indicating that primary trophoblasts continue differentiating in vitro. Molecular cloning revealed that PS beta G is an extremely polymorphic protein. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity in the 5' (designated PSG-5') and coding regions, but differ in sequences at the 3' region. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three mRNAs of 2.3, 2.2, and 1.7 kilobases in primary trophoblasts and human term placental tissue. Ribonuclease protection analysis demonstrated that primary trophoblasts expressed most of the placental PS beta G transcripts. However, culturing in vitro altered PS beta G gene expression, and the level of PS beta G transcripts containing a PSG95-3' sequence was preferentially increased in primary trophoblasts. Moreover, primary trophoblasts synthesized a 64K PS beta G polypeptide in variable amounts and three PS beta Gs of 72K, 62K, and 54K in roughly equal amounts, whereas purified human term placental PS beta G consists of a major polypeptide of 72K and two minor ones of 64K and 54K. PS beta G gene expression in primary trophoblasts was slightly reduced by 8-bromo-cAMP, but was markedly inhibited by sodium butyrate.