Development and characterization of a simian virus 40-transformed, temperature-sensitive rat antimesometrial decidual cell line.
The rat decidual tissue is formed by two cell populations, which express different genes and play diverse roles in pregnancy. Cells that decidualize in the antimesometrial region secrete several hormones and serve as a true endocrine gland. Isolation and maintenance of these decidual cells in primary culture is difficult. The goal of these experiments was to develop a cell line to serve as a model to study the expression and regulation of various genes specific to the antimesometrial decidual cells. Decidualization was induced in pseudopregnant rats. The antimesometrial decidua was dissected out, and cells were enzymatically dissociated and were cultured for 18 h at 37 C in RPMI-1640 medium containing 10% fetal bovine serum. Cells were washed repeatedly and then infected with a temperature-sensitive mutant of the simian virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified and harvested. Whereas primary cells in culture did not divide, the cloned decidual cell lines demonstrated transformed features; they multiplied at 33 C and formed multilayers. At the nonpermissive temperature (39 C), cell replication decreased, and after 4 days of culture the cells lost their transformed phenotype and continued to grow as a monolayer similar to primary cells. Cells under these conditions also assumed morphological characteristics similar to antimesometrial cells: polynucleated, large, and having cytoplasm filled with lipid droplets. Interestingly, cells cultured at 39 C that were shifted back to 33 C resumed rapid growth. To determine whether these cells also express messenger RNAs (mRNAs) found in normal antimesometrial decidual cells, the presence of activin beta A mRNA was investigated by Northern analysis and reverse transcription polymerase chain reaction. A single 6.8-kilobase activin beta A transcript was expressed abundantly at both 33 and 39 C, indicating that even when cells are rapidly dividing they express activin beta A. Activin beta B mRNA was also expressed in these cells, although in lower abundance, as were two binding proteins for activin, activin receptor II and follistatin. The activin beta A and beta B genes were responsive to cAMP stimulation in these cells. Since the hallmark of the antimesometrial decidual cells is the secretion of PRL-related hormones, the expression of decidual PRL-related protein and PRL-like protein B was examined. Northern analysis revealed a major 1.2-kilobase transcript of PRL-like protein B expressed equally at both temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)
Srivastava
R K
RK
Department of Physiology and Biophysics, University of Illinois at Chicago, Illinois 60612-7342, USA.
Gu
Y
Y
Zilberstein
M
M
Ou
J S
JS
Mayo
K E
KE
Chou
J Y
JY
Gibori
G
G
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D029404:Activin Receptors; D028341:Activins; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002455:Cell Division; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D046508:Culture Techniques / Q000379:methods; D017931:DNA Primers; D003656:Decidua / Q000166:cytology* / Q000378:metabolism*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006133:Growth Substances / Q000096:biosynthesis; D007265:Inhibins / Q000096:biosynthesis*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis; D011555:Pseudopregnancy; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017978:Receptors, Growth Factor / Q000096:biosynthesis*; D013539:Simian virus 40; D013696:Temperature
D017931:DNA Primers; D006133:Growth Substances; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D028341:Activins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007265:Inhibins; D017346:Protein Serine-Threonine Kinases; D029404:Activin Receptors