Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression.

Recent reports have demonstrated that prostaglandin F2alpha-induced generation of reactive oxygen species or their intermediates inhibits progesterone synthesis and may also serve as a trigger for apoptosis in the corpus luteum (CL). BCL-2, an inhibitor of apoptosis in a wide variety of cell types, has been reported to prevent oxidative stress-induced cell death. Thus, the present studies were conducted to determine whether levels of mRNA encoding BCL-2 and related members of this gene family (BAX and BCL-Xshort, which induce apoptosis; BCL-Xlong, a BCL-2 homologue that prevents apoptosis) differed in functional (Day 21 of pregnancy) versus regressed (Day 21 of the estrous cycle) CL in the bovine ovary. Levels of mRNAs encoding p53, a transcriptional regulator of the bcl-2 and bax genes, and interleukin-1beta-converting enzyme (ICE), a protein recently implicated in the induction of apoptosis whose expression may be enhanced by oxidative stress, were also assessed. Partial cDNA clones encoding bovine bax, bcl-x, p53, and Ice were isolated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with total RNA prepared from functional or regressed CL. A bovine bcl-2 cDNA could not be isolated from luteal tissue RNA despite the use of several primer pairs for amplification. Total RNA was then extracted from functional or regressed CL and analyzed by Northern blot analysis. The occurrence of apoptosis in regressed CL, as evidenced by the presence of internucleosomal DNA cleavage, was associated with a significant increase in both bax and Ice mRNA levels as compared with levels of bax and Ice expression in functional CL (p < 0.05, n = 3). There were no significant differences in bcl-x or p53 mRNA levels in functional versus regressed CL. Analysis of bcl-x mRNA by RT-PCR revealed that the long form was the primary, if not only, mRNA expressed in functional and regressed bovine luteal tissue. On the basis of data that increased expression of bax is associated with, and may be required for, apoptosis in ovarian granulosa cells and germ cells, we propose that BAX may play a similar role in apoptosis induction during luteal regression. Moreover, the increased Ice mRNA levels in regressed CL provides the first evidence that the ICE family of death proteases may be involved in luteolysis.

Modulation of CCK mRNA in cell lines in response to isoproterenol and retinoic acid.

Regulation of cholecystokinin (CCK) expression was studied in the human neuroepithelioma cell line SK-N-MCIXC and the rat medullary thyroid carcinoma cell line WE 4/2. The cells were treated with the beta-adrenergic agonist isoproterenol and retinoic acid, a natural derivative of vitamin A, which plays a role in cell growth and proliferation. Levels of CCK mRNA were determined after 6, 12 and 24 h drug treatments, with Northern blot analysis using human CCK riboprobes. In WE 4/2 cells no differences were observed in CCK mRNA levels, between control and isoproterenol treated cells, after 6, 12 or 24 h treatments. In SK-N-MCIXC cells isoproterenol increased CCK mRNA levels at all time points examined, the beta-adrenergic antagonist propranolol blocked this effect. SK-N-MCIXC cells were also treated with actinomycin D or cycloheximide in combination with isoproterenol. Actinomycin D decreased CCK mRNA levels. Cycloheximide increased CCK mRNA levels when compared to isoproterenol acting alone. Retinoic acid did not affect CCK mRNA levels in WE 4/2 cells. In SK-N-MCIXC cells, retinoic acid consistently decreased CCK mRNA level. CCK mRNA levels in SK-N-MCIXC cells treated with retinoic acid combined with either isoproterenol or phorbol-12-myristate-13 acetate, were not significantly different from cells treated with retinoic acid alone.

Comparison of mRNA levels for the PGF(2α) receptor (FP) during luteolysis and early pregnancy in the ovine corpus luteum.

Prostaglandin F(2α) (PGF(2α)) is the physiological signal that triggers luteolysis in the non-pregnant ewe. This is associated with a decline in circulating levels of progesterone, beginning around day 14 of a 16-17 day estrous cycle. Recently, the receptor for PGF(2α) (FP) was cloned from an ovine day 10 large luteal cell cDNA library. The purpose of this study was to measure relative abundance of FP mRNA as it may change with luteolysis during the luteal phase. Corpora lutea (CL) were collected from ewes on day 10, 12, 14 or 16 (n≥4/day; day 0=synchronized estrus); 12 h after PGE(2α)-treatment on day 10, 12 or 14 (n≥3/day) of the estrous cycle; or on day 16 of pregnancy (n=6). Pregnancy was confirmed by visualization of the conceptus. Blood samples were collected 12 h prior to and at the time of tissue collection to determine levels of progesterone. Serum concentrations of progesterone declined with the onset of luteolysis in control animals (day 14, day 16;P<0.05) as well as 12 h following PGF(2α)-treatment (day 10, day 12;P<0.05). Genomic DNA from these tissues was prepared and visualized by agarose gel electrophoresis. Internucleosomal fragmentation (indicative of apoptosis) was seen in CL from animals in which luteolysis had been initiated (all PGE(2α)-treated and day 16 control ewes), but not in ewes with functional CL. Total RNA isolated from each CL was separated through a denaturing 1% agarose gel, transferred to nylon membranes and hybridized to a radioactive ovine FP cDNA probe. Hybridization to a radiolabeled 18S ribosomal cRNA probe was used to confirm equal loading of RNA in each lane. By northern analysis, a major transcript was seen at ∼6.1 kb. A relatively high level of FP mRNA was measured in CL collected from control non-pregnant ewes during the mid luteal phase (day 10, 2.73±0.17; day 12, 2.47±0.91; FP/18S ratio), but varied among animals (3.09±1.59) on day 14. Administration of PGF(2α) resulted in the lowest amounts of FP mRNA on days 10, 12, and 14 (P<0.05). Amounts of FP mRNA were higher (P<0.05) on day 16 of pregnancy as compared to day 10 (by 1.9-fold) or to day 16 (by 5.9-fold) of the estrous cycle. From these observations we conclude that PGF(2α) or some event associated with luteolysis appears to down regulate amounts of the FP mRNA. Furthermore, pregnancy, and/or the antiluteolytic signals associated with maternal recognition of pregnancy may prevent the decline in the amount of FP mRNA.

Differential protein-DNA interactions at the promoter and enhancer regions of developmentally regulated U4 snRNA genes.

In the chicken genome there are two closely-linked genes, U4B and U4X, that code for different sequence variants of U4 small nuclear RNA (snRNA). Both genes are expressed with nearly equal efficiency in the early embryo, but U4X gene expression is specifically down-regulated relative to U4B as development proceeds. At the present time, little is known about the mechanisms that regulate differential expression of snRNA genes. We have now identified a novel chicken factor, PPBF, that binds sequence-specifically in vitro to the proximal regulatory region of the U4X gene, but not to the proximal region of the U4B gene. PPBF is itself regulated during development and may therefore be a key factor involved in differentially regulating U4X gene transcription relative to U4B. The U4X and U4B enhancers contain distinct sequence variants of two essential motifs (octamer and SPH). The Oct-1 transcription factor binds with similar affinities to both the U4X and U4B octamer motifs. However, a second essential snRNA enhancer-binding protein, SBF, has a 20- to 30-fold lower affinity for the SPH motif in the U4X enhancer than for the homologous SPH motif in the U4B enhancer. A potential role therefore exists for SBF, as well as PPBF, in the preferential down-regulation of the U4X RNA gene during chicken development.

Developmental and tissue-specific expression of U4 small nuclear RNA genes.

U4 RNA is one of several small nuclear RNAs involved in the splicing of mRNA precursors. The domestic chicken has two genes per haploid genome that are capable of encoding U4 RNA. The U4X RNA gene (which encodes a sequence variant of U4 RNA that was unknown prior to the cloning of the gene) and the U4B RNA gene were both expressed in vivo in each of seven adult and three embryonic chicken tissues examined. However, the ratio of U4B RNA to U4X RNA can vary more than sevenfold in both a tissue- and stage-specific manner.