Regulation of ovine pituitary glycoprotein hormone alpha subunit mRNA by 17 beta-estradiol in cell culture

Messenger RNA (mRNA) activity for the alpha glycoprotein subunit in ovine pituitary cell cultures was quantified using a wheat germ translation assay. [35S] Cysteine alpha translation product normally accounted for 0.3-0.9% of total 35S-product in translations of total culture RNA. Treatment of cultures with 1 nM 17 beta-estradiol (E2) decreased alpha mRNA activity by 50% and 92% after 1.8 and 8 days, respectively. Alpha mRNA activity decreased by 1/2 in 51 h, which is about three times slower than the rate of decrease reported for follicle-stimulating hormone (FSH) beta mRNA activity in similarly treated cultures (Alexander and Miller, 1982). The effective dose (ED50) for E2 in vitro was 30 pM (8 pg/ml), which is within the physiological range for ewes. These results indicate that E2 can act directly on the ovine pituitary to decrease alpha mRNA activity coordinately with mRNA activity for FSH beta.     

Complete sequence of ovine alpha s2-casein messenger RNA

The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein.     

An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization.

A progressive reduction in the size of rat metallothionein-1 mRNA following induction by copper chloride or dexamethasone was demonstrated on RNA blots, and was shown to be due to shortening of the poly(A)-tail. The rate of poly(A) removal was the same in rat liver and kidney following copper chloride induction, in rat liver following dexamethasone induction, and in mouse liver following copper chloride induction. In mouse liver metallothionein-1 and 2 mRNAs were shortened at the same rate. The reduction of the poly(A) tail was more rapid in the first 5 hours (approximately 20 nucleotides/h) but much slower (approximately 3 nucleotides/h) after the poly(A)-tail had been reduced to about 60 residues. Metallothionein mRNA molecules with poly(A) tail sizes less than 30-40 nucleotides were not observed. Exonuclease digestion of the poly(A)-tail is suggested, at least in the initial rapid phase. It is hypothesized that poly(A)-tails longer than 30 are required for mRNA stability and that much longer poly(A) tails may give newly synthesized mRNA molecules a competitive advantage in protein synthesis.     

High-resolution analysis of gro alpha mRNA poly(A) shortening: regulation by interleukin-1 beta.

We have previously shown that destabilization of gro alpha mRNA is associated with poly(A) shortening. In this study, we used high-resolution Northern blots to determine the rate and extent of gro alpha mRNA poly(A) shortening. gro alpha mRNA was found to undergo complete deadenylation within 2 h following withdrawal of IL-1. However, the process was not uniform: at 1 h following IL-1 withdrawal, gro alpha mRNA poly(A) lengths ranged from 0 to 180 nucleotides. There was an accumulation of deadenylated gro alpha mRNA which suggested that there may be another step before the mRNA is destroyed. Cycloheximide was found to block gro alpha mRNA degradation at the level of poly(A) shortening. Northern blots revealed a previously unrecognized periodic distribution of poly(A) lengths that was consistent with endonucleolytic cleavage between complexes of poly(A)-binding protein. The findings indicate that the degradation pathway of gro alpha mRNA is a slower version of the c-fos mRNA model, with the important additional feature that deadenylation and degradation are subject to physiologic regulation. This study provides a detailed picture of gro alpha mRNA poly(A) shortening and establishes a basis for further investigation of the mechanism by which IL-1 stabilizes specific mRNAs.     

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.     

A comparison of the anti-luteolytic activities of recombinant ovine interferon-alpha and -tau in sheep

Interferon tau (IFNT) is secreted by the trophectoderm of ruminant conceptuses during the peri-implantation period and serves an anti-luteolytic function. The question as to whether IFNT is superior as an anti-luteolytic agent to closely related Type I IFNs, such as IFN alpha (IFNA), which have a different function, remains unanswered. Thus, the aim of this study was to determine whether equivalent antiviral (AV) units of ovIFNA and ovIFNT are equipotent in extending estrous cycle length. Four distinct ovIFNA mRNA (ovIFNA1-4) were cloned from ovine lymphocytes. Recombinant ovine IFNs (ovIFNT4 and ovIFNA1) were prepared in the yeast Pichia pastoris. The AV activity of the purified IFNs was determined on a bovine cell line (MDBK) and on transformed ovine luminal uterine epithelial cells. Indwelling uterine catheters were fitted into crossbred ewes on Day 3 postestrus (Day 0 = estrus). Between Days 10 and 18 postestrus, ewes received twice-daily infusions of 0.7 x 10(7) IU of either ovIFNA1 or T4, plus serum albumin. Control ewes received serum albumin only. Daily blood samples were collected for progesterone determination, and ewes were monitored twice daily for estrus. Both ovIFNA (P = 0.04) and ovIFNT (P = 0.01) caused estrous cycle extension in nonpregnant ewes compared to controls when administered at equivalent AV doses. In conclusion, the uniqueness of IFNT as an anti-luteolytic agent most likely resides in its unique expression pattern rather than its special biopotency.     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estradiol on gonadotropin subunit messenger ribonucleic acid amounts during an induced gonadotropin surge in anestrous ewes

The preovulatory gonadotropin surge in the sheep was recently characterized by a divergent pattern of LH beta and FSH beta mRNAs immediately preceding this event. It is not clear whether this pattern is due to estradiol (E2), inhibin or other effectors. In this study, to determine if E2 may be involved in the divergent beta mRNA patterns seen during the surge, gonadotropin surges were induced in anestrous ewes (An) by E2 (An + E2) and several parameters were then measured. These included the amounts of alpha, LH beta, and FSH beta mRNAs, as assessed by solution hybridization assays, plus pituitary and serum gonadotropin concentrations. The values were compared with those observed in control, An ewes, to assess the effect of E2. The E2 treatment resulted in LH and FSH surges that appeared to be similar to the normal surges seen during the breeding season. Concomitantly, the E2 treatment lowered pituitary concentrations of FSH (P less than 0.05), while LH amounts did not change. Although the effect of E2 on gonadotropin subunit mRNA amounts varied depending upon the individual subunit, the changes that were observed paralleled changes reported during the preovulatory surge of the cycle. Specifically, alpha mRNA amounts increased significantly (P less than 0.001) while FSH beta mRNA amounts fell dramatically (P less than 0.001). Moreover, LH beta mRNA amounts were slightly increased, although not significantly by E2. These results demonstrate that E2 effects changes in the amounts of the gonadotropin subunit mRNAs during an induced gonadotropin surge in An ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secondary structure at the beginning of the poly(A) sequence of mouse beta-actin messenger RNA.

A portion of the beta-actin mRNA of mammalian cells is believed to lack a poly(A) tail, because of its failure to bind to oligo(dT)-cellulose. S1 mapping and Northern blot analysis of this mRNA shows it to contain a poly(A) sequence of about 60 nucleotides. Only about 20-40 nucleotides are available for interaction with oligo(dT). The rest is masked, presumably by base-pairing with a poly(U) stretch present in the 3' non-coding region of the mRNA. A similar configuration occurs in the bulk of the actin mRNA, which carries a poly(A) tail with sizes ranging from approximately 60 to 200 nucleotides.