Effects of estradiol on the prolactin surges and the feedback mechanisms of gonadotropins in pseudopregnant rats

The influences of estradiol on the prolactin (PRL) surges and on the secretion of gonadotropins (LH and FSH) were investigated in the pseudopregnancy (PSP) of acutely ovariectomized rats. The four following experimental groups were prepared: 1) intact PSP as a control, 2) ovariectomy was performed on day 0 of PSP (OVX), 3) a Silastic tube containing estradiol was implanted for day 1-4 into the OVX rats (OVX-E 1-4), and 4) the Silastic tube was implanted for day 5-8 by the same manner into the OVX rats (OVX-E 5-8). In the OVX group nocturnal (N) PRL surges were observed at 0500 h on days 4, 8 and 12 examined, and increased secretions of LH and FSH were noted. In the OVX-E 1-4 group, the N PRL surge was suppressed on day 4, and the suppressed N PRL surge did not occur on day 8, after the removal of the implanted tubes. Diurnal (D) PRL surges with LH surges were observed at 1700 h on day 4 in these rats. Similarly, more remarkable results were obtained on days 8 and 12 in the OVX-E 5-8 group than in the OVX-E 1-4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa and luteal cells by prolactin

Regulation by PRL of aromatase (P450arom) mRNA and protein and estradiol (E) biosynthesis was examined in granulosa cells during early stages of luteinization in vitro and in vivo. PRL caused a dose-dependent (10-1000 ng/ml) decrease in P450arom mRNA and E biosynthesis (greater than 99%) in luteinized rat granulosa cells in vitro, even when the cells were cultured in the presence of insulin and hydrocortisone (hormones known to synergize with PRL to induce proteins in mammary tissue) or in the presence of forskolin (a nonhormonal stimulator of cAMP). PRL also prevented the marked increases in aromatase mRNA and E biosynthesis stimulated by FSH and forskolin in nonluteinized preovulatory granulosa cells in culture. These effects of PRL on granulosa cells in culture were specific for aromatase and were not observed for other proteins, such as cholesterol side-chain cleavage cytochrome P450 (P450scc) and alpha 2-macroglobulin. PRL also decreased P450arom mRNA and protein during the early stages of luteinization in vivo. PRL administered to rats beginning day 1 postovulation to mimic hormone release during pseudopregnancy reduced the progressive increase in P450arom mRNA occurring in corpora lutea on days 3-4 in ovulated rats not treated with PRL. CB 154, a dopamine agonist that inhibits pituitary release of PRL, caused P450arom mRNA and protein to decrease 50% if given to pregnant rats on days 8-10 of gestation, but increased P450arom mRNA and protein if given to pregnant rats on days 10-12 of gestation. These diverse effects of PRL in pregnancy suggest that placental factors may modify the response of luteal cells to PRL during gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Disparity in the effects of two N-methyl nicotinamides on poly(ADP-ribose) synthetase and macromolecular synthesis in hepatomas

The inhibitory effects of nicotinamide analogs on the activity of poly(ADP-ribose)) synthetase were compared to effects on precursor incorporation into macromolecules in three lines of hepatoma cells (Morris hepatomas 5123C, 7777 and HTC). N'-methylnicotinamide was a less effective inhibitor of poly (ADP-ribose) synthetase than was 1-methylnicotinamide while both these compounds had smaller inhibitory effects on the enzyme than were seen with nicotinamide or 3-aminobenzamide. On the other hand, the incorporation of [3H]thymidine into DNA and of [3H]uridine into RNA were inhibited by N'-methylnicotinamide in the concentration range 2-20 mM but not by 1-methylnicotinamide. Under the conditions examined there were no significant effects on the incorporation of [14C]lysine and [3H]leucine in hepatoma cells. The data indicated that the inhibitory effect of N'-methylnicotinamide on nucleic acid synthesis may be unrelated to action on poly (ADP-ribose) synthetase.     

Stage-specific initiation of milk synthesis in the ovariectomized pregnant mouse

Stage-specific initiation of milk synthesis was investigated in mice ovariectomized on days 7, 9, 11 and 13 of pregnancy, and the lactogenic response of the mammary gland was monitored by using the synthesis of casein, lactose content and PRL binding as parameters. Pregnant mice ovariectomized on day 7 or 9 showed no increase in these parameters 24 h after operation. After day 11 of pregnancy, a clear response was induced by ovariectomy, characterized by a low level of casein synthesis, small amounts of lactose and PRL binding, which increased linearly in a time-dependent manner thereafter. These results indicate that pregnant mice after 11 days are able to initiate synthesis of casein and lactose in parallel. Mammary glands were cultured with insulin, cortisol and PRL. The mammary explants of pregnant mice at 7 and 9 days showed active synthesis of casein after a lag period of 2 days, suggesting that the mammary gland in early pregnancy is less sensitive to PRL, probably due to the absence of an increase in PRL binding after ovariectomy.     

Pyridine nucleotide cycling and poly(ADP-ribose) synthesis in resting human lymphocytes

NAD is a critical cofactor for the oxidation of fuel molecules. The exposure of human PBL to agents that cause DNA strand breaks to accumulate can deplete NAD pools by increasing NAD consumption for poly(ADP-ribose) formation. However, the pathways of NAD synthesis and degradation in viable PBL have not been carefully documented. The present experiments have used radioactive labeling techniques to trace the routes of NAD metabolism in resting PBL. The cells could generate NAD from either nicotinamide or nicotinic acid. PBL incubated with [14C]nicotinic acid excreted [14C]nicotinamide into the medium. Approximately 50% of a prelabeled [14C]NAD pool was metabolized during 6 to 8 hr in tissue culture. Basal NAD turnover was prolonged threefold to fourfold by 3-aminobenzamide (3-ABA), an inhibitor of poly(ADP-ribose) synthetase. Supplementation of the medium with 3-ABA also prevented the accelerated NAD degradation that ensued after exposure of PBL to deoxyadenosine plus deoxycoformycin at concentrations previously shown to cause DNA strand break accumulation. These results demonstrate that quiescent human PBL continually produce NAD and utilize the nucleotide for poly(ADP-ribose) synthesis.     

Mutant cells defective in poly(ADP-ribose) synthesis due to stable alterations in enzyme activity or substrate availability

We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.     

The role of the young in the control of the hormonal events during lactation and behavioral weaning in the golden hamster

This study was designed to determine the relationship between the behavioral and physiological changes occurring in the hamster over the course of lactation. Experimental (E) females were given litters of six 3-day-old pups on Day 13 of lactation and allowed to raise them. Control (C) females raised their own litters of six pups. Groups of E and C females were decapitated at 1000 and 1700 hr when their pups were either 8, 18, or 28 days old. Nursing was observed for 1 hr on the 3 days prior to autopsy. Nursing behavior disappeared by Day 28 in C females. E females exhibited nursing behavior at levels equal to those observed in C females until E pups were 28 days old and 38 days had elapsed since parturition. Despite the fact that E mothers continued to nurse pups on day 18 postpartum when pups were 8 days old, E pups showed lower growth rates than did C pups. Prolactin (PRL) levels remained elevated when E pups were 8 days old even though E pups did not grow normally. PRL levels decreased over time in both C and E females and reached baseline by Day 28 although nursing behavior was still elevated in E females. Thus, nursing behavior did not stimulate PRL release during late lactation. Estradiol (E₂) levels in C females remained at baseline until Day 28 when levels increased. LH, FSH, and Progesterone (P) levels in C females showed a dramatic diurnal pattern which disappeared by Day 28, when levels dropped. Levels of E₂, P, LH, and FSH in E females closely paralleled those of C females only when groups were compared with respect to pup age. Thus, behavioral weaning and the return to estrous cyclicity appear to be dependent upon the age of the pups rather than the time elapsed since parturition. However, milk production and PRL release appear to be more closely tied to the number of days postpartum and can be dissociated from the amount of nursing behavior observed.     

Endocrinology of pregnancy and early pregnancy detection by reproductive hormones in reindeer (Rangifer tarandus tarandus)

The endocrinology was studied throughout pregnancy in reindeer (Rangifer tarandus tarandus) located in Oulu, Finland (65 degrees N, 25 degrees E) with 13 captive, semi domestic adult females. Blood samples were analyzed for plasma progesterone (P4), estradiol (E2) and estrone sulphate (E1SO4), 15-ketodihydro-PGF2alpha (PG-metabolite) and pregnancy associated glycoproteins (PAG). The mean plasma P4 concentration peaked twice during gestation: at around 24 and three weeks prior to calving. In pregnant females the plasma PAG concentration increased over basal concentrations 21-30 days after the estimated day of conception and peaked at the time of calving. The concentrations of E2 and E1SO4 remained low until 60 days before calving when a rapid increase was found for both hormones. The mean plasma concentration of PG-metabolite increased throughout pregnancy to a maximum at parturition. The estimated mean (range) gestation length was 216 (212-220) days. Judged from measures on reproductive organs collected from 86 free-ranging, semi-domestic female reindeer of unknown age presented for slaughter at Roros, Norway (63 degrees N, 11 degrees E) in the second week of December 1999, it was concluded that the breeding season lasted from early September until the end of November. The results also showed that plasma PAG concentration could provide a tool for detection of pregnancy in reindeer.     

Changes in mRNA levels of poly(ADP-ribose) polymerase during activation of human lymphocytes

The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) polymerase (ADP-ribosyltransferase, EC 2.4.2.30) was found to be very low in quiescent human lymphocytes and to increase at least 10-fold between 1 and 2 dyas after stimulation with the mitogen phytohaemagglutinin, staying high for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of poly(ADP-ribose) polymerase) but was not affected significantly by aphidicolin. Incubation of activated cells with cycloheximide for 2 h increased the expression slightly. These data demonstrate that, during lymphocyte activation, the level of mRNA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or immunochemistry.     

Inhibitors of poly(ADP-ribose) synthesis enhance X-ray killing of log-phase Chinese hamster cells

Postirradiation incubation of V79 Chinese hamster cells with inhibitors of poly(ADP-ribose) synthesis was found to potentiate the killing of cells by X rays. Potentiation increased with incubation time and with concentration of the inhibitor. Preirradiation incubation had only a small effect. The enhanced response correlated well with the known extent of the inhibition of poly(ADP-ribose) synthesis. A radiation-sensitive line, V79- AL162 /S-10, was affected to a lesser extent than the normal cells. Cells repaired the radiation damage with which the inhibitors interacted within 1 hr, a process that has similar kinetics to what is observed when a postirradiation treatment with hypertonic buffer is used [H. Utsumi and M. M. Elkind , Radiat . Res. 77, 346-360 (1979)]. However, the sectors of damage affected by inhibitors of poly(ADP-ribose) synthesis and hypertonic buffer do not entirely overlap. The inhibitor nicotinamide enhanced the killing mainly of late S-phase cells and did not affect cells at the G1/S border. It is concluded that the repair process(es) involving poly(ADP-ribose) synthesis is important for cell survival in repair-competent cells and that the radiation-sensitive cells that were examined are partially deficient in a repair pathway in which poly(ADP-ribose) participates.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.     

ADP-ribosyltransferase from hen liver nuclei. Purification and characterization

Chromatin-bound ADP-ribosyltransferase from adult hen liver nuclei was purified to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose. The ADP-ribosyltransferase has a pH optimum at 9.0 and does not require DNA for reaction. The purified enzyme has a molecular weight of 27,500 +/- 500. Agmatine sulfate, arginine methyl ester, histones, and casein proved to be effective acceptors for the ADP-ribose molecule. Among histones, H3 was most active, followed by H2a, H4, and H2b, in that order, the lowest activity seen with H1. With all the acceptors tested, the rate of nicotinamide release was in excess of the ADP-ribosylation. However, changes in the ratio of nicotinamide release to ADP-ribosylation seemed to depend on concentrations of the acceptor used. ADP-ribose-whole histones X adducts formed by ADP-ribosyltransferase served as initiators for poly(ADP-ribose) synthesis when these adducts were incubated in the presence of NAD, DNA, Mg2+, and the purified poly(ADP-ribose) synthetase, in which poly(ADP-ribose) formation can occur.     

Induction of poly(ADP-ribose) polymerase gene expression in lectin-stimulated human T lymphocytes is dependent on protein synthesis

The poly(ADP-ribose) polymerase mRNA level in quiescent T lymphocytes was low, but was significantly higher than that in B lymphocytes or monocytes. When T lymphocytes were stimulated with phytohemagglutinin, a prompt increase in the mRNA level was observed from 4 hours after stimulation. The level of poly(ADP-ribose) polymerase mRNA reached a maximum in the late G1 phase about 1-2 days after lectin stimulation, and then decreased gradually returning to the basal level 10 days after lectin stimulation. Cycloheximide abrogated increase in poly(ADP-ribose) polymerase gene expression suggesting that a newly synthesized protein(s) was involved in poly(ADP-ribose) polymerase gene induction in lectin-stimulated T lymphocytes.     

Antagonistic action of a tumor promoter and a poly(adenosine diphosphoribose) synthesis inhibitor in radiation-induced transformation in vitro

Transformation of mouse C3H 10T1/2 cells by X-irradiation in vitro was blocked by the addition of 1 mM 3-aminobenzamide, an inhibitor of polyadenosine diphosphoribose (poly[ADP-ribose]) synthesis immediately after irradiation. 3-Aminobenzamide also inhibited an increase in the frequency of transformants caused by the addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, 7 days after irradiation. These results demonstrate a role for poly(ADP-ribose) synthesis during the initiation and promotion stages of transformation. From previous studies it is known that poly(ADP-ribose) synthesis is stimulated by the DNA damage caused by X rays during initiation. During promotion, however, 12-O-tetradecanoylphorbol-13-acetate acted as a mitogen but did not induce detectable DNA damage, and we could detect no stimulation of poly(ADP-ribose) synthetase. The roles of poly(ADP-ribose) during initiation and during promotion must, therefore, be significantly different.     

Influence of poly(ADP-ribose) synthesis inhibitors on the repair of sublethal and potentially lethal damage in gamma-irradiated mammalian cells

The influence of poly(ADP-ribose) synthesis inhibitors on mammalian cell radiosensitivity was investigated. Four different inhibitors were studied: 3-methoxybenzamide, 3-aminobenzamide, 6-aminonicotinamide and nicotinamide. When exponentially growing or plateau phase cells are incubated before irradiation with non-toxic concentrations of these compounds, their radiosensitivity is enhanced except in the case of nicotinamide. The poly(ADP-ribose) inhibitors do not modify the repair of sublethal damage, but totally suppress the repair of potentially lethal damage, as shown by the survival of CHO cells and of a human osteosarcoma cell line.