Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

人子宫内膜纤蛋白溶酶元激活因子及其抑制因子的分布与调控

人子宫内膜中存在组织型(tPA)及尿激酶型(uPA)两类纤蛋白溶酶元激活因子,其含量在增殖期高于分泌期。本文应用免疫组织化学定位证实uPA及tPA两类抗原存在于子宫内膜的腺体细胞和间质细胞中。应用SDS-PAGE分高蛋白质,继而应用纤蛋白-琼脂糖铺盖技术测得离体培养下间质细胞仅释放tPA,腺体细胞仅释放uPA,但两种细胞均分泌PA的抑制因子(PAI)。培液中加入孕酮,明显抑制PA和刺激PAI生成。雌二醇作用与孕酮相反。某些肽类激素hCG、PRL、GnRH及cAMP作用基本与雌二醇相同。但福司克林(FK)则刺激间质、腺体两种细胞产生tPA及少量uPA,抑制PAI生成。本工作表明人子宫内膜中存在PA及PAI作用相反的酶,受激素调控,其生理意义尚待进一步探讨。     

Absence of sexual dimorphism in the goat: Induction of luteinizing hormone discharge in the castrated male and female and in the intersex with estradiol benzoate

Gonadectomized male (n = 5) and female (n = 5) and intact intersex goats (n = 2) were injected i.m. with 50 mug 17(beta)-estradiol benzoate (EB). After treatment, there was a transient 6- to 9-hr decrease in circulating levels of LH followed by a preovulatory-like discharge of LH in all goats. Release peaked at 12 to 18 hr after EB treatment. The magnitude of discharge and the time from treatment until peak of release were not influenced by the goat's sex. These findings suggested that the positive feedback effects of estrogen on LH release were not sexually differentiated in the goat. Since tonic concentrations of LH prior to EB treatment were not different among the groups, the studies also suggested that the intersex goats lacked the inhibitory gonadal influences on gonadotropin release that characterize intact animals.     

Influence ofl-thyroxine,l-triiodothyronine,thyroid stimulating hormone,or estradiol on the cell kinetics of cultured mammary cancer cells

Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T₃),l-thyroxine (T₄), a thyroid-stimulating hormone (TSH), and/or estradiol (E₂: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G₂+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T₃, T₄, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S+G₂+M fraction as does E₂. Furthermore, our data indicate that E₂ and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics. This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium).     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

酪氨酸对大鼠子宫胞浆雌,孕激素受体的影响

采用放射受体分析法,测定了动情周期不同阶段及去卵巢大鼠子宫胞浆雌二醇和孕酮受体含量,并观察了子宫腔内注射酪、丝、苏三种氨基酸对子宫胞浆雌。醇、孕酮受体含量的影响。结果表明：（１）Ｌ－酪氨酸对动情前期、动情期、间情期大鼠子宫胞浆雌二醇和孕酮受体都具有明显的降低作用。（２）Ｌ－酪氨酸也降低去卵巢大鼠子宫胞浆雌二醇和孕酮含量,即这一作用不是通过影响卵巢激素分泌实现的。（３）Ｌ－苏氨酸仅可降低动情期和间情期大鼠子宫胞浆孕酮受体含量,而对相应周期雌二醇受体没有明显作用。（４）Ｌ－丝氨酸和Ｌ－苏氨酸对去卵巢大鼠子宫胞浆雌二醇和孕酮受体均无影响。     

Effects of prenatal morphine on hypothalamic metabolism of neurotransmitters and gonadal and adrenal activities,during the early postnatal period in the rat

It is noteworthy that exposure to opiates during fetal development results in permanent changes in adults related to morphological, behavioral and biochemical measures; however little is known concerning the effects of such drugs in early postnatal life. We investigated in newborn rats the effects of prenatal morphine-exposure on both—the hypothalamic metabolism of norepinephrine (NE), serotonin (5 HT) and neuropeptide Y (NPY)—the activity of the hypothalamo-pituitary gonadal and adrenal axes. In a previous study performed in newborns of untreated mothers, we reported some sex-dependent changes in the metabolism of NE, 5 HT and NPY in the hypothalamus and an early activation of the gonadostimulating function and of the corticostimulating one. In control newborns from saline-treated mothers, a slight increase in the hypothalamic metabolism of NE (males) and 5 HT (males and females) was observed and it was comparable in both sexes. On the other hand, the hypothalamic content of NPY was unaffected in early postnatal period in newborn males as well as in females. These changes observed on hypothalamic metabolisms are temporally correlated with the early postnatal activation of the corticostimulating function in neonates of both sexes and that of the gonadostimulating one, mainly in males. Prenatal morphine exposure altered the hypothalamic metabolism of 5 HT which was increased mainly in newborn females but did not affect either the metabolism of NE or the NPY content of the hypothalamus. The more drastic effect of the prenatal morphine treatment is the atrophy and hypoactivity of the adrenals in newborns of both sexes at birth time and during the early postnatal period. In contrast morphine did not impair postnatal surge of the plasma testosterone level in male pups as well as late and slight increase of plasma estradiol in female ones.     

Influence of elevated ambient temperature after breeding on plasma corticoids, estradiol and progesterone in gilts

Thirty Yorkshire gilts were used to determine the influence of elevated ambient temperature during days 8 to 16 after breeding on concentrations of progesterone, corticoids and estradiol in plasma. Gilts were mated to a boar between 0800 and 1000 hr of the first day (day 0) and each subsequent day of estrus. On day 5 or 6, 22 gilts were anesthetized and a cannula was placed in the anterior vena cava. Gilts were randomly allotted to either control (23+/-1 C) or hot (35+/-1 C for 12 hr and 32+/-1 C for 12 hr daily) environmental chambers on day 8. Gilts were bled twice daily (0800 and 2000 hr) on days 9 through 16, then once daily until day 28. A third group of noncannulated gilts was bred and assigned to the control chamber. All cannulated gilts were injected intravenously with 25 IU of ACTH on day 16, and frequent blood samples were collected. Concentrations of progesterone in plasma were similar for heat-stressed and control gilts that were pregant. However, progesterone in plasma was reduced during days 13 to 19 in nonpregnant heat-stressed gilts compared to the pregnant and nonpregnant control gilts. Concentrations of estradiol in plasma were greater in nonpregnant heat-stressed gilts than in nonpregnant control and all pregnant gilts on days 10, 11 and 12 after estrus. Concentrations of corticoids and progesterone in plasma after infusion of ACTH on day 16 after breeding were reduced in heat-stressed nonpregnant gilts compared to heat-stressed pregnant, control pregnant and control nonpregnant gilts. These data indicate that reduced reproductive performance which occurs after exposure of gilts to increased ambient temperature during days 8 to 16 after breeding may be related to altered endocrine function.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Estradiol pulses induce progestin receptors selectively in substance P-immunoreactive neurons in the ventrolateral hypothalamus of female guinea pigs

Low doses of estradiol, administered as pulses, are as effective as higher doses for priming ovariectomized (OVX) guinea pigs to display progesterone-facilitated lordosis. High doses of estradiol, administered by constant-release implants, induce progestin receptors in many substance P-immunoreactive (SP-IR) neurons in the ventrolateral hypothalamus (VLH), a site at which estradiol primes OVX guinea pigs to respond behaviorally to progesterone. To test the hypothesis that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P-containing neurons in the VLH, OVX females received estradiol implants 1 week prior to perfusion, or two pulses of estradiol- 17β, injected 39 and 11 h before perfusion. Colchicine was administered intracerebroventricularly prior to perfusion. No significant differences were observed in the total number of progestin receptor-immunoreactive (PR-IR) or substance P-immunoreactive cells in the VLH and VLH/ventromedial hypothalamus (VMH), respectively, of females receiving the two estradiol treatments. However, the percentage of PR-IR cells in the VLH also immunoreactive for SP was significantly higher in the estradiol pulse-treated (53%), than in the estradiol capsule-implanted animals (36%). These data suggest that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P-containing neurons in the VLH and are consistent with the hypothesis that substance P is involved in progesterone-facilitated lordosis in guinea pigs.