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.     

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.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney, prostate, seminal vesicles and muscle were analysed for 3H-testosterone, 3H-5alpha-dihydrotestosterone (5alphaDHT) and 3H-5alpha-androstanediol (Adiol). When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5alphaDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 mug/day) for 4 days decreased and 3H-5alphaDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 1/24 h/100 g body wt to 22.6 1/24 h/100 g body wt.     

A cyp19a1b‐gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens. genesis 47:67–73, 2009. © 2008 Wiley‐Liss, Inc.     

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.     

Plasma netrin-1 is a diagnostic biomarker of human cancers

Context:?The axon guidance cues netrin-1 is a secreted protein overexpressed in many different cancer tissues.

Objectives:?To determine whether plasma netrin-1 can be used as a diagnostic biomarker of human cancer.

Materials and Methods:?A total of 300 cancer plasma samples from breast, renal, prostate, liver, meningioma, pituitary adenoma, glioblastoma, lung, pancreatic and colon cancer patients were compared against 138 control plasma samples. Netrin-1 levels were quantified by ELISA and immunohistochemistry.

Results:?Plasma netrin-1 levels were significantly increased in breast, renal, prostate, liver, meningioma, pituitary adenoma, and glioblastoma cancers as compared to control samples.

Discussion and Conclusion:?Our results suggest that plasma netrin-1 can be used as a diagnostic biomarker for many human cancers.     

Relationship between leptin receptor and polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders, which is involved in the multi-system disease, and its etiology is still not clearly understood. It is currently considered that not only the genetic factors but also the environment factors play a crucial role in the pathogenesis of PCOS. Obesity plays an important role through the insulin, leptin and endocannabinoid system in the pathological process of PCOS, leading to more severe clinical manifestations. The aim of our present study is to investigate whether there is association between single nucleotide polymorphisms (SNPs) of Gln223Arg and Pro1019Pro in the leptin receptor gene (LEPR) and PCOS in a Korean population. Interestingly, a significant association was found between the Pro1019Pro in LEPR gene and PCOS, and a highly significant association was found between the Gln223Arg in LEPR gene and PCOS (P = 0.033, OR = 1.523, 95% confidence interval and P < 0.0001, OR = 0.446, 95% confidence interval). Moreover, genotype combination and haplotype analyses indicate that Gln223Arg and Pro1019Pro polymorphisms of LEPR are significantly associated with the risk of PCOS.