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1.
We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion. 相似文献
2.
To determine if soluble factors (other than steroids) secreted by bovine thecal cells may be involved in local regulation of follicular development, we examined the effects of thecal cell secretory products on the growth of granulosa cells obtained from the same follicles. DNA synthesis (assessed by the incorporation of 3H-thymidine) by granulosa cells plated on coverslips and cocultured with, but not directly in contact with, thecal cells in organ culture dishes in a serum-free medium was 5-fold greater than controls. The effect of the thecal cell-secreted products on DNA synthesis by granulosa cells was significantly higher than the maximum response produced by epidermal growth factor (EGF). Thecal cell-conditioned medium stimulated 3H-thymidine incorporation into the DNA of granulosa cells and a normal rat kidney cell line in a dose-dependent manner. The increases in 3H-thymidine incorporation into granulosa cell DNA subsequently lead to an increase in cell number. Preliminary characterization studies using ultrafiltration membranes indicated that the mitogenic factor was retained in the greater than 10,000 molecular weight fraction. The activity was stable to heating at 90 degrees C for 5 min and was not extracted in ether. The thecal cell-generated growth factor may act as a paracrine regulator of granulosa cell growth, thus providing the dominant follicle with autonomy over other follicles in the cohort. 相似文献
3.
Actions of growth factors in the follicle 总被引:1,自引:0,他引:1
In this paper we have examined the possibility that soluble factors produced by the thecal and granulosa cells may be essential local modulators of follicular development. The observations that insulin could influence both the growth and the differentiation of granulosa cells were important in establishing the concept that peptides could act as amplifiers of the actions of gonadotrophins. Insulin alone did not influence aromatase activity significantly but acted synergistically with FSH to augment aromatase activity in rat granulosa cells. Unlike aromatase activity, insulin alone was able to significantly stimulate 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, the maximum level achieved approaching that obtained with high concentrations of FSH. To determine if insulin could influence other parameters of granulosa cell function in addition to steroidogenesis, we measured a component of extracellular matrix, fibronectin, previously shown to be inhibited by FSH. Treatment with insulin independently inhibited the increase in fibronectin secretion observed in control cultures. Also, insulin alone was able to stimulate quiescent bovine granulosa cells to incorporate [3H]thymidine into DNA under serum-conditions. The concentration of insulin required in these experiments was higher than physiological levels suggesting that other insulin-like growth factors may be involved. Our work and that of others has shown that IGF1 can mimic the actions of insulin and is effective at lower concentrations. A possible source of IGF1 production in the follicle was sought initially by collecting rat granulosa cell conditioned medium, and assessing biological activity and immunoreactivity. Conditioned medium augmented the actions of FSH on aromatase activity and alone stimulated 3 beta-HSD, indicating the presence of insulin-like bioactivity. A positive reaction on immunoblots using specific antiserum confirmed the presence of immunoreactive IGF1. Conditioned medium from thecal cells contained a growth-promoting activity (TcGF) that did not augment FSH-induced aromatase activity. The production of growth factors locally within the follicle may represent the self-amplifying mechanism that enables the dominant follicle to complete its developmental program and ovulate. 相似文献
4.
Estrogen regulation of progestin biosynthetic enzymes in cultured rat granulosa cells 总被引:2,自引:0,他引:2
The mechanism by which estrogens enhance gonadotropin-stimulated ovarian progestin production was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) in cultured rat granulosa cells. Cells from immature hypophysectomized, estrogen-treated rats were cultured for 3 days with follicle-stimulating hormone (FSH) and/or estrogens. Pregnenolone production was measured in the presence of cyanoketone which inhibits 3 beta-HSD activity. Activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. Some cells were also primed with FSH to induce luteinizing hormone (LH) receptors for studies on the effects of estrogens on LH-modulated parameters. Pregnenolone production by cultured granulosa cells was stimulated by FSH, while treatment with diethylstilbestrol (DES) or estradiol further enhanced the gonadotropin action. Treatment with FSH increased 3 beta-HSD activity. Similarly, concomitant treatment with DES further enhanced 3 beta-HSD activity in a dose-dependent manner with an apparent ED50 of 10(-8) M. Also, treatment with estrogens alone increased 3 beta-HSD activity. The increases in enzyme activity induced by estrogen alone or in combination with FSH were not associated with changes in the apparent Km values. FSH also stimulated 20 alpha-HSD activity by 2-fold in these cells, while concomitant treatment with DES did not affect the FSH action. In FSH-primed cells, LH stimulated pregnenolone production while the LH action was enhanced by concomitant treatment with the estrogens. Likewise, LH stimulated the activity of 3 beta-HSD, while concomitant DES treatment further augmented LH action. LH did not stimulate 20 alpha-HSD activity when added alone or in combination with DES. Thus, the estrogen enhancement of the gonadotropin-stimulated progesterone production in cultured rat granulosa cells is associated with increases in pregnenolone biosynthesis and the activity of the 3 beta-HSD enzyme, without affecting the 20 alpha-HSD activity. 相似文献
5.
Patricia E. Saragüeta Guillermo M. Lanuza J. Lino Baraao 《Molecular reproduction and development》1997,47(2):170-174
Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of 3H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10−11 M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED50 = 1.58 ± 0.22 × 10−10 M) and native GnRH (ED50 = 1.4 ± 0.3 × 10−6 M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10−8 M could prevent the inhibition elicited by 10−7 M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc. 相似文献
6.
Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions. 相似文献
7.
Alejandro A. Colman Lerner Daniel F. Salamone M. Eugenia Chiappe J. Lino Baraao 《Journal of cellular physiology》1995,164(2):395-403
A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compaed BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [3H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [3H]-progesterone to 5α-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-1 (IGF-I), and transforming growth factor-b? (TGF-b?) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [3H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-b? when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-b? displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues. © 1995 Wiley-Liss, Inc. 相似文献
8.
Luteinization stimulator (LS) is an intrafollicular compound which was shown to be released by granulosa cells under in vitro conditions with stimulatory effects on immature granulosa cell differentiation. This study was undertaken to determine the effects of various endocrine agents which are involved in the regulation of ovarian function on LS secretion by porcine granulosa cells isolated from 5-8-mm follicles (LGC). Cell conditioned media (CM) obtained after the 4-day culture of LGC were tested in the culture of immature (small) granulosa cells (SGC). The activity of LS released into the LGC conditioned medium was estimated by measuring progesterone (P4) produced by SGC in the presence of CM. Stimulation of P4 secretion was observed after addition of media from cultures treated by LHRH (10(-4) mol.l-1), epinephrine (10(-5) mol.l-1), LH (1 microgram.ml-1), dbcAMP (0.5 and 2.0 micrograms.ml-1) or insulin (1.0-5.0 micrograms.ml-1). Norepinephrine (10(-5) and 10(-7) mol.l-1), estradiol (0.1 and 1.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml-1) did not change steroidogenic activity of CM. Epinephrine and norepinephrine (10(-5) and 10(-7) mol.l-1), LH (1 microgram.ml-1), dbcAMP (2.0 microgram.ml-1) and estradiol (1 microgram.ml-1) alone enhanced P4 production by SGC, whereas LHRH (10(-3) and 10(-4) mol.l-1), insulin (1.0-5.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml.-1) did not have any effect. These observations suggest that the process of LS secretion in developing follicles is subject to a specific hormonal control. 相似文献
9.
Little is known regarding the role of insulin-like growth factor 2 (IGF2) and the regulation of the IGF2 receptor (IGF2R) during follicular development. Granulosa cells were collected from small (1-5 mm) and large (8-22 mm) bovine follicles and were treated with IGF2 for 1-2 days in serum-free medium, and steroid production, cell proliferation, specific (125)I-IGF2 binding, and gene expression were quantified. IGF2 increased both estradiol and progesterone production by granulosa cells, and cells from large follicles were more responsive to the effects of IGF2 than those from small follicles. Abundance of aromatase (CYP19A1) mRNA was stimulated by IGF2 and IGF1. The effective dose (ED(50)) of IGF2 stimulating 50% of the maximal estradiol production was 63 ng/ml for small follicles and 12 ng/ml for large follicles, and these values were not affected by FSH. The ED(50) of IGF2 for progesterone production was 20 ng/ml for both small and large follicles. IGF2 also increased proliferation of granulosa cells by 2- to 3-fold, as determined by increased cell numbers and (3)H-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for (125)I-IGF2 existed in granulosa cells, and 2-day treatment with estradiol, FSH, or cortisol had no significant effect on specific (125)I-IGF2 binding. Also, FSH treatment of small- and large-follicle granulosa cells had no effect on IGF2R mRNA levels, whereas IGF1 decreased IGF2R mRNA and specific (125)I-IGF2 binding. Granulosa cell IGF2R mRNA abundance was 3-fold greater in small than in large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R, thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R. 相似文献
10.
Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medium (16-25 mm), and large (25-48 mm) follicles were collected from cyclic mares (n = 14), cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells. 相似文献
11.
Asangla Ao Robert P. Erickson John R. D. Stalvey 《Molecular reproduction and development》1993,35(2):159-164
The factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle-stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8-fold within a 2-day period. In serum-free medium, human luteinized granulosa cells were able to incorporate 3H-thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, 3H-thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH- and estradiol-treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze-thawing, was stable for several weeks at – 20°C, became unstable at 4°C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight >30,000 dalton. We suggest that an inhibitory growth factor produced by human luteinized granulosa cells could be involved in the differentiation of growing follicles to corpus luteum. © 1993 Wiley-Liss, Inc. 相似文献
12.
R W Brueggemeier K Tseng N E Katlic M A Beleh Y C Lin 《Journal of steroid biochemistry》1990,36(4):325-331
Estrogen metabolism was evaluated in freshly isolated kidney and liver microsomes and in primary kidney cell cultures from Syrian hamsters, a potential experimental model for examining the possible role(s) of estrogens in tumor initiation and development. Initial velocity studies of the conversion of estradiol to 2-hydroxyestradiol, as determined by the 3H2O release assay with the substrate [2-3H]estradiol, resulted in similar apparent Kms of estrogen 2-hydroxylase of 2.85 and 6.25 microM for liver and renal microsomes, respectively. The apparent Vmax for freshly prepared liver microsomes was 0.13 nmol.mg-1.min-1, while that for renal microsomes was 0.040 nmol.mg-1.min-1. Evaluation of estrogen metabolism was also performed in primary cell cultures of hamster kidney cells, consisting of 75% epithelial cells. [6,7-3H]Estradiol (10 microM) was incubated for 0, 24 and 48 h in primary kidney cell cultures, and the organic soluble metabolites analyzed by reverse-phase HPLC. The cultures from untreated, castrated hamsters metabolize [3H]estradiol to yield small quantities of estrone and significant amounts of polar metabolites, while no catechol estrogens were isolated. Estrogen metabolism by diethylstilbestrol-treated (DES-treated) hamster kidney cell cultures also provided small quantities of estrone and no evidence of catechol estrogens. Additionally, larger amounts of additional polar metabolites were isolated in the cultures from DES-treated hamsters. Finally, levels of estrogen 2-hydroxylase were detected in these cultures using the 3H2O release assay. Thus, the short-term primary kidney cell cultures from the Syrian hamster are capable of metabolizing estrogens. Furthermore, the enzymatic processes appear to be available for the conversion of any catechol estrogens formed into more polar metabolites. These investigations in intact cells, capable of performing all biochemical processes, complement both in vivo and subcellular biochemical studies and may aid in elucidating the roles of estrogens and estrogen metabolism in the initiation and development of estrogen-induced, estrogen-dependent kidney tumors in the Syrian hamster. 相似文献
13.
Regulation of follicular development by diethylstilboestrol in ovaries of immature rats 总被引:1,自引:0,他引:1
Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (less than 200 microns, 200-400 microns and greater than 400 microns) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes. Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36-48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries. These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully. 相似文献
14.
Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function. 相似文献
15.
Growth hormone (GH) and insulin-like growth factors (IGFs) are recognized as regulators of ovarian function. This study was designed to compare the effect of GH and IGFs added alone or together on porcine theca interna and granulosa cells proliferation and steroidogenesis. Moreover, the effect of GH on IGF-I secretion was examined. Cells were isolated from medium size follicles and cultured in vitro for 48 h in serum free medium. Estradiol and IGF-I medium concentrations were determined by radioimmunoassays. Proliferation was evaluated by alamar blue assay and by radiolabelled thymidine incorporation. GH increased IGF secretion by granulosa cells while decreased its secretion by theca cells. Proliferation of both cell types was stimulated by IGF-I and IGF-II (30 ng/ml) and modestly inhibited by GH (100 ng/ml). Insulin-like growth factor II increased, in a statistically significant manner, estradiol secretion by both cell types, while IGF-I stimulated estradiol secretion to a greater extent by granulosa then by theca cells. The synergistic action of GH and IGFs on estradiol secretion was stimulatory in theca cells and inhibitory in granulosa cells. These data demonstrate that despite its direct action on estradiol secretion by granulosa and theca cells, GH also modulated estradiol secretion induced by IGFs. Differences in the estradiol production in response to GH alone and the effect of the synergistic action of GH and IGFs suggest that different cellular mechanisms for these hormones are triggered in each cell type. 相似文献
16.
Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts 总被引:7,自引:0,他引:7
At concentrations of 25 ng/ml in serum-free medium, somatomedin C (SM-C) and insulin stimulated 3H-thymidine incorporation in adult human fibroblasts 4- and 1.5-fold, respectively. The presence of 0.25% human hypopituitary serum (HHS), which by itself had little effect, enhanced the mitogenicity of both SM-C and insulin. Furthermore, 10(-7)M dexamethasone dramatically potentiated SM-C stimulation (70-fold) and insulin stimulation (28-fold) of 3H-thymidine incorporation. With dexamethasone and 0.25% HHS, significant stimulation of DNA synthesis was seen at 2.5 ng/ml for both SM-C and insulin. The effects of SM-C and insulin on 3H-thymidine incorporation were additive. These 3H-thymidine incorporation results were clearly supported by cell replication studies. On the other hand, SM-C and insulin had equivalent, nonadditive effects on RNA and protein synthesis and protein degradation. Half-maximal effects were seen for both peptides on all three metabolic processes at 2-5 ng/ml. In contrast to their synergism with SM-C in the stimulation of DNA synthesis and cell replication, HHS and dexamethasone did not enhance SM-C stimulation of RNA or protein synthesis or protein degradation. These data indicate that SM-C and insulin stimulate DNA, RNA, and protein synthesis, protein degradation, and cell replication in adult human fibroblasts at nanomolar concentrations, suggesting that each peptide is capable of acting through its own receptor. Both SM-C and insulin are also capable of synergism with low concentrations of serum and dexamethasone in the stimulation of DNA synthesis and cell replication. It is proposed that SM-C and insulin both participate in the regulation of cell growth and metabolism in vivo. 相似文献
17.
Ethanol inhibits hormone stimulated hepatocyte DNA synthesis 总被引:4,自引:0,他引:4
Insulin, glucagon, and epidermal growth factor (EGF) addition stimulated DNA synthesis in primary hepatocyte cell cultures prepared from adult rat liver. The addition of ethanol (20-200mM) to the culture medium resulted in a substantial reduction in DNA synthesis as measured by 3H-thymidine incorporation and autoradiography. This effect was specific for differentiated hepatocytes compared to fibroblasts and two other human hepatoma cell lines. These studies demonstrate in a cell culture system that one of the major properties of ethanol is the inhibition of hepatocyte DNA synthesis. 相似文献
18.
L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium. 相似文献
19.
The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells 总被引:6,自引:0,他引:6
The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge. 相似文献