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1.
The ability of rabbit endometrium to synthesize estrogen was investigated. Aromatase activity was measured by incubating the viable tissue fragments with [3H] testosterone at 37 degrees C and quantitated the [3H]estrogens at the end of 6 h incubation. The activity was not detectable in endometrium and myometrium of non-pregnant rabbits. In pregnant rabbit, the activity increased from 2 to 12 fmol/h/g of tissue in endometrium obtained from both gravid and nongravid uterine horns 4-8 days after mating. Aromatase activity in endometrium at implanted sites on the 8th day after mating reached up to 550 fmol/h/g of tissue. Hormonal regulation of the aromatase activity was studied directly in cultured rabbit endometrial stromal cells. Both progesterone and medroxyprogesterone acetate stimulated the activity with 2- to 25-fold increase over the control values (0.2-1 fmol/h/mg protein). The stimulation of aromatase activity by progestin was found to be both time- and dose-dependent. Estrogen, Bu2cAMP, forskolin, HCG or relaxin had no effect on the activity. Aromatase activity in glandular epithelial cells was neither detectable nor affected by progestin. These results indicate that aromatase activity in rabbit endometrium is concentrated in stromal cells and progestin stimulates the activity. However, estrogen, forskolin and relaxin which enhanced the stimulation of the activity by progestin in human endometrium had no effect on aromatase in rabbit endometrium. The increase of aromatase activity observed after the onset of conception may be physiologically important to increase the local estrogen content for decidualization of the endometrium. 相似文献
2.
Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium 总被引:1,自引:0,他引:1
Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction. 相似文献
3.
Activation of bovine and baboon primordial follicles in vitro 总被引:5,自引:0,他引:5
Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles. 相似文献
4.
Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 x 10(-7) M), (3) with cortisol (1 x 10(-7) M) and (4) with thyroxine plus cortisol (1 x 10(-7) M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16:1(cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal delta 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine. 相似文献
5.
Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts 总被引:14,自引:1,他引:13
The process of mammalian implantation has been investigated using an in vitro model system wherein the trophoblast cells of mouse blastocysts attach to and outgrowth on tissue culture plates containing a complex medium. We now report that two extracellular matrix glycoproteins, fibronectin and laminin, when individually precoated on tissue culture plates promoted in vitro attachment and outgrowth of mouse blastocysts in serum-free medium. The kinetics of attachment and outgrowth processes in the presence of either of these two proteins were identical to that observed in complex, serum-containing medium. In contrast, plates containing a collagen matrix or pretreated with a variety of other serum proteins or various lectins failed to support in vitro attachment and outgrowth of blastocysts. Because all components of the culture medium are defined and both fibronectin and laminin are known components of the basement membrane of the endometrium, this in vitro system offers considerable advantages over the serum supplemented system to study in vitro implantation. 相似文献
6.
Sharma M Shubert DE Sharma M Lewis J McGarrigle BP Bofinger DP Olson JR 《Chemico-biological interactions》2003,146(3):237-249
Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium. 相似文献
7.
Slices of human endometrium obtained from hysterectomy specimens were cultured for 48 hours in an organ culture medium supplemented with ethanol (control, vehicle), 17-β-estradiol (.5 μg/ml), or progesterone (.5 μg/ml). Uncultured endometrial tissue, cultured tissues, and the media were assayed for prostaglandin F (PGF) by a radioimmunoassay technique. Hematoxylin and eosin and periodic acid-Schiff stain histologic controls were done on all tissues. The concentrations of PGF in picograms/milligram, corrected for percent recovery, in the differently treated tissues were: preculture 298; culture control 2210; estrogen-treated 2680; progesterone-treated 1260. All differences except those between estrogen and control (p >; .10) and progesterone and control (p < .10) are significant at the p = .02 level or better. Progesterone appears to inhibit PGF synthesis which occurs during in vitro culture of human endometrium; estrogen tended to increase PGF synthesis in this system. 相似文献
8.
Testosterone stimulates the primary to secondary follicle transition in bovine follicles in vitro 总被引:1,自引:0,他引:1
The mechanisms controlling the initiation and early stages of follicular growth are poorly understood. Our laboratory developed a serum-free culture system that supports spontaneous and wholesale activation of primordial follicles in pieces of cortex dissected from the ovaries of fetal calves and fetal baboons. However, very few follicles activated in vitro progressed to the secondary stage. To determine whether androgens can promote the primary to secondary follicle transition, pieces of fetal bovine ovarian cortex were cultured in serum-free medium in the absence or presence of testosterone (T, 10(-7) and 10(-6) M) or estradiol (E(2), 10(-6) M) for 10 days. Cortical pieces were then fixed and embedded in plastic for serial sectioning and morphometric analysis; fresh cortical pieces fixed on Day 0 served as uncultured controls. Freshly isolated cortical pieces contained mostly primordial follicles, whereas after 10 days in vitro, most primordial follicles had activated, differentiating into primary follicles as expected. Neither T nor E(2) affected the number of primordial and primary follicles compared with controls (P > 0.05). However, T (10(-7) and 10(-6) M) increased the number of secondary follicles (P < 0.05), whereas E(2) had no effect, suggesting that the effect of T was not due to conversion of T to E(2). In the second experiment, the optimal concentration of T for preantral follicle growth was determined. A range of lower doses of T (10(-10)-10(-7) M) increased the number of secondary follicles in cultured cortical pieces in a dose-dependent manner, with 10(-7) M T being the most effective (P < 0.05). In the third experiment, addition of a specific androgen receptor blocker, flutamide, inhibited the stimulatory effects of T on the primary to secondary follicle transition (P < 0.05), suggesting a receptor-mediated action of T. Localization of androgen receptors by immunohistochemistry revealed immunostaining for the androgen receptor in ovarian stromal cells and increasing immunoreactivity in follicle cells as follicular development progressed from primordial and primary to secondary to antral follicles, suggesting the involvement of the androgen receptor in bovine folliculogenesis. In summary, our results show that T promotes the growth of bovine follicles activated in vitro and suggest that its stimulatory effect is mediated through androgen receptors in the stroma and/or follicular cells. 相似文献
9.
Gary N. Piquette Barry G. timms 《In vitro cellular & developmental biology. Plant》1990,26(5):471-481
Summary Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet
certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method
was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were
isolated from estrous rabbits and cultured for 6 d in 5% serum-supplementedd-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study
showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct
cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders
and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous
structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments
of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17β-hydroxysteroid dehydrogenase. However,
only GC contained delta 5-3β hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical
characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM
cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free
medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro.
This work was supported in part by Grant No. 202-3192 from the University of South Dakota Parsons Fund 相似文献
10.
de la Fuente I Alcalde I Gamboa OL Garrosa M Gayoso MJ 《Histology and histopathology》2012,27(1):95-102
Schwann cells (SCs) are basic elements for cell therapy and tissue engineering in the central and peripheral nervous system. Therefore, the development of a reliable method to obtain SC cultures is required. For possible therapeutic applications the cultures need to produce a sufficiently large number of SCs with a high level of purity in a relatively short period of time. To increase SC yield and purity we pre-degenerated pieces of 1-2 mm of adult rabbit sciatic nerves by incubating them for seven days in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and NRG1-β1. Following pre-degeneration the nerve pieces were dissociated and then cultured for 6 or 15 days in the same culture medium. After 6 days of culture we obtained around 9.5x103 cells/mg with approximately 94% SCs (S-100 positive) purity. After 15 days of culture the yield was about 80x103 cells/mg and the purity was approximately 75%. Pre-degeneration and subsequent culture of small pieces of adult nerve with NRG1-β1 supplemented medium increased the number of SCs and restricted the overgrowth of fibroblast-like cells. 相似文献
11.
A serum-free vaginal explant culture system was established to investigate the in vitro effect of estrogen on the growth of mouse vaginal epithelium. Vaginal explants were isolated from 40-day-old, ovariectomized BALB/cCrgl mice and cultured in a basal unsupplemented medium or in basal medium plus various doses of 17 beta-estradiol. Explants were processed for histology at the end of culture periods or were given 4-hour pulses of tritiated thymidine at various times and processed for autoradiography. Vaginal epithelium increased 3- to 5-fold in thickness and 2-fold in the number of epithelial cell layers during 72 hours of culture without estrogen; addition of estrogen did not significantly influence epithelial growth. Keratinization of vaginal epithelium occurred within 48 hours of culture in the absence of estrogen, and again addition of estrogen did not accelerate its appearance. Covering the explants with collagen decreased the estrogen-independent growth of vaginal epithelium. Autoradiography showed that ca. 70-90% of basal epithelial cells entered S phase during the initial 4 hours of culture and that this number declined rapidly after 48 hours to ca. 20%. Addition of 1.8 nM 17 beta-estradiol significantly decreased the labelling index of basal cells at 48 hours, but did not affect the labelling index at 24 and 72 hours. Stromal cells were not labelled at any time. Thus, DNA synthesis, cellular proliferation, and differentiation (keratinization) of vaginal epithelium in organ culture occurred without estrogen and were not stimulated by the addition of estrogen. 相似文献
12.
In vitro growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue. 总被引:5,自引:0,他引:5
A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue. 相似文献
13.
Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis. 相似文献
14.
Isachenko V Mallmann P Petrunkina AM Rahimi G Nawroth F Hancke K Felberbaum R Genze F Damjanoski I Isachenko E 《PloS one》2012,7(3):e32549
At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips. 相似文献
15.
The human endometrium is a dynamic tissue that undergoes cyclic changes under the influence of steroid hormones as well as numerous local paracrine and autocrine factors. Heat shock 70 kDa protein (HSPA5; also known as GRP78/BiP), a molecular chaperone within the endoplasmic reticulum, plays crucial roles in normal cellular processes as well as in stress conditions, in which it is a central regulator for the unfolded protein response (UPR). We hypothesized that HSPA5 expression level is variable throughout the menstrual cycle in human endometrium and that estrogen signaling cross-talks with UPR signaling by interacting with HSPA5. HSPA5 expression throughout the menstrual cycle was evaluated in vivo in normal human endometrium. Using in vitro techniques, we then assessed the bidirectional regulation of HSPA5 and estrogen signaling in human endometrial glandular (Ishikawa) and stromal cells (ESC). HSPA5 immunoreactivity in endometrial glandular and stromal cells was cycle-dependent, and was significantly higher in phases of the menstrual cycle when estradiol (E(2)) levels are known to be the lowest compared with the rest of the cycle (P < 0.001). E(2) did not affect HSPA5 expression after 8-24 h incubation in Ishikawa cells and ESC in vitro. However, tunicamycin-induced HSPA5 expression was significantly lowered in these cells when pretreated with E(2) (P < 0.01 and P < 0.05, respectively). On the other hand, tunicamycin decreased E(2) up-regulated alkaline phosphatase activity (P < 0.001). In conclusion, there is cycle-dependent HSPA5 expression with a possible inverse correlation between HSPA5 expression and E(2) levels in human endometrium. We suggest that estrogen signaling cross-talks with the UPR cascade by interacting with HSPA5, as supported by our in vitro findings. 相似文献
16.
H Honjo M Urabe K Naitoh Y Ogino J Kitawaki J Yasuda T Yamamoto H Okada 《Human cell》1989,2(3):254-259
Estrone sulfate (E1-S) has been shown to be quantitatively the most important estrogen in peripheral blood. But, the physiological and/or pathological role of E1-S is not yet clarified. At present, we tried to clarify it using tissue cultures. In tissue cultures of human endometrium, secretory endometrium showed higher activity of estrone sulfatase (E1----E1-S) than proliferative endometrium. Progesterone added in the medium induced an increase of estrone sulfotransferase in the proliferative endometrium. The results suggest a reducing effect of estrogen by progesterone in secretory endometrium in physiological conditions. Estrogen dependent malignant tumors (breast cancer, endometrial cancer) have high estrone sulfatase. It converts E1-S to E1 (----E2) which are abundant in these tumors. Ishikawa cell line increased estrone sulfotransferase activity with progesterone, somewhat like the physiological conditions. From out study in vivo, there is a possibility of some ameliorative effects of E1-S on the central nervous system of patients with senile dementia (Alzheimer's type). Effects of E1-S on central nerves were investigated using tissue cultures. 相似文献
17.
N Nakamura Y Nishizawa S Noguchi N Uchida B Sato K Matsumoto 《Journal of steroid biochemistry》1987,27(1-3):459-464
Shionogi carcinoma 115 (SC115) had been accepted for 20 yr as an androgen-dependent mouse mammary tumor. However, we recently found that the growth of SC115 tumors in vivo is also stimulated by pharmacological doses of estrogen through estrogen receptor. In the present study, action mechanisms of androgen or high doses of estrogen in the growth stimulation of SC115 were examined using a cloned cell line (SC-3) derived from the SC115 tumor. In serum-supplemented [2% steroid-free fetal calf serum-Eagle's minimum essential medium (MEM)] and serum-free [HAM F-12: MEM (1:1, v/v) containing 0.1% bovine serum albumin] media, testosterone (Test, 10(-9)-10(-6) M) significantly increased both cell number and DNA synthesis of SC-3 cells (by up to 10-fold), whereas oestradiol-17 beta (10(-12)-10(-6) M) had no such effects; the Test-induced growth was completely inhibited by the addition of a 100-fold molar excess of cyproterone acetate (CA). The serum-free medium cultured with SC-3 cells in the presence or absence of 10(-8) M Test was collected [conditioned medium (CM) or conditioned medium without Test (CM-)], and then Test in CM was removed by Gel filtration using Sephadex G-100 or inactivated by the addition of a 100-fold molar excess of CA. In the serum-free culture system, the addition of the CM without Test activity significantly enhanced both number of SC-3 cells and DNA synthesis in the cells, whereas CM(-) had no such effects. The present findings suggest that growth-stimulatory activities of androgen and high doses of estrogen on SC115 cells are mediated by growth factor(s), secreted from SC115 cells through androgen receptor and from some of nontransformed cells through estrogen receptor, respectively. 相似文献
18.
H. -P. Hohn E. Winterhager L. C. Busch M. M. Mareel Prof. Dr. Dr. H. -W. Denker 《Cell and tissue research》1989,257(3):505-518
Summary This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typcial for early pregnancy, to continue in vitro. This system appears to compare favorably with in vitro models so far proposed for the study of hormonal control of uterine function or for the investigation of cell-biological aspects of embryo implantation. The specific aim in the presented system is to provide approximate normal epithelium-stroma interrelationships. Fragments of endometrium consisting of epithelium and stroma were obtained during early pseudopregnancy and cultured on a gyratory shaker. Morphology was investigated by light microscopy, transmission and scanning electron microscopy. During the first two days the epithelium grows over the exposed stroma regenerating a complete epithelial lining. No central necrosis is found in the stroma for up to 6 days, and the tissue keeps its organotypic architecture although certain morphological differences can be observed between regenerated versus original epithelium. In the regenerating portion a stage-specific cell differentiation and the reformation of a basal lamina are missing. Progesterone substitution preserves cell morphology and allows to maintain, in vitro, the stage-specific pattern of cell organelles. Most characteristic is the induction of extensive fusion of epithelial cells. These large symplasms are comparable in size and structure to those formed in pregnancy in the implantation chamber in vivo. Only the superficial parts of the original (not the regenerated) epithelium are capable of progesterone-induced large-scale fusion. This organotypical culture system appears to be of potential value for in vitro studies on hormone action and on endometrial receptivity for embryo implantation.Abbreviations
d.p.c.
dies post coitum
-
d p. hCG
days after injection of hCG (dies post injectionem hCG)
-
FBS
fetal bovine serum
-
hCG
human chorionic gonadotropin
Preliminary results were presented by Denker et al. (1984) and by Hohn et al. (1984) 相似文献
19.
Specimens of proliferative and secretory human endometrium were incubated under organ culture or superfusion conditions and the levels of PGF2 alpha in the medium were measured by radioimmunoassay. Basal rates of PGF2 alpha output during short-term superfusions and long-term (1-2 day) batch incubations, performed on the same tissue specimens, were similar. Basal output of PGF2 alpha by proliferative endometrium (230-280 ng/mg protein X d) was significantly higher than that of secretory tissue under both experimental conditions. Estradiol (10(-8) M) increased PGF2 alpha output significantly (4-fold) only in secretory endometrium under organ culture conditions; Progesterone (10(-7) M) decreased it significantly (to 1/2-1/4 of the basal level) in both types of endometria during long-term incubations and in proliferative endometrium during superfusion. Glands isolated from proliferative and secretory endometrium produced PGF2 alpha during superfusion at a rate comparable to that of endometrial tissue under similar conditions. PGF2 alpha output by glands isolated from secretory endometrium increased significantly (3-fold) when estradiol was added to the superfusion medium. 相似文献
20.
Punyadeera C Kamps R Defrère S Dijcks F de Goeij A Ederveen A Dunselman G Groothuis P 《The Journal of steroid biochemistry and molecular biology》2008,112(1-3):102-109
We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle, 17beta-estradiol (17beta-E2, 1nM), oestrogen receptor (ER) antagonist ICI 164.384 (40nM), and 4-OH-tamoxifen (40nM), raloxifene (4nM), lasofoxifene (4nM) and acolbifene (4nM). Protein expression of ERalpha, ERbeta1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17beta-E2 increased the fraction of Ki-67 positive cells (p<0.001) by 55% in glands compared to the control. Raloxifene (4nM) increased (p<0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p<0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium. 相似文献