Objective: To investigate the influence of diethylstilbestrol (DES) on the mRNA expressions of the androgen receptor (AR), estrogen receptor α (ERα), proliferating cell nuclear antigen (PCNA), and actin alpha 1 (ACTα1) in the gubernaculums testis of newborn mice and explore their action mechanisms. METHODS: A total of 140 male Kunming mice were randomly divided into a blank control, a dimethyl sulfoxide (DMSO) control, and 5 experimental groups to be treated subcutaneously with normal saline, DMSO, and DES at 0.02, 0.1, 0.5, 10 and 50 μg per kg of the body weight per day, respectively, at gestation days 9－17. On the first day after birth, the animals were sacrificed and the gubernaculums testis collected for detection of the mRNA expressions of AR, ERα, PCNA and ACTα1 by RT-PCR. RESULTS: Compared with the DMSO control, the experimental groups, particularly the DES 10 and 50 μg groups, showed significant increases in the mRNA expression of ERα (RE2 = 0.825, P <0.05), but remarkable decreases in those of AR, PCNA and ACTα1 (RA2 = 0.713, RP2 = 0.946, RT2 = 0.960, P <0.01), all in a dose-dependent manner. CONCLUSIONS The AR, ERα, PCNA, and ACTα1 mRNA are expressed in the gubernaculum testis of normal newborn mice, and their expression levels may be influenced by intervention with different concentrations of DES during the gestation. Exogenous estrogens may affect the proliferation and contraction of gubernaculum testis cells and consequently the normal development of the testis or even the whole male reproductive system by influencing the metabolism of ER and/or AR.
Actins ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Diethylstilbestrol ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Estrogen Receptor alpha ; metabolism ; Estrogens, Non-Steroidal ; pharmacology ; Genitalia, Male ; Gubernaculum ; drug effects ; metabolism ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Receptors, Androgen ; metabolism ; Testis ; drug effects ; metabolism

OBJECTIVETo study whether effect of aspirin plus low-dose diethylstilbestrol is more effective and safer than high diethylstilbestrol dose alone on prevention of ovariectomy-induced osteopenia and dyslipidemia.

METHODSThirty-eight 4-month-old female SD rats were divided into baseline (BAS) group (n=6), sham operation group (n=8) and ovariectomy (OVX) group (n=24). The OVX group was further divided into vehicle treatment group (n=8), diethylstilbestrol (30 μg/kg•d) treatment group (OVX+D30 group, n=8), and aspirin (9 mg/kg•d) plus diethylstilbestrol (10 μg/kg•d) treatment group (OVX+A-D10 group, n=8). Their left tibiae were collected for the bone histomorphometric analysis in undecalcified sections. Left femurs were collected for the bone mineral density measurement.

RESULTSThe body weight and serum cholesterol were increased, while uterine weight and cancellous bone mass were decreased in OVX rats compared with the SHAM group. Cancellous bone mass was significantly increased, while body weight and bone resorption parameters were decreased in both A-D10 and D30 treatment group compared with OVX group. The rats treated with A-D10 showed significantly increased in bone formation parameters and decreased in serum triglyceride compared with the D30-treated rats.

CONCLUSIONAspirin plus low-dose diethylstilbestrol can effectively prevent osteopenia by reducing bone resorption, and is thus a better treatment modality for preventing dyslipidemia than high-dose diethylstilbestrol alone.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; therapeutic use ; Aspirin ; pharmacology ; therapeutic use ; Biomarkers ; blood ; Body Weight ; drug effects ; Bone Density ; Bone Diseases, Metabolic ; blood ; prevention & control ; Bone and Bones ; drug effects ; Diethylstilbestrol ; pharmacology ; therapeutic use ; Drug Evaluation, Preclinical ; Drug Therapy, Combination ; Dyslipidemias ; blood ; prevention & control ; Estrogens, Non-Steroidal ; pharmacology ; therapeutic use ; Female ; Organ Size ; drug effects ; Ovariectomy ; Rats ; Uterus ; drug effects

OBJECTIVETo establish a primary culture of the testis gubernacular cells of Kunming mice, observe the morphological characteristics of the cells, and explore the effects of exogenous estrogens (EEs) on the development of the testis gubernacula in vitro.

METHODSWe removed the gubernacula from 3-day-old mice with the surgical magnifier and cultured the gubernacular cells. Then we detected the cell viability by trypan blue and cell morphology by HE staining. The subcultured cells were randomly divided into a blank control, a DMSO (0.1%, v/v) control, and 4 experimental groups (given 0.01, 0.10, 1.00 and 10.00 micdrog/ml of diethylstilbestrol [DES] dissolved in DMSO, respectively). After treated for 12, 24 and 48 hours, the gubernacular cells were observed for morphological changes and proliferation inhibition by CCK-8.

RESULTSMost of the cultured gubernacular cells were fibroblasts, and a few were epithelioids. The primary cells showed a viability of 85%-90%. Dose- and time-dependent inhibition of cell proliferation was found in the four experimental groups at three different times, with statistically significant differences (P < 0.01).

CONCLUSIONGubernacular cells can be cultured in vitro. EEs inhibit the proliferation of gubernacular cells in a dose- and time-dependent manner. An in- sight into the effects EES on cultured gubernacular cells is an effective approach to the study of their influence on the development of the reproductive system.

Animals ; Cells, Cultured ; drug effects ; Diethylstilbestrol ; pharmacology ; Estrogens, Non-Steroidal ; pharmacology ; Male ; Mice ; Mice, Inbred Strains ; Spermatic Cord ; cytology ; drug effects ; Testis ; cytology ; drug effects

AIMTo assess the effect of estradiol-17beta (E(2)) and bisphenol A (BPA) administered chronically by implanting a silicone tube throughout pregnancy and lactation on male pups' reproductive system in ICR mice.

METHODSFemale mice were implanted with a tube filled with 10 ng, 500 ng, 1 microg, or 10 microg of E(2), or 100 microg or 5 mg of BPA, before mating. The tube was kept in the mice throughout pregnancy and lactation, until the pups had weaned at 4 weeks of age. During the period, E(2) was released from the tube at 120 pg or 6, 12 or 120 ng/day, and BPA at 1.2 or 60 microg/day.

RESULTSMost of the mice given 1 microg and 10 microg of E(2) did not maintain their pregnancy. However, the other groups showed high rates of birth, more than 70%. At age of 4 weeks, the male pups were killed. Body weight and reproductive organ weights (testes, epididymides and accessory reproductive glands) in the treated groups did not differ from the control values, whereas the percentage of seminiferous tubules in the testis with mature spermatids was significantly lower in the groups given 10 ng and 500 ng of E(2) and 5 mg of BPA than that in the control.

CONCLUSIONChronic exposure to E(2) and BPA might disrupt spermatogenesis in male pups.

Animals ; Benzhydryl Compounds ; Birth Rate ; Estradiol ; pharmacology ; Estrogens, Non-Steroidal ; pharmacology ; Female ; Genitalia, Male ; drug effects ; pathology ; Lactation ; Male ; Mice ; Phenols ; pharmacology ; Pregnancy ; Spermatogenesis ; drug effects

OBJECTIVETo explore the phytoestrogenic effects of ten kinds of Chinese medicine including flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus ligustri lucidi, fructus lycii, radix clycyrrhizae, herba cistanches, herba epimedii, fructus psoraleae and semen cuscutae.

METHOD240 female Kunming mice weighting 9 - 12 g were randomly divided into two main groups A and B. A group was divided into 12 small groups: 1 solvent control group, 1 diethylstilbestrol control group and 10 Chinese medicine groups. B group was also divided into 12 small groups: 1 solvent control group, 1 diethylstilbestrol control group and 10 Chinese medicine antagonistic groups. Mice in ten antagonistic groups were administered both Chinese medicine and diethylstilbestrol everyday. After administered(op) for 4 days, blood was collected and serum was separated. The effect of the pharmacological serum on proliferation rate of MCF-7 (ER+) was analyzed by MTT-assay.

RESULTIn A group, proliferation rates of MCF-7 cells treated with serum from eight Chinese medicine groups including flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus lycii, herba cistanches, herba epimedii, fructus psoraleae and semen cuscutae were coued markedly increase respectively. While serum from fructus ligustri lucidi group could markedly decrease the proliferation rate of MCF-7 cells. In B group, the increased proliferation rate of MCF-7 cells caused by diethylstilbestrol was significantly reduced in seven Chinese medicine antagonistic groups including flos carthami, radix cyathulae, radix salviae miltiorrhizae, radix clycyrrhizae, herba epimedii, fructus psoraleae and semen cuscutae. While the increased proliferation rate could be markedly enhanced in herba cistanches group.

CONCLUSIONSix kinds of Chinese medicine such as flos carthami, radix cyathulae, radix salviae miltiorrhizae, herba epimedii, fructus psoraleae and semen cuscutae show both estrogenic effects (when administered indepently) and antiestrogenic effects (when administered together with diethylstilbestrol). Such bidirectional effects depends on the internal estrogen level.

Animals ; Breast Neoplasms ; metabolism ; pathology ; Carthamus tinctorius ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Diethylstilbestrol ; pharmacology ; Drug Antagonism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Estrogens, Non-Steroidal ; pharmacology ; Female ; Humans ; Mice ; Phytoestrogens ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Random Allocation ; Receptors, Estrogen ; metabolism ; Salvia miltiorrhiza ; chemistry ; Serum

Bisphenol A (BPA), a ubiquitous environmental contaminant, has been shown to cause developmental toxicity and carcinogenic effects. BPA may have physiological activity through estrogen receptor (ER) -alpha and -beta, which are expressed in the central nervous system. We previously found that exposure of BPA to immature mice resulted in behavioral alternation, suggesting that overexposure of BPA could be neurotoxic. In this study, we further investigated the molecular neurotoxic mechanisms of BPA. BPA increased vulnerability (decrease of cell viability and differentiation, and increase of apoptotic cell death) of undifferentiated PC12 cells and cortical neuronal cells isolated from gestation 18 day rat embryos in a concentration-dependent manner (more than 50 micrometer). The ER antagonists, ICI 182,780, and tamoxifen, did not block these effects. The cell vulnerability against BPA was not significantly different in the PC12 cells overexpressing ER-alpha and ER-beta compared with PC12 cells expressing vector alone. In addition, there was no difference observed between BPA and 17-beta estradiol, a well-known agonist of ER receptor in the induction of neurotoxic responses. Further study of the mechanism showed that BPA significantly activated extracellular signal-regulated kinase (ERK) but inhibited anti-apoptotic nuclear factor kappa B (NF-kappaB) activation. In addition, ERK-specific inhibitor, PD 98,059, reversed BPA-induced cell death and restored NF-kappaB activity. This study demonstrated that exposure to BPA can cause neuronal cell death which may eventually be related with behavioral alternation in vivo. However, this neurotoxic effect may not be directly mediated through an ER receptor, as an ERK/NF-kappaB pathway may be more closely involved in BPA-induced neuronal toxicity.
Animals ; Apoptosis/drug effects ; Blotting, Western ; Cell Differentiation/drug effects ; Cell Survival/drug effects ; Estradiol/analogs & derivatives/pharmacology ; Estrogens, Non-Steroidal/*toxicity ; Flavonoids/pharmacology ; NF-kappa B/metabolism ; Neurons/*drug effects/physiology ; PC12 Cells ; Phenols/*toxicity ; Rats ; Receptors, Estrogen/metabolism ; Tamoxifen/pharmacology

Phospholipid hydroperoxide glutathione peroxidase(PHGPx), an antioxidative selenoprotein, is modulated byestrogen in the testis and oviduct. To examine whetherpotential endocrine disrupting chemicals (EDCs) affectthe microenvironment of the testes, the expression patternsof PHGPx mRNA and histological changes were analyzedin 5-week-old Sprague-Dawley male rats exposed to severalEDCs such as an androgenic compound [testosterone (50,200, and 1,000microg/kg)], anti-androgenic compounds [flutamide(1, 5, and 25mg/kg), ketoconazole (0.2 and 1mg/kg), anddiethylhexyl phthalate (10, 50, and 250mg/kg)], andestrogenic compounds [nonylphenol (10, 50, 100, and 250mg/kg), octylphenol (10, 50, and 250mg/kg), and diethyl-stilbestrol (10, 20, and 40microg/kg)] daily for 3 weeks via oraladministration. Mild proliferation of germ cells andhyperplasia of interstitial cells were observed in the testesof the flutamide-treated group and deletion of thegerminal epithelium and sloughing of germ cells wereobserved in testes of the diethylstilbestrol-treated group.Treatment with testosterone was shown to slightly decreasePHGPx mRNA levels in testes by the reverse transcription-polymerase chain reaction. However, anti-androgeniccompounds (flutamide, ketoconazole, and diethylhexylphthalate) and estrogenic compounds (nonylphenol,octylphenol, and diethylstilbestrol) significantly up-regulated PHGPx mRNA in the testes (p<0.05). Thesefindings indicate that the EDCs might have a detrimentaleffect on spermatogenesis via abnormal enhancement ofPHGPx expression in testes and that PHGPx is useful as abiomarker for toxicity screening of estrogenic or anti-androgenic EDCs in testes.
Androgen Antagonists/pharmacology ; Animals ; Diethylhexyl Phthalate/pharmacology ; Diethylstilbestrol/pharmacology ; Endocrine Disruptors/*pharmacology ; Estrogens, Non-Steroidal/pharmacology ; Flutamide/pharmacology ; Glutathione Peroxidase/*biosynthesis/genetics ; Histocytochemistry ; Ketoconazole/pharmacology ; Male ; Phenols/pharmacology ; RNA, Messenger/*biosynthesis/genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogenesis/drug effects ; Testis/*drug effects/*enzymology ; Testosterone/pharmacology

OBJECTIVETo explore the effects of zearalenone (ZEA) on proliferation and apoptosis in estrogen-dependent human breast cancer MCF-7 cells and the likely underlying molecular mechanisms.

METHODSCell viability was determined by MTT assay and cell cycle distribution by cytometry. Apoptosis was detected by Cell Death Detection ELISA and cytometry, respectively. The expressions of bax and bcl-2 were examined using multiple RT-PCR and Western-blot both at mRNA and protein level, respectively.

RESULTSThe current study confirmed the previous studies that ZEA could stimulate proliferation in MCF-7 cells with inducing a profound increase in S phase and a modest increase in G(2)/M phase that was accompanied by a decrease in G(0)/G(1) phase. ZEA could inhibit apoptosis in MCF-7 cells following estrogen ablation at a range of concentrations of 2 nmol/L -96 nmol/L. Western blot and RT-PCR analysis revealed that the anti-apoptotic bcl-2 was upregulated at both protein and mRNA level, together with the downregulation of pro-apoptotic bax.

CONCLUSIONZEA should have possessed comparative estrogenic activity and could promote the progression of MCF-7 cells through the cell cycle by a decreasing in the G(0)/G(1) phase and by a significant increasing in S-phase. The pro-proliferative activity of ZEA was due to inhibition of apoptosis through regulation of bax/bcl-2 expression.

Apoptosis ; drug effects ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estrogens, Non-Steroidal ; pharmacology ; Female ; Flow Cytometry ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Zearalenone ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics

OBJECTIVETo study the effects of phytoestrogen alpha-zearalanol (alpha-ZAL) on normal human breast.

METHODSTen specimens of normal human breast tissues were subcutaneously implanted into 30 athymic nude mice aged 9-10 weeks, one for 3 mice. These mice were then randomly divided into three groups: control group (without hormone treatment, n = 10), 1 mg/kg alpha-ZAL group (n = 10), and 5 mg/kg alpha-ZAL group (n = 10). All breast tissues were taken out 6 weeks later. Immunohistochemistry was used to determine the protein expressions of proliferating cell nuclear antigen (PCNA), inhibiting apoptosis gene Bcl-2, estrogen receptor (ER), and progesterone receptor (PR). Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the expression levels of estrogen sulfotransferase (EST) mRNA and bridging integrator protein-1 (BIN1) mRNA. Morphological features of grafts before and after treatment were also observed.

RESULTSAlpha-ZAL had no significant effects on Bcl-2, PCNA, ER, and PR expression of mammary epithelial cells in graft specimens. Alpha-ZAL upregulated BIN1 mRNA expression in grafts, but had no significant effect on ESTmRNA expression.

CONCLUSIONSAlpha-ZAL does not affect the morphology, proliferating, and apoptosis of epithelial cells in normal human breast tissues implanted into nude mice, but it may increase the gene expression of tumor-inhibiting BIN1, suggesting that alpha-ZAL may have potential proteotive effect on normal human breast.

Adult ; Animals ; Breast ; chemistry ; drug effects ; Estrogens, Non-Steroidal ; pharmacology ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phytoestrogens ; pharmacology ; Proliferating Cell Nuclear Antigen ; analysis ; Random Allocation ; Receptors, Estrogen ; analysis ; Receptors, Progesterone ; analysis ; Zeranol ; pharmacology

Genistein, a soybean-originated isoflavone, is widely consumed by humans for putative beneficial health effects but its estrogenic activity may affect adversely the development of male reproductive system. Five-week-old ICR mice were purchased and fed with a soybean-based Purina Chow diet until 6 months of age. The animals were exposed by gavage to genistein (2.5 mg/kg/day) or 17beta-estradiol (7.5 microgram/kg/day) for five weeks. Corn oil was used for the negative control. The animals were fed the caseinbased AIN-76A diet throughout the experimental periods. There were no significant differences in body and organ weights of mice among experimental groups. No significant differences in sperm counts and sperm motile characteristics were found between the control and the genistein groups. Treatment of 17beta-estradiol caused a significant decrease in epididymal sperm counts compared to the control (p<0.05). The level of phospholipid hydroxide glutathione peroxidase in the epididymis of mice exposed to genistein was significantly higher than that of the control mice (p<0.05). 17beta-estradiol treatment caused a reduction of germ cells in the testis and hyperplasia of mucosal fold region in the prostate of mice. Genistein treatment did not cause any lesion in the testis, epididymis, and prostate. These results suggest that dietary uptake of genistein at adult stage of life may not affect male reproductive system and functions.
Animals ; Estradiol/metabolism ; Estrogens, Non-Steroidal/*pharmacology ; Genistein/*pharmacology ; Genitalia, Male/*drug effects/pathology ; Glutathione Peroxidase/genetics/metabolism ; Histocytochemistry/veterinary ; Male ; Mice ; Mice, Inbred ICR ; Organ Size/drug effects/physiology ; Prostate/drug effects/pathology ; RNA/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; *Soybeans ; Sperm Count/veterinary ; Sperm Motility/drug effects/physiology