OBJECTIVETo investigate the impact of prenatal exposure to diethylstilbestrol (DES) on the specific receptor LGR8 of insulin-like factor 3 (INSL3) in the mouse gubernaculum testis, and that of exoestrogens on descensus testis in mice.

METHODSA total of 120 pregnant KM mice aged 8 to 10 weeks were assigned to a normal, a blank control and 4 DES groups of equal number, the blank controls injected subcutaneously with dimethyl sulfoxide plus normal saline, and the DES groups with DES at 0.1, 1, 10 and 100 microg/kg body weight, respectively, from embryonic day 9 (ED9) through ED17. Immunohistochemistry and RT-PCR were used to detect the expressions of LGR8 protein and mRNA in the gubernaculum testis of the ED18 fetuses and PND20 (postnatal day 20) offspring of the mice.

RESULTSHistological analysis showed that the gubernaculum testis of the ED18 fetuses were well developed in both the normal and control groups, with an inner mesenchymal core and muscular outer layer. In contrast, the gubernaculum testis were poorly developed in the experimental groups, morphologically abnormal and without visible dividing line between the mesenchymal tissue and the muscular outer layer. No obvious differences were found in the gubernaculum testis development of the neonates between the normal and experimental groups. Positive immunostaining was seen in the mesenchymal core and muscular outer layer, but mainly in the latter. The expression of LGR8 was weaker in the experimental groups than in the normal group (P < 0.05), but that of LGR8 mRNA was increased in the high-dose (10 and 100 microg/kg) DES groups (P < 0.05). No obvious mutations were observed in the PCR products in any of the experimental groups.

CONCLUSIONPrenatal exposure to diethylstilbestrol affected the expression of LGR8 mRNA in the mouse gubernaculum testis, which suggests that diethylstilbestrol may induce cryptorchidism by interfering with the INSL3-LGR8 signaling system and consequently the development of the gubernaculum testis.

Animals ; Diethylstilbestrol ; pharmacology ; Female ; Male ; Mice ; Mice, Inbred Strains ; Pregnancy ; Receptors, G-Protein-Coupled ; metabolism ; Testis ; drug effects ; embryology ; metabolism

AIMTo study the effect of prenatal exposure to diethylstilbestrol (DES) and the role of actin and proliferating cell nuclear antigen (PCNA) on testicular gubernaculum development in fetal male Kunming mice.

METHODSPregnant mice were randomly assigned to 6 groups and injected with DES subcutaneously from gestational day 9 (E9) to day 17 (E17) at doses of 0, 25, 50, 100, 200 microg.kg-1.d-1 in 0.2 mL dimethyl sulfoxide (DMSO). On E17 they were sacrificed and fetuses quickly removed for fixation. Male fetuses were sliced on serial coronal plane. Histological changes were observed under the light microscope (LM) and ultrastructural changes with the scanning and transmission electron microscopes (SEM and TEM). The expression intensity of actin and PCNA in the gubernacula was quantitated by immunohistochemistry.

RESULTSThe mortality of the fetuses was higher in the DES-treated groups than that in the DMSO and saline controls (P<0.05). Under LM the gubernacula were seen to be poorly developed with smaller bulbs. On SEM the bulbs lose the clear demarcation between the mesenchymal inner core and the muscular outer layer and looked like a small cone instead of the normal cylindrical appearance. On TEM there were some smaller disordered myofibrils and sparse cytoplasmic organelles in the gubernacular muscular cells of the treated groups. The expression intensity of actin and PCNA in the gubernacula was significantly weaker in the treated groups than that in the DMSO and saline controls (P<0.05).

CONCLUSIONDES induces underdevelopment of the gubernacula in a dose-dependent manner in fetal male mice and down regulates the actin and PCNA expression.

Animals ; Diethylstilbestrol ; pharmacology ; Dose-Response Relationship, Drug ; Estrogens, Non-Steroidal ; pharmacology ; Female ; Fetal Death ; chemically induced ; Immunohistochemistry ; Male ; Mice ; Microscopy, Electron, Scanning ; Pregnancy ; Testis ; cytology ; drug effects ; embryology

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

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

AIMTo investigate the effect of diethylstilbestrol (DES), one of the most potent endocrine disruptors, on the metabolism of polyamines in hamster epididymis.

METHODSMale golden hamsters of 7-week-old were kept under a light and dark cycle of 14 h and 10 h for 1 week to stimulate maximally the gonadal function. DES was injected subcutaneously at doses of 0.01 mg . kg(-1) . day(-1), 0.1 mg . kg(-1) . day(-1) and 1 mg . kg(-1) . day(-1) for one week.

RESULTSDES treatment caused a significant decrease in the weight of epididymis. The activity of epididymal ornithine decarboxylase (ODC) increased 1 day after DES treatment, kept at a high level for 4 days and then decreased to nearly normal level at day 7. The activity of spermidine/spermine N1-acetyltransferase (SSAT) also increased transiently after DES treatment. The contents of putrescine, spermidine, spermine and N(1)-acetylspermidine were increased 1 day approximately 4 days after DES treatment and restored to normal at day 7. All these changes showed a marked difference between the caput and the cauda.

CONCLUSIONThe polyamine biosynthesis in the hamster epididymis can be affected by DES, a xenoestrogen. DES may probably affect polyamine metabolism in the epididymis by regulating the rate-limiting enzymes involved in the polyamine biosynthesis.

Acetyltransferases ; metabolism ; Animals ; Cricetinae ; Diethylstilbestrol ; pharmacology ; Epididymis ; anatomy & histology ; drug effects ; metabolism ; Male ; Mesocricetus ; Organ Size ; drug effects ; Ornithine Decarboxylase ; metabolism ; Polyamines ; metabolism ; Putrescine ; metabolism ; Spermidine ; analogs & derivatives ; metabolism ; Spermine ; metabolism

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

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

The cajanine (longistylin A-2-carboxylic acid) is isolated and identified from extracts of Cajanus cajan L. (ECC) , which structure is similar to diethylstilbestrol. The regulation properties of the cajanine and other four extracts of Cajanus cajan L. (32-1, 35-1, 35-2, and 35-3) were tested in human osteoblast-like (HOS) TE85 cells and marrow-derived osteoclast-like cells. By using MTT assay to test the change of cell proliferation, 3H-proline incorporation to investigate the formation of collagen, and by measuring alkaline phosphatase (ALP) activity, bone formation in HOS TE85 cell was evaluated after pretreated for 48 hours. Bone marrow cells were cultured to examine the derivation of osteoclast cells (OLCs), which were stained with tartrate-resistant acid phosphatase (TRAP). The long term effect (pretreated for 18 days) on promoting mineralized bone-like tissue formation was tested by Alizarin red S staining in HOS TE85 cells. After the treatment with cajanine (1 x 10(-8) g x mL(-1)) for 48 hours, cell number increased significantly (57.7%). 3H-Proline incorporation also statistically increased (98.5%) in those cells. Significant change of ALP activity was also found (P < 0.01) in 35-1 and 35-3 treated cells (they were 66.2% and 82.4% in the concentration of 1 x 10(-8) g x mL(-1), respectively). The long term (18 days) effects of 32-1 and 35-3 on promoting mineralized bone-like tissue formation in HOS TE85 cell were obvious. There were much more red blots over the field of vision compared with that of control group. After the treatment of cajanine, derived-osteoclast cells appeared later and much less compared with control. The inhibition of cajanine was 22.8% while it was 37.9% in 32-1 treated cells in the dose of 1 x 10(-7) g x mL(-1). It is obvious that cajanine and ECCs promoted the osteoblast cells proliferation and mineralized bone-like tissue formation in HOS TE85 cells, while inhibited derivation of osteoclast cells. All of these suggested that cajanine has the estrogen-like action on osteoblast and osteoclast, which could be developed as anti-osteoporosis drugs.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Bone Neoplasms ; metabolism ; pathology ; Cajanus ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Diethylstilbestrol ; analogs & derivatives ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Osteoblasts ; drug effects ; Osteoclasts ; cytology ; metabolism ; Osteogenesis ; drug effects ; Osteosarcoma ; enzymology ; pathology ; Phytoestrogens ; isolation & purification ; pharmacology ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar

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 the mechanisim of Yi Qi Tong Lin Chingji in treating benign prostatic hyperplasia.

METHODThe expressions of VEGF, bFGF and TGF beta 1 in prostatic hyperplasia model rats were examined by immunohistochemistry. 8 rats were in contrastive group, 24 influenced by Yi Qi Tong Ling Chingji and Prostacar.

RESULTThe expressions of VEGF and bFGF were significantly difference, but the expression of TGF beta 1 was not significant. (P < 0.01). The expression of VEGF and bFGF were significant in contrastive group to the high-dose of Yi Qi Tong Ling Chingji and Prostacar group (P < 0.05) but were not significant to the low-dose of Yi Qi Tong Ling Chingji group. There was no significant difference between high-dose of Yi Qi Tong Ling Chingji and Prostacar group and three growth factors.

CONCLUSIONYi Qi Tong Ling Chingji inhibit the expression of VEGF and bFGF in BPH so as to decrease the volume of prostate.

Aconitum ; chemistry ; Animals ; Cinnamomum aromaticum ; chemistry ; Diethylstilbestrol ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fibroblast Growth Factor 2 ; metabolism ; Male ; Plants, Medicinal ; chemistry ; Prostate ; metabolism ; pathology ; Prostatic Hyperplasia ; chemically induced ; metabolism ; pathology ; Rats ; Rats, Wistar ; Rehmannia ; chemistry ; Testosterone Propionate ; Transforming Growth Factor beta ; metabolism ; Transforming Growth Factor beta1 ; Vascular Endothelial Growth Factor A ; metabolism