Effect of prenatal exposure to diethylstilbestrol on gubernacular development in fetal male mice

Aim: To 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. Methods: Pregnant 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 μg·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. Histologi-cal changes were observed under the light microscope (LM) and ultrastructural changes with the scanning and transmission electron microscopes (SEM & TEM). The expression intensity of actin and PCNA in the gubernacula was quantitated by immunohistochemistry. Results: The 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). Conclusion: DES induces underdevelopment of the gubernacula in a dose-dependent manner in fetal male mice and down regulates the actin and PCNA expression.

μ-and m-calpain expression and activity changes following diethylstilbestrol injection in the rat anterior pituitary

Little is known about changes in calpain activity in the pituitary gland. In the present study,μ- and m-calpain activity changes were detected in the rat anterior pituitary following intraperitoneal injection of diethylstilbestrol. Double-immunofluorescence labeling confirmed colocalization of μ- and m-calpain in prolactin-secreting cells （lactotrophs）. Western blot analysis revealed significantly increased expression of both calpains, which accompanied upregulated cytosol and membrane zymographic activities at 12 weeks following diethylstilbestrol injection, compared with rats injected with sunflower oil. Moreover, following estrogen injection, pituitary gland pathological damage gradually worsened with increasing time. Results demonstrated that estrogen regulated calpain expression and activity, and both calpains participated in the pathophysiological processes of the pituitary gland. Ubiquitous calpain expression could serve as an effective target for anti-estrogen drugs.

Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring

Maternal exposure to estrogenic xenobiotics or phthalates has been implicated in the distortion of early male reproductive development, referred to in humans as the testicular dysgenesis syndrome. It is not known, however, whether such early gestational and/or lactational exposure can influence the later adult-type Leydig cell phenotype. In this study, Sprague-Dawley rats were exposed to dibutyl phthalate （DBP; from gestational day （GD） 14.5 to postnatal day （PND） 6） or diethylstilbestrol （DES; from GD14o5 to GD16.5） during a short gestationalllactational window, and male offspring subsequently analysed for various postnatal testicular parameters. All offspring remained in good health throughout the study. Maternal xenobiotic treatment appeared to modify specific Leydig cell gene expression in male offspring, particularly during the dynamic phase of mid-puberty, with serum INSL3 concentrations showing that these compounds led to a faster attainment of peak values, and a modest acceleration of the pubertal trajectory. Part of this effect appeared to be due to a treatment-specific impact on Leydig cell proliferation during puberty for both xenobiotics. Taken together, these results support the notion that maternal exposure to certain xenobiotics can also influence the development of the adult-type Leydig cell population, possibly through an effect on the Leydig stem cell population.

Simultaneous Determination of Hexoestrol, Diethylstilbestrol, Estrone and 17-Beta-estradiol in Feed by Gas Chromatography-mass Spectrometry

A method was developed for the simultaneous determination of four kinds of estrogens （hexoestrol, diethylstilbestrol, estrone, and 17-beta-estradiol） in feed by gas chromatography-mass spectrometry （GC-MS）. After the sample was extracted by ethyl ether and cleaned-up on HLB phase extraction column, four kinds of estrogens were derived and quantified in gas chromatographymass spectrometry. The results showed that the linear detectable ranged from 2.5 ng· mL-1 to 250 ng· mL-1for hexoestrol and from 5 ng· mL-1 to 500 ng· mL-1 for three other estrogens with the correlation coefficients （R2） were no less than 0.990. The recoveries were in the range of 76.34%-96.33% and the relative standard deviation was no more than 22.7%. The limits of quantitation （LOQ） for all analytics were between 10 ug· kg^-1 and 20 ug· kg^-1. The method was accurate and sensitive and could meet the actual requirements for the analyses of feed samples.

Prevention of Osteopenia and Dyslipidemia in Rats after Ovariectomy with Combined Aspirin and Low-dose Diethylstilbestrol

Objective To 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. Methods Thirty-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 ug/kg.d） treatment group （OVX＋D30 group, n=8）, and aspirin （9 mg/kg.d） plus diethylstilbestrol （10 ug/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. Results The 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. Conclusion Aspirin 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.

Quantitative proteomic determination of diethylstilbestrol action on prostate cancer

Diethylstilbestrol （DES） has a direct cellular mechanism inhibition on prostate cancer. Its action is independent from the oestrogen receptors and is preserved after a first-line hormonal therapy. We aimed to identify proteins involved in the direct cellular inhibition effects of DES on prostate cancer. We used a clonogenic assay to establish the median lethal concentration of DES on 22RV1 cells. 22RV1 cells were exposed to standard and DES-enriched medium. After extraction, protein expression levels were obtained by two-dimensional differential in-gel electrophoresis （2D-DIGE） and isotope labelling tags for relative and absolute quantification （iTRAQ）. Proteins of interest were analysed by quantitative RT-PCR and western blotting. The differentially regulated proteins （P〈0.01） were interrogated against a global molecular network based on the ingenuity knowledge base. The 2D-DIGE analyses revealed DES-induced expression changes for 14 proteins （〉 1.3 fold; P〈0.05）. The iTRAQ analyses allowed the identification of 895 proteins. Among these proteins, 65 had a modified expression due to DES exposure （i.e., 23 overexpressed and 42 underexpressed）. Most of these proteins were implicated in apoptosis and redox processes and had a predicted mitochondrial expression. Additionally, ingenuity pathway analysis placed the OAT and HSBP1 genes at the centre of a highly significant network. RT-PCR confirmed the overexpression of OAT （P=0.006） and HSPB1 （P=0.046）.

Expression of Sex-Related Genes in Chicken Embryos During Male-to-Female Sex Reversal Exposure to Diethylstilbestrol

Sex emerges out of a delicate dance between a variety of promale, anti-male, and possibly profemale genes. To investigate the role that sex-related genes play in sex determination and gonadal differentiation of fowl, we constructed a male-to- female sex-reversal model of chick induced by diethylstilbestrol （DES） at onset of incubation （E0）. The results of semi- quantitative PCR showed that the expression of Sf1, the orphan nuclear receptor steroidogenic factor-1 gene, was put forward from E7d to E5d and up-regulated during E5-7d; the Dmrt1, the double sex and the Mab-3 related to transcription factor 1 gene, was down-regulated during E3-7d. Meanwhile, anti-Müllerian hormone gene （Amh） expressed at a similar level in the genetic females and sex-reversal females before E7d, while no expression products of the three female-specific genes Wpkci, Fet1 and Foxl2 were detected in male-to-female embryos. These findings suggest that the expression of some certain sex-related genes, induced by the exogenous estrogen during period of sex determination and gonadal differentiation, results in the male-to-female sex reversal. Moreover, high activity of Sf1 gene during E5-7d might be related to the profemale process, while low activity of Dmrt1 gene during E3-5d might be anti-male. The expression activity of Amh gene might only contribute to the promale process after E7d, however, it is possibly not an anti-female gene in chick embryos.

Electrochemical Determination of Diethylstilbestrol in Feeds at Polyethylenimine-nanogold Modified Electrode

In this paper, a new method for the modification of glass carbon electrode (GCE) by polyethylenimine (PEI) and nano-gold (Au-colloid) was established to explore sensitive techniques for voltammetric determination of diethylstilbestrol. Compared with bare GCE, the peak current at the potential of 0.45 V is increased notably at PEI and PEI-nanogold modified electrode both by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). This kind of electrode allows the detection of low concentration of diethylstilbestrol in the range of 0.03～12 mg/L by DPV measurement. Other optimizations for experimental conditions were also discussed in detail.

Effect of diethylstilbestrol on polyamine metabolism in hamster epididymis

<abstract>Aim: To investigate the effect of diethylstilbestrol (DES), one of the most potent endocrine disruptors, on the metabolism of polyamines in hamster epididymis. Methods: Male 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. Results: DES treatment caused a significant decrease in the weight of epididymis. The activity of epididymal ornithine decar boxylase (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 N1-acetylspermidine were increased 1 day -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. Conclusion: The 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.

Diethylstilbestrol in Fish Tissue Determined Through Subcritical Fluid Extraction and with GC-MS

As the key point in sex hormone analysis, sample pre-treatment technology has attracted scientists' attention all over the world, and the development trend of sample preparation forwarded to faster and more efficient technologies. Taking economic and environmental concerns into account, subcritical fluid extraction as a faster and more efficient method has stood out as a sample pre-treatment technology. This new extraction technology can overcome the shortcomings of supercritical fluid and achieve higher extraction efficiency at relatively low pressures and temperatures. In this experiment, a simple, sensitive and efficient method has been developed for the determination of diethylstilbestrol(DES) in fish tissue using subcritical 1,1,1,2-tetrafluoroethane(R134a) extraction in combination with gas chromatography-mass spectrometry(GC-MS). After extraction, freezing-lipid filtration was utilized to remove fatty co-extract. Further purification steps were performed with C_(18) and NH_2 solid phase extraction(SPE). Finally, the analyte was derived by heptafluorobutyric anhydride(HFBA), followed by GC-MS analysis. Response surface methodology(RSM) was employed to optimizing the extraction condition, and the optimized was as follows: extraction pressure, 4.3 MPa; extraction temperature, 26℃; amount of co-solvent volume, 4.7 m L. Under this condition, at a spiked level of 1, 5, 10 μg kg^(-1), the mean recovery of DES was more than 90% with relative standard deviations(RSDs) less than 10%. Finally, the developed method has been successfully used to analyzing the real samples.

Long-term application of diethylstilbestrol upregulates expressions of μ-and m-calpains in pituitary intermediate lobe of female Wistar rats

BACKGROUND： During formation of prolactin neoplasia, how cells and its structure in adenohypophysis affect prolactin cells should be further studied. Intermediate lobe can be regarded as a driving region to release prolactin （PRL） and may promote formation of prolactin neoplasia in pituitary anterior lobe. OBJECTIVE： To observe the effect of diethylstilbestrol （DES） on the expressions of μ and m-calpains in pituitary intermediate lobe of female Wistar rats. DESIGN： Observational contrast animal study. SETTING： Beijing Neurosurgical Institute. MATERIALS： A total of 21 female Wistar rats, 3 weeks old weighing 70-80 g were housed with free access to tap water and standard pellet food. They were kept in a CL-grade condition, at （24±1）℃ and a humidity of （55±5）%, and with a 12 hours day-night cycle. Caprine anti-μ- and m-calpains antibodies were provided by Santa Cruz Biotechnology, CA, USA; rabbit-anti-PRL antibodies by Dako, Denmark; rabbit-anti-ACTH antibody by Boster Company, Wuhan. METHODS： The experiment was carried out in Pathophysiological Department and Animal Laboratory, Beijing Neurosurgical Institute from August 2006 to January 2007. ① Rats were randomly divided into groups with 7 in each group, including vehicle control group, in which rats were injected intraperitoneally with sun-flower seed oil （1 mL/kg, twice a week） for 16 weeks; DES group, where animals were administered with DES （5 mg/kg, twice a week） for 16 weeks; DES ＋ vehicle control group, in which DES was administered for 12 weeks at the same dose with those in DES group, and then was discontinued and replaced by sun-flower seed oil （1 mL/kg, twice a week） for the following 4 weeks. ② At 16 weeks later, pituitary tissue was dealt with HE staining and PRL immunohistochemical examination to observe evoke of tumor; meanwhile, immunohistochemical examination was used to observe expression of PRL of pituitary anterior lobe, expressions of μ- and m-calpains of pituitary intermediate lobe and distribution of adrenocorticotropin. MAIN OUTCOME MEASURES： ① Expression of PRL of pituitary anterior lobe, expressions of μ- and m-calpains of pituitary intermediate lobe and distribution of adrenocorticotropin. ② Morphological observation of pituitary tissue. RESULTS： All 21 rats were involved in the final analysis. ① Results of immunohistochemical examination： Morphological changes of neoplasia in DES group were strongly positive to PRL, and this suggested that formation of prolactin adenoma was observed in pituitary tissue. As compared with vehicle control group, expression of adrenoeorticotropic hormone （ACTH） was increased in both DES group and DES ＋ vehicle control group. In addition, expressions of μ- and m-calpains in pituitary intermediate lobe were higher in DES group than that in vehicle control group. Otherwise, expressions of m-calpains in pituitary intermediate lobe was decreased in DES ＋ vehicle control group, but expression of μ-calpains was still increased. ② Morphological observation of pituitary tissue： Gland tubes were orderly arranged in rats in vehicle control group. Anterior pituitary gland in rats of DES group demonstrated an apparent disappearance of gland tubes and a relatively large-scaled vasculature formation, namely the vascular lake lined by tightly arranged endothelial cells. Local integrated tumor cell arrangements were also detected. In addition, the border between the IL and the anterior lobe was locally blurred. The definite tumor-like changes in pituitary tissueswere confirmed in 6 of 7 female Wistar rats in DES group, and one spontaneous occurrence of tumor formation was found in vehicle control group. In DES ＋ vehicle control group, DES withdrawal led to the subtile emergence of gland tube cavity, although tumor-like cells still existed in 4 of 7 rats, suggesting occurrence of the tumor regression due to the withdrawal of DES. CONCLUSION： A long-term application of DES can enhance the expressions of ubiquitours neutral cysteine protease in pituitary intermediate lobe and this suggests that both of them play a key role in release of hormone and formation of prolactin neoplasia through directly promoting PRL expression and release of neighboring pituitary intermediate lobe.

Spectrophotometric detection of diethylstilbestrol on the basis of the plasmon resonance absorption of silver nanoparticles

In this study,a spectrophotometric detection method for diethylstilbestrol(DES)was proposed by reducing silver nitrate(AgNO3)to obtain silver nanoparticles(AgNPs)in the medium of ammonia and sodium hydroxide.It was found that the resulting AgNPs have plasmon resonance absorption(PRA)characteristic at 415 nm,and the PRA is proportional to the increase of DES concentration in the range of 4.0×10-8-1.0×10-5M with the detection limit(3σ)of 1.2×10-7M.Most of the coexisting substances at high concentrations did not affect the detection of real samples,such as tablets.The recovery was in the range of 96.01%-107.41% and the RSD was lower than 4.7%.This method can be successfully applied to control preparation quality of DES.

Re-establishment of Spermatogenesis by Diethylstilbestrol after 2,5-Hexanedione-induced Irreversible Testicular Atrophy in Rats

To investigate the effects of diethylstilbestrol （DES） in reestablishing spermatogenesis and the mechanism by which estrogen works on spermatogenesis, rats were exposed to 1% 2,5-HD for 5 week. Then 0.1 mL of DES was given （s.c.） at a rate of 0.3 μg/kg, 30 μg/kg, 3 mg/kg every other day for 2 weeks respectively （DES group） while the other rats received ethyldeate only. Plasma testosterone （T） and LH were measured on the 8th week after the treatment. The rats were killed at the 18th week. The left testis was histopathologically examined. In all the rats in the DES groups, spermatogenesis was re-established and the rats in the 30 μg/kg group showed the best results. Serum T was suppressed markedly in rats of 30 μg/kg and 3 mg/kg groups while T was only mildly inhibited in 0.3 μg/kg group, without significant difference found in serum LH. It is concluded that the nearly complete testicular atrophy could be reversed by DES treatment in rats. Estrogen plays an important part in spermatogenesis, and the role of estrogen in spermatogenesis is more than suppressing the hypothalamo-pituitary-testis axis.

Development for the lung tumor model of newborn mice induced by diethylstilbestrol

Objective： To explore a economical and effective method for building lung tumor model induced by diethylstibestrol （DES）. Methods： The carcinogenic effect of neonatal mice treated by DES was studied. The newborn mice were divided into DES, Urethan （U） and U ＋ DES groups. U group was given in 500 mg/kg dose by ip at postnatal 14 day, DES group was administered by ip at the 1 d, 8 d, 15 d in the dose of 1/7, 2/7 and 4/7 LD50 of the day when they were injected respectively for DES （I）, DES （M）, DES （H） groups. Until 26 weeks, they were anatomized and checked the formation of tumors. The organ index, tumor incidence ratio and mean number of tumors were calculated. Results： Lung tumors were apparently induced in tested neonatal mice. The incidence of lung tumor of DES （L, M, H） groups were 16.7%, 22.4% and 43.1% respectively, the U ＋ DES （L, M, H） groups were 70.4%, 90.9% and 70.8% respectively, and the U group was 53.1%. The mean numbers of lung tumors of U ＋ DES （L, M） groups were higher than those of the DES （L, M） groups respectively （P 〈 0.05）. Conclusion： The higher ratio of lung tumor incidence had been induced by DES and U joined action to neonatal mice, which may be a useful and economical method to establish a lung tumor model induced by DES.

DES磁性分子印迹材料的制备及其吸附分离性能

利用溶剂热法制备纳米Fe 3 O 4,并将其分散在正硅酸乙酯(TEOS)水解液中,在表面沉积一层SiO_(2).用甲基丙烯酰氧基丙基三甲氧基硅烷(MPS)对SiO_(2)-Fe 3 O 4表面改性,得到MPS-SiO_(2)-Fe 3 O 4.将甲基丙烯酸(MAA)与己烯雌酚(DES)以摩尔比4∶1加入乙腈溶剂中,进行12 h自组装,再加入MPS-SiO_(2)-Fe 3 O 4、乙二醇二甲基丙烯酸酯(EGDMA)和偶氮二异丁腈(AIBN),进行超声分散30 min,65℃条件下机械搅拌,反应24 h,制得DES磁性分子印迹聚合物(MMIP).采用透射电镜、振动样品磁强计和吸附试验等方法进行表征分析.结果表明:MMIP的饱和磁化强度为268 kA/m,无磁滞现象,矫顽力为0,表现出超顺磁性,在磁铁作用下17 s就可与溶液分离;室温下,MMIP对DES的静态最大吸附量为7.1 mg/g,动态吸附时MMIP在60 min时可以达到静态最大吸附量的90%以上;MMIP对DES的印迹因子为3.70,因而对DES有较大的识别能力;MMIP可重复使用6次以上,具有良好的再生循环性能.

Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

An indirect competitive enzyme-linked immunosorbent assay （icELISA） based on polyclonal antibody for the estrogen diethylstilbestrol （DES） was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol （DES-CP） and mono-O-carboxymethyldiethylstilbestrol （DES-CM） with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carder proteins bovine serum albumin （BSA） by mixed-anhydride method for immunogen and conjugated with ovalbumin （OVA） by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection （LLD） of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.

Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol

Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.

Recovery of fertility in quinestrol-treated or diethylstilbestroltreated mice:Implications for rodent management

Fertility control is an alternative strategy to traditional culling for the management of rodent pests.Previous studies have demonstrated that quinestrol is a potential contraceptive for male rodents,but the recovery of fertility in quinestrol-treated rodents has not been evaluated.This study used C57BL/6J mice to evaluate the recovery rate of male fertility after the administration of quinestrol.Diethylstilbestrol(DES),a non-steroid estrogenic compound,was used for comparison.Different groups of mice were treated with 1 mg/kg quinestrol,1 mg/kg DES,or castor oil separately for 7 days.These mice were then killed on days 8,22 and 50 respectively.Our results indicated that the weight of epididymides and seminal vesicles decreased significantly on days 8 and 22 in quinestrol/DES-treated mice,with extensive histological changes in the seminiferous tubules.Sperm concentrations in the cauda epididymal fluid were significantly reduced on days 8 and 22 in both quinestrol and DES treatment groups and on day 50 for the DES,but not the quinestrol group.Further analysis revealed that DES-treated mice exhibited a higher proportion of abnormal sperm accumulation in the epididymis,indicating that the normal sperm transportation to the cauda epididymis was blocked.Our results indicate that the anti-fertility effects on male mice given quinestrol were of shorter duration than for those receiving DES at the dose of 1 mg/kg body weight.

Reference gene validation for quantification of gene expression during final oocyte maturation induced by diethylstilbestrol and di-(2-ethylhexyl)-phthalate in common carp

Final oocyte maturation is the key step to successful spawning and fertilization.Quantitative real-time PCR（q PCR） is the technique of election to quantify the abundance of functional genes in such study. Reference gene is essential for correct interpretation of q PCR data. However, an ideal universal reference gene that is stable under all experimental circumstances has not been described. Researchers should validate their reference genes while performing q PCR analysis. The expression of 6 candidate reference genes： 18 s r RNA,28 s r RNA, Cathepsin Z, Elongation factor 1-α, Glyceraldehyde-3-phosphate dehydrogenase andβ-actin were investigated during final oocyte maturation induced by different compounds（DES and DEHP） in common carp（Cyprinus carpio）. Four softwares（Bestkeeper, ge Norm,Norm Finder and Ref Finder） were used to screen the most stable gene in order to evaluate their expression stability. The results revealed that EF1α was highly stable expressed when final oocyte maturation was induced by DES, while gapdh was the most stable gene when final oocyte maturation was induced by DEHP. Stable expressed reference gene selection is critical for all q PCR analysis to get accurate target gene m RNA expression information.

饲料中乙烯雌酚含量的检测方法研究

本研究旨在开发一种高效、经济的方法,利用高效能液相层析仪(HPLC)配合光二极管侦测器(PDA),对饲料中乙烯雌酚含量进行分析。为了避免在低浓度情况下的误判,同时采用高效液相层析质谱仪(HPLC/MS)进行了再确认。在实验中,饲料样品以甲醇为萃取液,经过振荡、离心、浓缩上清液,残渣用70%甲醇溶解,通过Oasis■HLB卡匣去除干扰物质,最后用甲醇进行冲提,冲提液经高效液相层析仪检测。实验结果表明,对于饲料添加0.5、1、3、5 ppm的情况,回收率在80.8%到107.5%。仅采用光二极管侦测器检测时,最低检测限为0.03 ppm;而采用质谱侦测器检测时,最低检测限可达0.005 ppm。研究结果表明,所开发的高效液相层析仪饲料中乙烯雌酚检测方法具有便捷、经济、可行的特点,可作为饲料中乙烯雌酚检验的一种有效方法。