Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17 beta-estradiol levels in mural granulosa cell cultures.

Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (P<0.05) and alpha-zearalenol (P< 0.001). In preliminary trials on 17 beta-estradiol formation, at the same testing concentration, higher levels of 17 beta-estradiol were found in the presence of alpha-zearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).     

Melatonin has dose-dependent effects on folliculogenesis, oocyte maturation capacity and steroidogenesis

Chemo and/or radiotherapy applied to young cancer patients most often have severe effects upon female fertility. Today, few options are available to protect ovarian function in females. However, these options are either ineffective, belong to the field of experimental research or/and are not applicable to all patients. Drugs that could protect the oocyte and its surrounding feeder cells from damage can be of great importance. Melatonin, being an important indirect antioxidant and a powerful direct free radical scavenger could be such a reagent. This paper reports the direct effects of different melatonin concentrations (range: 1 nM to 2 mM) on folliculogenesis and oogenesis of in vitro cultured mouse ovarian follicles. Early secondary mouse follicles were cultured in vitro for 12 days under different melatonin regimes. Every fourth day, survival rates were scored, follicles were morphologically evaluated and medium was collected for steroid analyses. On day 12, in vitro ovulation was induced by hCG/EGF. Eighteen hours later, oocytes were measured, oocyte maturation was evaluated and normality of spindle and chromosomes ascertained. Results obtained in this study indicated that 2mM melatonin is toxic. One mM negatively influenced oocyte maturation capacity. In the presence of 100 microM melatonin, androstenedione and progesterone were increased whereas estradiol was not influenced. Lower melatonin concentrations had no effect on the evaluated parameters. These data indicate an effect of melatonin on theca cell steroidogenesis. For prophylactic use, a dose of 10 microM could be suitable to reduce oxidative stress in cultured follicles.     

The role of purines in the maintenance of meiotic arrest in mouse oocytes

Meiotic arrest of mammalian oocytes within ovarian follicles is maintained by a specific factor(s) within the follicle. There is strong evidence that cAMP plays an important role in the control of meiosis. Purines have also been implicated in the maintenance of meiotic arrest in vivo. Hypoxanthine and/or adenosine have been identified in pig and mouse follicular fluid and exert a meiosis-arresting action on mouse oocytes in culture. While adenosine apparently need not be metabolized to exert its action on oocyte maturation, the action of hypoxanthine is apparently due to the production of guanyl and/or xanthyl compounds by the oocyte-cumulus cell complex. The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid and bredinin, induced maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine. Hypoxanthine and adenosine are not toxic to oocytes, because oocytes undergo normal fertilization and pre- and post-implantation development following exposure to these molecules in vitro. It is not known how gonadotropins stimulate the resumption of meiosis within the follicle, but there are several possibilities: (1) the intrafollicular level of an oocyte maturation inhibitor is decreased; (2) the oocyte is uncoupled from surrounding follicle cells; (3) an inhibitory molecule is secreted or metabolized by the oocyte; and/or (4) a positive stimulus is produced by the follicle that overrides the presence of inhibitory molecules. Preliminary evidence suggests that cumulus cells may produce a positive stimulus that induces the maturation of cultured cumulus cell-enclosed oocytes. Whether germinal vesicle breakdown in vivo results from a positive induction, a loss of inhibitory input, or a combination of these two mechanisms remains to be determined.     

Evaluation of leptin, interleukin-1beta, tumor necrosis factor-alpha and vascular endothelial growth factor in serum and follicular fluids of women undergoing controlled ovarian hyperstimulation as prognostic markers of ICSI outcome

BACKGROUND: Cytokines play an important but controversial role during ovarian folliculogenesis for the development of mature and fertilizable oocytes. In this study, leptin, interleukin-1beta (IL1beta), tumor necrosis factor-alpha (TNFalpha) and vascular endothelial growth factor (VEGF) in serum and follicular fluids (FF) of women undergoing ovarian hyperstimulation were evaluated as prognostic markers of the outcome of intracytoplasmic sperm injection (ICSI) cycles. MATERIALS AND METHODS: Ninety-five ICSI cycles were included in the study. The cytokines were measured in serum and FF samples with enzyme immunoassay methods. RESULTS: The cytokine concentrations in serum were not significantly correlated with the cytokine concentrations in FFs. Serum IL1beta was inversely-correlated with the number of retrieved oocytes. Serum TNFalpha was negatively-correlated with fertilization rate. In FFs, TNFalpha was positively-correlated with leptin. Leptin and VEGF in FFs were negatively-associated with pregnancy outcome. CONCLUSION: Leptin and VEGF concentrations in FFs may serve as prognostic markers of success after ovarian hyperstimulation and ICSI.     

Evaluation of in vitro biotransformation of propranolol with HPLC, MS/MS, and two bioassays

The majority of human drugs enter aquatic systems after ingestion and subsequent excretion in the form of parent compounds and metabolites. Environmental exposure to drug metabolites has not been reported so far. The goal of the present study was to apply the in vitro method of biotransformation of compounds with S9 fraction to the ecotoxicological analysis. beta-adrenoceptor antagonist propranolol was metabolized with S9 rat liver fraction. The parent compound was quantified with HPLC, and the metabolites were identified with QToF MS. Propranolol was metabolized rapidly, during the first hour its level decreased by 80 and 50% of the initial 20 and 100 mg L(-1), respectively. Ten peaks were observed on the HPLC-RF chromatogram. Four peaks were identified with QToF MS/MS propranolol (m/z = 260), N-desisopropylpropranolol (m/z = 218), hydroxypropranolol (m/z = 276), and hydroxy N-desisopropranolol glycol (m/z = 235). Then the ecotoxicity of the reaction mixture was studied with two bioassays Spirotox with the protozoan Spirostomum ambiguum and Thamnotoxkit F with the anostracean crustacean Thamnocephalus platyurus. Propranolol is twofolds more toxic to Spirotox than to Thamnotoxkit F with 24 h-EC50 = 1.77 mg L(-1) and 24 h-LC50 = 3.86 mg L(-1), respectively. No statistically significant differences were found between the toxicity of the reaction mixtures after S9 biotransformation and the propranolol solution. These results indicate that the biological activity of the metabolites is similar to that of the parent drug. The presented method of in vitro biotransformation of drugs with S9 fraction followed by HPLC and ecotoxicity tests, may be used as screening method for evaluation of the toxicity of drug metabolites.     

Estrogen receptor alpha overexpressing mouse antral follicles are sensitive to atresia induced by methoxychlor and its metabolites

Methoxychlor (MXC) and its metabolites bind to estrogen receptors (ESRs) and increase ovarian atresia. To test whether ESR alpha (ESR1) overexpressing (ESR1 OE) antral follicles are more sensitive to atresia compared to controls, we cultured antral follicles with vehicle, MXC (1-100 μg/ml) or metabolites (0.1-10 μg/ml). Results indicate that MXC and its metabolites significantly increase atresia in ESR1 OE antral follicles at lower doses compared to controls. Activity of pro-apoptotic factor caspase-3/7 was significantly higher in ESR1 OE treated antral follicles compared to controls. ESR1 OE mice dosed with MXC 64 mg/kg/day had an increased percentage of atretic antral follicles compared to controls. Furthermore, pro-caspase-3 levels were found to be significantly lower in ESR1 OE ovaries than controls dosed with MXC 64 mg/kg/day. These data suggest that ESR1 OE ovaries are more sensitive to atresia induced by MXC and its metabolites in vitro and in vivo compared to controls.     

液相色谱-串联质谱法测定人血浆中兰索拉唑及其代谢物浓度

目的建立液相色谱-串联质谱同时测定人血浆中兰索拉唑及其代谢物兰索拉唑砜、5-羟基兰索拉唑浓度的方法。方法用Agilent SB-C18色谱柱,流动相为0.002 mol.L-1乙酸铵(用甲酸调pH为4)-乙腈(60∶40,v/v),流速为0.3 mL.min-1。用正离子电离,多离子反应监测进行定量分析。结果兰索拉唑、兰索拉唑砜、5-羟基兰索拉唑线性范围分别为5～3044,1.5～550,2～549.5 ng.mL-1,三者日内、日间精密度均小于10%,三者的提取回收率为81.55%～98.74%(RSD<10%)。结论本方法灵敏、准确、快速,可用于人血浆中兰索拉唑及其代谢物浓度的测定和药代动力学研究。     

LC/MS/MS analysis of vesnarinone and its principal metabolites in plasma and urine

An LC/MS/MS assay was developed and successfully used to quantitate vesnarinone and its principal metabolites (OPC-8230, OPC-18136, and OPC-18137) in human plasma and urine. Samples were pre-treated with liquid–solid extraction followed by simultaneous monitoring of primary and daughter ions which were used for the identification and quantitation of the analytes on LC/MS/MS. This assay offers advantages of specificity, speed and greater sensitivity over the previously developed HPLC-UV assay. The lower limit of quantitation is 500 ng ml⁻¹ for vesnarinone and 20 ng ml⁻¹ for OPC-8230, OPC-18137, and OPC-18136 in plasma. Methodology is similar for the estimation of these analytes in urine with the lower limit of quantitation being 500 ng ml⁻¹ for vesnarinone and 100 ng ml⁻¹ for each metabolite. Ascorbic acid was added to stabilize the analytes from degradation. This LC/MS/MS method was developed to overcome many practical problems associated with the HPLC method. The LC/MS/MS method offers the flexibility of analyzing additional metabolites and changing the linearity range to accommodate the differences in linear range (200–10 000 ng ml⁻¹ for vesnarinone and 20–1000 for metabolites) for the analytes.     

大鼠腔前卵泡体外培养方法的建立

目的 建立大鼠腔前卵泡的体外培养方法。方法 取性未成熟大鼠的卵巢,按照酶消化-机械结合法分离腔前卵泡,然后采用动态氧气法(初始孵育时氧气压力为4%,以后每隔24 h氧气压力增加1%,直至最后的氧气压力为11%)、向培养基中添加维生素C(VC)进行培养,观察获得的卵泡数量、形态,及对卵泡发育、激素生成、卵子发生的影响;并将体外卵母细胞的成熟情况与体内卵母细胞的生长情况进行比较,以判断体外培养方法是否成功。结果 酶消化-机械结合法及培养体系可获得大量基底膜完整的腔前卵泡;卵泡和卵母细胞直径显著增加,卵泡存活率91.14%、有腔卵泡形成率25.82%、卵丘细胞-卵母细胞复合体(COCs)排出率38.38%;体外培养卵泡分泌雌二醇(E2)和孕酮(P)与体内分泌特征一致。结论 本实验方法可以获得大量高质量的腔前卵泡,且对卵泡的长期培养发育无明显影响,与大鼠体内卵泡的发育一致。表明本实验成功建立大鼠腔前卵泡的体外培养方法。     

Effect of mono-(2-ethylhexyl) phthalate on bovine oocyte maturation in vitro

The effect of mono(2-ethylhexyl) phthalate (MEHP) on bovine oocyte maturation in vitro was examined. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with various levels of MEHP for 24h, and then examined for the degree of cumulus expansion and the stage of maturation. A higher percentage of oocytes remained at the germinal vesicle (GV) stage after exposure to 75 and 100 micro M MEHP treatments (13.8 and 44.9% of oocytes, respectively) than the control (2.1% of oocytes). The proportion of oocytes that progressed to the metaphase II (MII) stage was significantly decreased with 25 micro M (59.6% of oocytes), 50 micro M (19.8%), 75 micro M (21.3%), and 100 micro M (3.1%) treatments than the control (77.3%). MEHP did not affect the process of cumulus expansion. For denuded oocytes, MEHP treatment of 50-100 micro M resulted in a significantly higher rate of oocytes remained at the GV stage compared to the control (53.4, 80.2, 88.4, and 5.4%, respectively). The rate of MII formation was significantly decreased with 10 micro M (60.9%) and 25 micro M (22.5%) MEHP treatments compared to control (68.9%). Furthermore, with 50, 75 or 100 micro M MEHP, no oocyte reached the MII stage. When COCs were cultured for 24h with 50 or 100 micro M MEHP and then cultured for an additional 24h in MEHP-free medium, most of the oocytes reached the MII stage (71.1 and 64.5%, respectively).Taken together, these results indicate that MEHP, at doses lower than those reported in blood transfusion patients, could negatively modulate bovine oocyte meiotic maturation in vitro, suggesting possible risks for human and other mammalians reproductive health.     

Midkine and cytoplasmic maturation of mammalian oocytes in the context of ovarian follicle physiology

Midkine (MK) was originally characterized as a member of a distinct family of neurotrophic factors functioning in the CNS. However, it was later discovered that MK is abundantly expressed in ovarian follicles. Since then, the physiological roles of this molecule in the ovary have been steadily investigated. During the in vitro maturation (IVM) of oocytes MK was shown to promote the cytoplasmic maturation of oocytes, as indicated by post-fertilization development. This effect of MK could be mediated via its pro-survival (anti-apoptotic) effects on the cumulus-granulosa cells that surround oocytes. The oocyte competence-promoting effects of MK are discussed in the context of the recently discovered involvement of MK in the full maturation of ovarian follicles. MK was at the frontline of a new paradigm for neurotrophic factors as oocytetrophic factors. MK may promote the developmental competence of oocytes via common signalling molecules with the other neurotrophic factor(s). Alternatively or concomitantly, MK may also interact with various transmembrane molecules on cumulus-granulosa cells, which are important for ovarian follicle growth, dominance and differentiation, and act as a unique pro-survival factor in ovarian follicles, such that MK promotes oocyte competence. MK, along with other ovarian neurotrophic factors, may contribute to the optimization of the IVM system.

Linked Articles

This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4     

Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression

The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression.     

13C/12C isotope ratio MS analysis of testosterone, in chemicals and pharmaceutical preparations

The 13C/12C ratio can be used to detect testosterone misuse in sport because (semi)-synthetic testosterone is supposed to have a 13C abundance different from that of endogenous natural human testosterone. In this study, gas chromatography/combustion isotope ratio mass spectrometry (GC/C/IRMS) analysis for the measurement of the delta 13C/1000 value of testosterone from esterified forms of 13 pharmaceutical preparations, six reagent grade chemicals and three bulk materials (raw materials used in pharmaceutical proarations) obtained world-wide was investigated after applying a strong acidic solvolytic procedure. Mean delta 13C/1000 values of non esterified (free) testosterone from chemicals and bulk materials of several testosterone esters were in the range: -25.91/-32.82/1000 while the value obtained for a (semi)-synthetic, reagent grade, free testosterone was -27.36/1000. The delta 13C/1000 results obtained for testosterone from the pharmaceuticals investigated containing testosterone esters were quite homogeneous (mean and S.D. of delta 13C/1000 values of free testosterone: 27.43 +/- 0.76/1000), being the range between -26.18 and -30.04/1000. Values described above were clearly different from those reported by several authors for endogenous natural human testosterone and its main metabolites excreted into the urine in non-consumers of testosterone (delta 13C/1000 range: from -21.3 to -24.4/1000), while they were similar to those of urinary testosterone and metabolites from individuals treated with testosterone esters and testosterone precursors. This finding justifies the fact that administration of these pharmaceutical formulations led to a statistical decrease of carbon isotope ratio of urinary testosterone and its main metabolites in treated subjects.     

UPLC-MS/MS同时测定血浆样品中他莫昔芬及代谢产物的浓度和应用

目的:建立UPLC-MS/MS法测定人体血浆样品中他莫昔芬及代谢产物的浓度和应用.方法:采用Waters AcQuity型超高效液相色谱联用API4000型三重四级杆质谱仪,样品处理采用固相萃取的方法,采用Thermo hypurity C18( 150 mm×2.1 mm,5 μm)色谱分析柱,流动相为10 mmol/L甲酸胺-乙腈(40:60,V/V),流速为0.3 mL/min,进样体积为10 μL,柱温为30℃,样品室温度为5℃,质谱应用ESI源、正离子模式、多反应监测(MRM)方式检测他莫昔芬及各代谢产物的浓度.结果:他莫昔芬、NDTam和TamNox线性范围为1～400 ng/mL,4OH Tam线性范围为0.4～160 ng/mL,4-OHND-Tam线性范围为2～800 ng/mL,可以满足样本浓度定量检测的需要,方法稳定、特异性高,并成功地应用到用药后他莫昔芬及各代谢产物血浆浓度的检测.结论:该方法简便、准确、重复性好,可以准确地定量人体血浆他莫昔芬和代谢产物的浓度,除了为临床血药浓度监测及他莫昔芬药效学研究提供服务外,也可为遗传药理学科研提供技术方法.     

Transgenerational toxicity of Zearalenone in pigs

Zearalenone (ZEN) is a mycotoxin that can be a contaminant of food and feed commodities. ZEN acts as a xenoestrogen and is considered an endocrine disruptor. Since estrogens influence oogenesis during fetal growth, the effect of ZEN on oocytes was investigated in the F1-generation. Pregnant and lactating pigs were exposed to feed naturally contaminated with ZEN (200, 500 and 1000μg/kg feed). Ovaries of F1-animals were examined for follicle development, expression of estrogen converting enzymes and estrogen receptors, and oocyte quality. In F1-newborns, ZEN did not affect follicle dynamics, but follicle integrity decreased with increasing ZEN concentrations. Expression of estrogen receptor beta mRNA increased following ZEN exposure, whereas expression of genes coding for estrogen converting enzymes remained unchanged. In F1-prepubertal gilts, follicular atresia and oocyte maturation with subsequent embryo development remained unchanged. In conclusion, ZEN reduced the quantity of healthy follicles, which may lead to premature oocyte depletion in adulthood.     

Acetylcholine controls mouse oocyte maturation via downregulation of cAMP

1. In mice, acetylcholine (ACh) plays an important role in oocyte activation and embryonic development. However, the role of ACh in mouse oocyte maturation has not been investigated. 2. In the present study, the effects of 100 μmol/L and 1 mmol/L ACh on maturation processes of murine germinal vesicle (GV) intact oocytes (GV oocytes) exposed to 10 and 100 μmol/L 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, were evaluated morphologically and immunologically. It has been shown that IBMX inhibits the resumption of meiosis by preventing cAMP breakdown. 3. In the present study, at the start of in vitro culture 100% of oocytes were at the GV stage. After 18 h culture, 95 ± 3, 0 and 85.8 ± 10.2% of oocytes had passed the GV stage in the control, IBMX and IBMX + ACh groups, respectively. The IBMX-induced inhibition of the maturation process was significantly attenuated by approximately 90% by ACh in groups treated with 10 μmol/L IBMX + 100 μmol/L ACh and 100 μmol/L IBMX + 1 mmol/L ACh. Although cAMP levels were high in oocytes treated with 100 μmol/L IBMX, levels were reduced in groups treated simultaneously with 100 μmol/L ACh. Furthermore, compared with mature oocytes, ACh-treated GV oocytes exhibited significantly lower (by approximately 2.3-fold) or absent Ca(2+) peaks. 4. The results of the present study indicate that maturation of GV oocytes, arrested by IBMX treatment, is resumed following ACh treatment and that this effect is due to downregulation of cAMP rather than changes in intracellular Ca(2+) levels.     

GC/MS/MS detection of pyrrolic metabolites in animals poisoned with the pyrrolizidine alkaloid riddelliine

Pyrrolic metabolites from pyrrolizidine alkaloids (PAs) were detected in liver and dried blood samples using a gas chromatography/tandem mass spectrometry (GC/MS/MS) selected product-ion-monitoring method. A calibration curve was constructed using a protein-metabolite conjugate spiked into dried bovine blood. These spiked samples served as a model for tissues from animals poisoned by the toxic metabolite of PAs. Tissue samples from pigs fed various amounts of the PA alkaloid riddelliine (from Senecio riddellii) were analyzed for pyrrolic metabolites, and the results were applied to the calibration curve to provide a measure of the degree of PA poisoning. Pyrrolic metabolites were detected in liver and blood samples of all poisoned animals at levels between 2 and 64 ppm. Although differences in metabolite levels could be discerned under the reported experimental conditions, the amount detected did not correlate with the dose of riddelliine given; and livers fixed with formalin gave greatly reduced recovery than those same livers either frozen or freeze dried.     

Detection and identification of ACP-105 and its metabolites in equine urine using LC/MS/MS after oral administration

ACP-105 is a novel nonsteroidal selective androgen receptor modulator (SARM) with a tissue-specific agonist effect and does not have side effects associated with the use of common androgens. This research reports a comprehensive study for the detection of ACP-105 and its metabolites in racehorses after oral administration (in vivo) and postulating its structures using mass spectrometric techniques. To obtain the metabolic profile of ACP-105, a selective and reliable LC-MS/MS method was developed. The chemical structures of the metabolites were determined based on their fragmentation pattern, accurate mass, and retention time. Under the current experimental condition, a total of 19 metabolites were detected in ACP-105 drug administered equine urine samples. The study results suggest the following: (1) ACP-105 is prone to oxidation, which gives corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (2) along with oxidation, there is a possibility of elimination of water molecule (dehydration) from the third position of the tropine moiety, resulting in the dehydrated analogs of corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (3) from the study on the metabolites using LC-MS/MS, it is clear that the fragmentation pattern is identical and a great number of fragment ions are common in all the metabolites and the parent drug. (4) The ACP-105 and its metabolites were detected for up to 72 h; thus, the result is a valuable tool for evaluating its use and/or misuse in sport.