E.J. Schoevers

Exposure of Oocytes to the Fusarium Toxins Zearalenone and Deoxynivalenol Causes Aneuploidy and Abnormal Embryo Development in Pigs

Abstract Fungi of the Fusarium species can infect food and feed commodities and produce the mycotoxins zearalenone (ZEA) and deoxynivalenol (DON). Since both toxins have been reported to reduce fertility, the mechanisms of ZEA and DON on inhibition of oocyte maturation were examined. Pig oocytes were matured in the presence of ZEA (a mycotoxin with estrogenlike activity), 17beta-estradiol, and DON (all 3.12 μmol/L). Zearalenone, 17beta-estradiol, and DON inhibited oocyte maturation and caused approximately 34% of the oocytes to form an aberrant spindle. Different ratios of ZEA:DON did not lead to a more severe inhibition of oocyte maturation. Both mycotoxins caused abnormal formation of the meiotic spindle. The developmental competence of oocytes matured in the presence of mycotoxins was further investigated after in vitro fertilization. Presence of ZEA (3.12 μmol/L) during maturation reduced the percentages of oocytes that cleaved and formed a blastocyst to about 12%, compared with 25% of control oocytes. Maturation in the presence of equimolar concentrations of DON was not compatible with development. The ploidy of blastomeres from blastocysts derived from mycotoxin-exposed oocytes was analyzed with fluorescent in situ hybridization. All blastocysts, even those from the control group, contained at least one blastomere with abnormal ploidy, but the variation in the percentages of aneuploid blastomeres was significantly larger in embryos from oocytes exposed to mycotoxins. It is concluded that ZEA and DON can lead to abnormal spindle formation, leading to less fertile oocytes and embryos with abnormal ploidy, and that the effects of ZEA and DON are not synergistic.

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.

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development

The present study was conducted to evaluate the function of cumulus cells during bovine IVF Oocytes within cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology, fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.

Involvement of Bicarbonate-Induced Radical Signaling in Oxysterol Formation and Sterol Depletion of Capacitating Mammalian Sperm During In Vitro Fertilization

ABSTRACT This study demonstrates for the first time that porcine and mouse sperm incubated in capacitation media supplemented with bicarbonate produce oxysterols. The production is dependent on a reactive oxygen species (ROS) signaling pathway that is activated by bicarbonate and can be inhibited or blocked by addition of vitamin E or vitamin A or induced in absence of bicarbonate with pro-oxidants. The oxysterol formation was required to initiate albumin dependent depletion of 30% of the total free sterol and >50% of the formed oxysterols. Incubation of bicarbonate treated sperm with oxysterol-binding proteins (ORP-1 or ORP-2) caused a reduction of >70% of the formed oxysterols in the sperm pellet but no free sterol depletion. Interestingly, both ORP and albumin treatments led to similar signs of sperm capacitation: hyperactivated motility, tyrosin phosphorylation, and aggregation of flotillin in the apical ridge area of the sperm head. However, only albumin incubations led to high in vitro fertilization rates of the oocytes, whereas the ORP-1 and ORP-2 incubations did not. A pretreatment of sperm with vitamin E or A caused reduced in vitro fertilization rates with 47% and 100%, respectively. Artificial depletion of sterols mediated by methyl-beta cyclodextrin bypasses the bicarbonate ROS oxysterol signaling pathway but resulted only in low in vitro fertilization rates and oocyte degeneration. Thus, bicarbonate-induced ROS formation causes at the sperm surface oxysterol formation and a simultaneous activation of reverse sterol transport from the sperm surface, which appears to be required for efficient oocyte fertilization.

Seroprevalence of porcine reproductive and respiratory syndrome virus in Dutch weaning pigs

To determine whether under Dutch field conditions PRRSV infection occurs in weaning pigs before the finishing period, a cross-sectional study was performed on 32 breeding farms to estimate the seroprevalence of antibodies directed against PRRSV in 4- to 5-week-old and 8- to 9-week-old pigs. Farms were visited twice within 5 months, and during each sampling an average of 20 sera were randomly collected from a unit of 4- to 5-week-old and a unit of 8- to 9-week-old pigs. The sera (n = 2568) were tested in the IDEXX-ELISA for the presence of antibodies directed against PRRSV. The seroprevalence of PRRSV in 4- to 5-week-old pigs and 8- to 9-week-old pigs varied between both samplings for each farm. The seroprevalence in the younger pigs was significantly higher than in the older pigs for both samplings (p < 0.05), suggesting the presence of maternal antibodies. In addition, a longitudinal study was performed to evaluate the IDEXX-ELISA in detecting maternal antibodies directed against PRRSV and to determine the rate of decline of these antibodies in field sera. From serological results of eight litters, an average decay function was computed to quantify the maternal immunity to PRRSV. A seroprevalence in 8- to 9-weeks-old pigs of > or = 0.20 was calculated to indicate an active immune response to PRRSV. In the cross-sectional study in the pigs twenty-three percent of the units with 8- to 9-week-old pigs were considered to have an active serological response against PRRSV. We conclude that most Dutch pigs are seronegative for PRRSV at the start of the finishing period, since the results of this study showed that 77% of the units with 8- to 9-week-old pigs had a seroprevalence < 0.20.

Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle.

Deoxynivalenol (DON, vomitoxin) is a secondary metabolite and mycotoxin produced by Fusarium species that occurs with a high prevalence in cereals and grains intended for human and animal consumption. Pigs are considered to be the most sensitive animal species and exposure to DON results in reduced feed intake, reduced performance and cause alterations in the expression of markers of inflammation and cell cycle regulation. The objective of this study was to determine how DON possibly affects the oocyte developmental potential in vitro at concentrations which correspond to those observed in practice. To evaluate DON toxicity during specific stages of oocyte meiosis, cumulus-oocyte complexes were exposed to 0.02, 0.2, or 2 microM DON. Exposure to the highest DON concentration inhibited cumulus expansion and induced cumulus cell death. After exposure for 42 h, DON at all concentrations reduced Metaphase II formation and led to malformations of the meiotic spindle. Despite spindle malformations, exposure to different concentrations of DON did not lead to increased percentages of blastomeres with abnormal ploidy in embryos. Spindle malformation occurred by DON exposure during formation of meiotic spindles at Metaphase I and II, but embryo development was also reduced when oocytes were exposed to DON during Prophase I. Together, these results indicate that exposure to DON via contaminated food or feed can affect oocyte developmental competence by interfering directly with microtubule dynamics during meiosis, and by disturbing oocyte cytoplasmic maturation through other as yet undetermined mechanisms.

Chemotherapy of human head and neck cancer xenografts with three clinically active drugs: cis -platinum, bleomycin and methotrexate

Human head and neck tumours were successfully transplanted in athymic nude mice. In 14 xenograft lines the effect of 1 to 3 clinically active agents could be tested. Maximum tolerated doses were given daily for 3-7 days. Growth delay was estimated in terms of the number of volume doubling times gained by the treatment. Cis-platinum and bleomycin appeared to be effective agents. In all 6 lines in which cis-platinum was examined, growth delay sometimes followed by complete regression was achieved. In 6/7 lines a response to bleomycin was observed. There was wide variation in sensitivity to cis-platinum and bleomycin among the different lines. Methotrexate, effective in 40-60% of patients with head and neck cancer, essentially showed no activity. Methotrexate produced a minimal growth delay in 1/11 lines treated. Two of the patients from whom xenografts were obtained responded to methotrexate treatment. The observed lack of activity of methotrexate against these tumour xenografts indicates that this model has limitations in the screening of new anticancer agents.

Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology

Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients’ exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human.