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The need for faster diagnosis and more accurate treatment decisions in separation-related problems (SRPs) in dogs is urgent, and a more precise behavioral phenotyping and the development of biomarkers may be of great value. Vasopressin could be a potential non-invasive biomarker of anxiety in dogs with SRPs, but reliable measurement of its concentration is challenging. Here, we compared the behavior and salivary concentrations of copeptin, an arginine vasopressin surrogate, in dogs with SRPs (Case group, n = 13) and with no problems (Control group, n = 15) as they were introduced to a novel environment and subjected to a short episode of separation and reunion with the owner. Dogs in the Case group had greater odds of showing locomotory or oral behaviors during the pre- and post-separation than Controls, while the odds were significantly lower during separation. They also had greater odds of being persistent in seeking attention and proximity from the stranger during reunion. Overall, dogs with SRPs were more likely to express an anxiety-like state during the entire test than Controls, with separation from the owner, and even its anticipation, possibly accounting for this group difference. Although salivary copeptin concentrations did not differ between the two groups, a different trend was detected in Cases and Controls that is worth exploring in further validation studies involving a larger sample.

The aryl hydrocarbon receptor (AhR) has been extensively characterized as an environmental sensor with major roles in xenobiotic-induced toxicity. Evidence is accumulating that these functions serve as adaptive mechanisms overlapping its physiological roles. We previously described a critical role of constitutive AhR activation for the correct progress of mammalian oocyte maturation but the signaling pathway through which AhR controls maturation remains unclear. The aim of this study was to investigate whether the AhR interacts with the epidermal growth factor receptor (EGFR) and p42/44 extracellular regulated kinases (ERK1/2), both key factors in the signaling network that finely regulates the oocyte maturation. As experimental model we used bovine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM). Blocking ERK1/2 signaling in COCs during IVM with the specific EGFR inhibitor AG1478 or the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 downregulated the expression of the AhR-target gene Cyp1a1. Inhibition of AhR activity was associated with a reduction in the oocytes’ ability to progress in meiosis resumption. In contrast, exposure to the AhR antagonist resveratrol reduced both CYP1A1 expression and the oocytes’ maturation competence, without affecting ERK1/2 signaling. These findings strongly indicate the EGFR/ERKs signaling network as an upstream regulator of the AhR activation in COCs, offering a new understanding of the finely tuned physiological mechanism leading to oocyte maturation. This information may provide fresh opportunities for improving oocyte in vitro maturation, and therefore boosting the efficiency of assisted reproduction techniques in mammals.

Simple Summary Intensive growth and development of the piglet’s small intestine is a process that is faster than growth of the whole organism; moreover, it begins early in the prenatal period, accelerating soon after birth and continuing into the post-weaning phase. In fact, during gestation, the gut experiences important morpho-functional changes facilitated by hormones, growth factors, and luminal products. All of these changes are essential to ensure a correct gut function in order to protect animal health and welfare, to ensure optimal productive and reproductive performance, and reduce antimicrobial use. Abstract During the prenatal, neonatal, and weaning periods, the porcine gastrointestinal tract undergoes several morpho-functional, changes together with substantial modification of the microbial ecosystem. Modifications of the overall structure of the small intestine also occur, as well as a rapid increase of the volume, mainly in the last period of gestation: intestinal villi, starting from jejunum, appears shortly before the sixth week of gestation, and towards the end of the third month, epithelial cells diversify into enterocytes, goblet cells, endocrine, and Paneth cells. Moreover, in the neonatal period, colostrum induces an increase in intestinal weight, absorptive area, and brush border enzyme activities: intestine doubles its weight and increases the length by 30% within three days of birth. During weaning, intestinal environment modifies drastically due to a replacement of highly digestible sow milk by solid feed: profound changes in histological parameters and enzymatic activity are associated with the weaning period, such as the atrophy of the villi and consequent restorative hypertrophy of the crypts. All these modifications are the result of a delicate and precise balance between the proliferation and the death of the cells that form the intestinal mucosa (i.e., mitosis and apoptosis) and the health conditions of the piglet. An in-depth knowledge of these phenomena and of how they can interfere with the correct intestinal function can represent a valid support to predict strategies to improve gut health in the long-term and to prevent weaning gut alterations; thus, reducing antimicrobial use.

Endocrine disruptors (EDs) are compounds known to promote transgenerational inheritance of adult-onset disease in subsequent generations after maternal exposure during fetal gonadal development. This study was designed to establish whether gestational and lactational exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) at environmental doses promotes transgenerational effects on reproductive health in female offspring, as adults, over three generations in the mouse. Gestating F0 mouse dams were exposed to 0, 0.05, 5mg/kg/day DEHP in the diet from gestational day 0.5 until the end of lactation. The incidence of adult-onset disease in reproductive function was recorded in F1, F2 and F3 female offspring. In adult F1 females, DEHP exposure induced reproductive adverse effects with: i) altered ovarian follicular dynamics with reduced primordial follicular reserve and a larger growing pre-antral follicle population, suggesting accelerated follicular recruitment; ii) reduced oocyte quality and embryonic developmental competence; iii) dysregulation of the expression profile of a panel of selected ovarian and pre-implantation embryonic genes. F2 and F3 female offspring displayed the same altered reproductive morphological phenotype and gene expression profiles as F1, thus showing transgenerational transmission of reproductive adverse effects along the female lineage. These findings indicate that in mice exposure to DEHP at doses relevant to human exposure during gonadal sex determination significantly perturbs the reproductive indices of female adult offspring and subsequent generations. Evidence of transgenerational transmission has important implications for the reproductive health and fertility of animals and humans, significantly increasing the potential biohazards of this toxicant.

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency.

Background Glomerular filtration rate (GFR) is the most sensitive indicator of initial renal function decline during chronic kidney disease (CKD), but conventional protocols for measuring GFR are labor‐intensive and stressful for the dog. Objectives To assess the diagnostic potential for detecting CKD with simplified GFR protocols based on iohexol plasma clearance. Animals Seventeen CKD‐positive and 23 CKD‐negative dogs of different breeds and sex. Methods Prospective nonrandomized study. Plasma iohexol was measured 5, 15, 60, 90, and 180 minutes after injection. Glomerular filtration rate was calculated using 5 samples (GFR5) or simplified protocols based on 1, 2, or 3 samples. The GFR5 and simplified GFR were compared by Bland‐Altmann and concordance correlation coefficient (CCC) analysis, and diagnostic accuracy for CKD by receiver operating characteristic curves. A gray zone for each protocol was bounded by the fourth quartile of the CKD‐positive population (lower cutoff) and the first quartile of the CKD‐negative population (upper cutoff). Results All simplified protocols gave reliable GFR measurements, comparable to reference GFR5 (CCC >0.92). Simplified protocols which included the 180‐minutes sampling granted the best GFR measure (CCC: 0.98), with strong diagnostic potential for CKD (area under the receiver operating characteristic curve ± SE: 0.98 ± 0.01). A double cutoff including a zone of CKD uncertainty guaranteed reliable diagnosis outside the gray area and identified borderline dogs inside it. Conclusions The simplified GFR protocols offer an accurate, hands‐on tool for CKD diagnosis in dogs. The gray zone might help decision‐making in the management of early kidney dysfunction.

Background: Di(2-ethylhexyl) phthalate (DEHP) and its metabolites are known to affect lipid metabolism and adipogenesis, mainly by activation of peroxisome proliferator-activated receptors (PPARs). Exposure to DEHP has been linked with testicular impairment and male subfertility. However, the effects of DEHP on female reproductive health and metabolism have not been studied in detail. Objective: We examined the effects of dietary DEHP exposure on metabolism and fertility in female mice. Methods: In two independent approaches, female C3H/N mice were exposed to DEHP (0.05, 5, or 500 mg/kg of body weight per day) via their diet for 8 weeks, and we recorded food intake, weight gain, and litter size. After exposure, liver, visceral fat, and plasma from F0 females (study I) and F0 dams and their F1 offspring (study II) were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results: In study I, DEHP-exposed F0 females (all dose groups) had a significant increase in body weight, food intake, and visceral adipose tissue compared with controls. In the 500-mg DEHP group, PPARα and PPARγ transcripts were significantly changed in liver tissue. In the same group, PPARγ mRNA was significantly reduced in liver but not in fat tissue. In addition, leptin and FABP4 (fatty acid binding protein 4) mRNA were increased in adipose tissue, whereas adiponectin was decreased. In study II, we detected a 100% abortion rate in F0 dams in the 500-mg group. F1 offspring exposed in utero and during lactation had an increase in visceral fat tissue and body weight. Conclusion: Fertility was impaired in mice exposed to high doses of DEHP, and body weight and visceral fat deposits were increased in mice exposed to environmentally relevant doses. Although F1 mice were exposed to DEHP only in utero and during lactation, we observed metabolic changes in the offspring of diet-exposed females.

There is a large body of literature describing effects of environmental chemicals (ECs), many of them anthropogenic with endocrine-disrupting properties, on development in rodent laboratory species, some of which lead to impaired reproduction and adverse health. This literature joins extensive human epidemiological data and opportunistic wildlife findings on health effects of ECs. In contrast, the effect of endocrine disruption on foetal development and reproductive performance in domestic species is less extensively documented. This applies both to domestic farm and to companion species even though the former is critical to food production and the latter share our homes and many aspects of the modern developed human lifestyle. In domestic species, the nature of chemicals exposure in utero and their consequences for animal health and production are poorly understood. A complication in our understanding is that the pace of development, ontogeny and efficiency of foetal and maternal hepatic and placental activity differs between domestic species. In many ways, this reflects the difficulties in understanding human exposure and consequences of that exposure for the foetus and subsequent adult from epidemiological and largely rodent-based data. It is important that domestic species are included in research into endocrine disruption because of their (i) wide variety of exposure to such chemicals, (ii) greater similarity of many developmental processes to the human, (iii) economic importance and (iv) close similarities to developed world human lifestyle in companion species.

We investigated the effects of maternal exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) and the organic industrial compounds polychlorinated biphenyls (PCBs), singly and combined, on the reproductive function of male mouse offspring. Mice dams were exposed throughout pregnancy and lactation to 1μg PCBs (101+118)/kg/day, 50μg DEHP/kg/day, or the DEHP/PCB mixture in the diet. The mixture induced permanent alterations in adult F1 males' reproductive health in a way, differently from the single compounds. Depending on the endpoint, we observed: (1) synergy in altering the gross and histological morphology of the testis; (2) antagonism on the expression levels of genes involved in pituitary-gonadal cross-talk; (3) non-interactions on sperm parameters and testosterone production. This study illustrates the complex action of a DEHP/PCB mixture, leading to a unique panel of effects on the male reproductive system, indicating the need for research on the reproductive hazards of combined endocrine disruptors.

The arylhydrocarbon receptor (AhR) mediates the adverse effects of dioxin-like compounds. However, it has also been reported that the AhR may exert a role in ovarian physiology. In the present study, porcine cumulus–oocyte complexes (COCs) were maturedin vitroin the presence of 10% follicular fluid. Expression of AhR and its partner, AhR nuclear translocator occurs in immature COCs. Afterin vitromaturation (IVM), an up-regulation of AhR and cytochrome P450 1A1 (CYP1A1; the main AhR-target gene) was observed. To explore the role of the AhR during IVM, we exposed the COCs to 50 μM β-napthoflavone (βNF). The treatment induced a marked up-regulation of CYP1A1 mRNA, indicating both constitutive and inducible AhR activity. However, in contrast to what was observed in other cell types, no sign of toxicity was observed in COCs. To investigate if components of porcine follicular fluid may exert a protective role against AhR ligands, we exposed porcine COCs to βNF, in the absence of follicular fluid. In these conditions, a marked decrease in the percentage of matured oocytes, concomitant with an increase in oocyte degeneration, was observed. Furthermore, βNF increased apoptosis in cumulus cells in the absence of follicular fluid, whereas βNF has no effects when COCs were treated in the presence of porcine follicular fluid (pFF). In conclusion, these results suggest the presence of unknown endogenous AhR-ligand(s) during porcine IVM and that a dysregulation of this mechanism may result in ovotoxicity by inducing apoptosis in cumulus cells. However, this phenomenon is interrupted by the presence of follicular fluid, indicating a putative protective role for follicular fluid components against exogenous insults.

Cellular and molecular mechanisms mediating the effect of polychlorinated biphenyls on oocyte in vitro maturation: Polychlorinated biphenyls (PCBs) are stable, lipophilic compounds that accumulate in the environment and in the food chain. Recent studies provide evidence that exposure to PCBs can cause reproductive problems. PCBs have been identified in the ovarian follicle of women and other mammals and many data in the literature clearly indicate that both follicles and oocytes are particularly susceptible to these pollutants. In the present review we describe the multifaceted effects of PCBs on mammalian oocyte maturation in detail. Published studies clearly indicate that PCB congeners, both singly or as complex mixtures, disrupt mammalian oocyte maturation and subsequent embryo development. Specifically, data point out to the ability of PCBs to interfere with the organization of the microtubules cytoplasmic network resulting in an altered compartmentalization of the ooplasm. Furthermore, a critical role of cumulus cells in mediating PCB ovotoxicity has been observed, most likely related to a disregulation in intracellular communication between the germinal and the somatic compartment. Finally, since coplanar PCBs, induce gene expression via a ligand-dependent transactivating factor, the aryl hydrocarbon receptor, this signalling pathway is also reviewed with respect to understanding the toxic mechanisms of these compounds.

Oviduct epithelial cells are important for the nourishment and survival of ovulated oocytes and early embryos, and they respond to the steroid hormones estrogen and progesterone. Endocrine-disrupting polyhalogenated aromatic hydrocarbons (PHAH) are environmental toxins that act in part through the ligand-activated transcription factor arylhydrocarbon receptor (AhR; dioxin receptor), and exposure to PHAH has been shown to decrease fertility. To investigate effects of PHAHs on the oviduct epithelium as a potential target tissue of dioxin-type endocrine disruptors, we have established a novel telomerase-immortalized oviduct porcine epithelial cell line (TERT-OPEC). TERT-OPEC exhibited active telomerase and the immunoreactive epithelial marker cytokeratin but lacked the stromal marker vimentin. TERT-OPEC contained functional estrogen receptor (ER)-alpha and AhR, as determined by the detection of ER-alpha- and AhR-specific target molecules. Treatment of TERT-OPEC with the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant increase in the production of the cytochrome P-450 microsomal enzyme CYP1A1. Activated AhR caused a downregulation of ER nuclear protein fraction and significantly decreased ER-signaling in TERT-OPEC as determined by ERE-luciferase transient transfection assays. In summary, the TCDD-induced and AhR-mediated anti-estrogenic responses by TERT-OPEC suggest that PHAH affect the predominantly estrogen-dependent differentiation of the oviduct epithelium within the fallopian tube. This action then alters the local endocrine milieu, potentially resulting in a largely unexplored cause of impaired embryonic development and female infertility.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors α and β (ERα and ERβ) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1–0.0001 μg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17β-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 μg/ml. Groups tre...

To date, approximately 60 chemicals have been identified as endocrine disruptors: exogenous agents that interfere with various aspects of natural hormone physiology. The potential reproductive and health hazards of these environmental chemicals have recently generated concern among the scientific community, policy makers and general public. The present review presents and discusses the available evidence that environmental chemicals are causing ovarian toxicity in various species, with particular attention to farm animals. The impact of chronic exposure to endocrine disruptors via food and drinking water cannot be neglected when studying fertility problems in these species. This review focuses attention on the superfamily of organochlorine chemicals, persistent organic pollutants (POPs), because of their persistence in the environment, ability to concentrate up the food chain, continued detection in environmental matrices and ability to be stored in the adipose tissue of animals and humans. Published data clearly indicate that POPs disrupt mammalian oocyte maturation and follicle physiology in every species studied so far, including farm animals. However, as most of the data available still derive from experiments performed on laboratory species or in vitro models, great care should be taken when extrapolations to other species or environmental situations are attempted.

The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH β subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH β subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species.

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread group of endocrine disrupting compounds in the ecosystem. High concentrations of these substances are known to be present in sewage sludge from industrial, agricultural, and domestic origin that is spread in increasing amounts on arable land and pasture as fertilizer and is found in water, representing an increasing risk for the reproductive health of farm animals. Objective of this study was to determine the impact of PCBs on maturation and developmental competence of cattle oocytes. Since PCBs are a family of 209 molecules present in the environment as a mixture, Aroclor-1254, a pool of more than 60 congeners, was used in these experiments as its composition is considered to be environmentally relevant. Cumulus-oocytes complexes were exposed during IVM to serial concentrations of Aroclor-1254 (between 1 microg/ml and 0.0001 microg/ml) and compared with control groups. Aroclor decreased the percentage of oocytes that reached metaphase II stage after 24 hr, at doses as low as 0.01 microg/ml. Groups treated with 0.001 microg/ml or above, showed an impaired fertilization rate and a dramatic increase of polyspermy. Moreover, exposure during maturation resulted in a reduced proportion of oocytes that cleaved and developed until blastocyst stage although no differences in embryo cell numbers were observed. The present study indicates that very low PCBs concentrations are sufficient to disrupt bovine oocyte maturation, its fertilization, and developmental competence. These results also provide a set of reference data for the assessment of the risk posed by these substances to animal reproductive health, though further work will be necessary to equate in vitro doses to in vivo exposures.

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.

The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the maturation and subsequent development of cattle oocytes in vitro were evaluated in three experiments. Cumulus\p=n-\oocytecomplexes (COC) were collected from cattle ovaries and matured for 20\p=n-\24h in control medium or in medium containing 50 ng EGF ml \m=-\1, 100 ng IGF-I ml\m=-\1, EGF + IGF-I, or 10% (v/v) fetal calf serum plus 0.1 i.u. human menopausal gonadotrophin ml \m=-\ 1 (hMG). In Expt 1, treatment with EGF + IGF-I stimulated cumulus expansion, the metabolism of pyruvate and glutamine, and nuclear maturation. In Expt 2, only the metabolic measurements from oocytes that reached metaphase II were considered, and EGF + IGF-I stimulated pyruvate metabolism to the same extent as serum + hMG. In Expt 3, the oocytes were fertilized after maturation culture, and the resultant embryos cultured for up to 8 days. The cleavage was greater in the EGF and EGF + IGF-I groups than in the controls but less than in the serum + hMG group. Moreover, the number of blastocyst cells at 7 days after insemination and the proportion of cleaved embryos that developed to the blastocyst stage by day 8 was greater in the serum + hMG group than in the control group indicating that maturation treatment can affect early embryonic development. In conclusion, EGF + IGF-I can stimulate cumulus expansion, oxidative metabolism, nuclear maturation and cleavage after fertilization of bovine oocytes in vitro. The relative effects of the treatments on oocyte pyruvate metabolism in Expts 1 and 2 generally paralleled their effects on cleavage and subsequent development in Expt 3, suggesting that mitochondrial function is related to developmental potential. Further investigation is required to determine which component(s) of serum or gonadotrophin treatment is responsible for the effects on subsequent embryonic development.

Oocyte developmental competence is correlated with antral follicle count through ill-defined mechanisms. Oocytes from ovaries with fewer than 10 mid-antral follicles of 2 to 6 mm in diameter (low group) show reduced competence compared with those from ovaries with more than 10 follicles (high group). To unravel mechanisms underlying this phenomenon, this work explored the role of follicular fluid microRNAs (miRNAs, short non-coding RNAs regulating gene expression at the post-transcriptional level). A total of 3 pools of 300 µL of follicular fluid (FF) were collected from mid-antral follicles of low (L) and high (H) groups, respectively. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisEqn 2000 (Illumina Inc., San Diego, CA, USA). Differentially expressed miRNAs were identified with R package edgeR (http://bioconductor.org/packages/release/bioc/html/edgeR.html). Target genes of differentially expressed miRNAs were predicted with DIANA miRPath using homologous human miRNA and gene union options (P 0.05). We found that 27 miRNAs were differentially expressed (false discovery rate ≤0.001): 17 were up-regulated in L and 10 in H. Up-regulated miRNAs in L group were predicted to target 121 genes, 39 of which are specific for ovarian function (e.g. BCL2, FOXO3, KIT, TP53, and PTK2). The GO analysis indicated that these genes were kinases, anti-apoptotic and oncogenic factors, and enriched stress-activated MAPK cascade mediated by oxygen reactive species, G1/S transition checkpoint, cellular response to interleukin-1, and negative regulation of cellular adhesion. Overexpressed miRNAs in H group were predicted to target 92 genes, 22 of which (e.g. MAPK, APC, JNK, PKA) are involved in folliculogenesis. These genes were represented by kinases, apoptotic and cytoskeleton remodelling factors, and enriched very important ovarian processes such as cell cycle, ephrin receptor, smoothened and phosphatidylinositol 3-kinase activity GO processes. Only 7 target genes were common between the 2 groups, 2 of which were important in ovarian functionality (CDKN1A and ITGA5). Interestingly, overexpressed miRNAs in both groups regulate several genes involved in processes apparently not related to folliculogenesis. Finally, regulation can be exerted also by low levels of specific miRNAs such as miR-320, which was reduced in L group and is known to be associated with premature ovarian senescence in women and decreased developmental potential of mouse oocytes. Our results indicate that the different oocyte quality is associated with a different miRNA blueprint, which may alter the expression of several genes relevant for intra- and extra-ovarian processes. Further studies will be necessary to determine if FF miRNAs can act outside the ovary and if their levels can be detected in the bloodstream thereby becoming possible noninvasive, real-time markers to determine oocyte quality in living animals. This study was supported by FP7-KBBE-2012-FECUND-312097.

Several studies indicate that in utero and perinatal exposure to polychlorinated biphenyls (PCBs) induces adverse reproductive effects, but it remains unclear whether such effects may be transmitted to subsequent generations. We therefore investigated the association between maternal exposure to PCBs and reproductive health in male and female offspring over three generations. Mouse dams were fed 0, 1, 10, and 100 mg/kg/day of a PCB mixture (101 1 118) during pregnancy and lactation. PCB levels were measured in the tissues of both dams and offspring. PCB concentrations at all doses investigated were greater in the offspring than in the dams (p £ 0.0001) confirming that the progeny were exposed as a result of maternal exposure. In F1 offspring, exposure to PCBs resulted in reductions in (1) testis weight (p £ 0.05) and seminiferous tubule diameter (p £ 0.05), (2) sperm viability (p £ 0.0001) and developmental capacity (p £ 0.05), (3) ovary weight (p £ 0.05), (4) oocyte developmental capacity (p £ 0.05), and (5) increased follicular atresia (p £ 0.0001). In females, adverse effects were observed only in the F1 animals. In contrast, male offspring exhibited reduced sperm viability and altered seminiferous tubule distribution up to the third generation, showing intergenerational transmission. In summary, our data indicate that exposure to PCBs at the time of gonadal sex determination perturbed, significantly, the reproductive physiology of male and female offspring in adulthood. Furthermore, male reproductive deficiencies may be observed in at least two further generations. These findings have significant implications for reproductive health and fertility of animals and humans.

The present study examined the effects in mice of exposure to di(2-ethyl-hexyl) phthalate (DEHP) throughout pregnancy and lactation on the development and function of the pituitary-gonadal axis in male and female offspring once they have attained adulthood. Groups of two to three dams were exposed with the diet from gestational d 0.5 until the end of lactation, at 0, 0.05, 5, and 500 mg DEHP/kg · d. The experiment was repeated three times (total: seven to 10 dams per treatment). The 500-mg dose caused complete pregnancy failure, whereas exposure to doses of 0.05 and 5 mg did not affect pregnancy and litter size. In total, about 30 male and 30 female offspring per group were analyzed. Offspring of the DEHP-treated groups, compared with controls, at sexual maturity showed: 1) lower body weight (decrease 20–25%, P < 0.001); 2) altered gonad weight (testes were ∼13% lighter and ovaries ∼40% heavier; P < 0.001); 3) poor germ cell quality (semen was ∼50% less concentrated and 20% less viable, and ∼10% fewer oocytes reached MII stage, P < 0.001); 4) significant lower expression of steroidogenesis and gonadotropin-receptor genes in the gonads; and 5) up-regulated gonadotropin subunit gene expression in the pituitary. In conclusion, our findings suggest that, in maternally exposed male and female mice, DEHP acts on multiple pathways involved in maintaining steroid homeostasis. Specifically, in utero and lactational DEHP exposure may alter estrogen synthesis in both sexes. This, in turn, induces dysregulation of pituitary-gonadal feedback and alters the reproductive performance of exposed animals.

Evidence is accumulating that environmental chemicals (ECs) including endocrine-disrupting compounds (EDCs) can alter female reproductive development, fertility and onset of menopause. While not as clearly defined as in the male, this set of abnormalities may constitute an Ovarian Dysgenesis Syndrome with at least some origins of the syndrome arising during foetal development. ECs/EDCs have been shown to affect trophoblast and placental function, the female hypothalamo-pituitary–gonadal axis, onset of puberty and adult ovarian function. The effects of ECs/EDCs are complex, not least because it is emerging that low-level, ‘real-life’ mixtures of ECs/EDCs may carry significant biological potency. In addition, there is evidence that ECs/EDCs can alter the epigenome in a sexually dimorphic manner, which may lead to changes in the germ line and perhaps even to transgenerational effects. This review summarises the evidence for EC, including EDC, involvement in female reproductive dysfunction, it highlights potential mechanisms of EC action in the female and emphasises the need for further research into EC effects on female development and reproductive function.

In recent years, the development of methods for the genetic manipulation of domestic species has generated a rapidly increasing demand for pre-attachment embryos. The limited prolificacy of these species makes superovulation and surgical recovery of embryos necessary. However, these techniques are too expensive and labour-intensive to be used routinely for supplying enough material for experimental or commercial applications. This has provided the thrust for an unprecedented effort to develop methods for the culture of embryos derived from in vitro maturation and fertilization of oocytes collected from slaughtered animals. Offspring generated in vitro have been obtained using cattle, goats, pigs and sheep, but the efficiency and reliability of the techniques and the quantity of the embryos vary between species. At present, the best results can be obtained in ruminants, while pig embryos have proved to be more difficult to generate. Although many obstacles have been overcome simply by empirical trials and observations, the availability of high numbers of easily accessible embryos has also led to a substantial advance in our knowledge of their physiology. This has therefore widened the range of experimental models that can effectively be used in developmental studies, especially since, in some cases, models using these species may be more relevant to human embryology than those using rodents.

International audience; Anthropogenic pollutants comprise a wide range of synthetic organic compounds and heavy metals, which are dispersed throughout the environment, usually at low concentrations. Exposure of ruminants, as for all other animals, is unavoidable and while the levels of exposure to most chemicals are usually too low to induce any physiological effects, combinations of pollutants can act additively or synergistically to perturb multiple physiological systems at all ages but particularly in the developing foetus. In sheep, organs affected by pollutant exposure include the ovary, testis, hypothalamus and pituitary gland and bone. Reported effects of exposure include changes in organ weight and gross structure, histology and gene and protein expression but these changes are not reflected in changes in reproductive performance under the conditions tested. These results illustrate the complexity of the effects of endocrine disrupting compounds on the reproductive axis, which make it difficult to extrapolate between, or even within, species. Effects of pollutant exposure on the thyroid gland, immune, cardiovascular and obesogenic systems have not been shown explicitly, in ruminants, but work on other species suggests that these systems can also be perturbed. It is concluded that exposure to a mixture of anthropogenic pollutants has significant effects on a wide variety of physiological systems, including the reproductive system. Although this physiological insult has not yet been shown to lead to a reduction in ruminant gross performance, there are already reports indicating that anthropogenic pollutant exposure can compromise several physiological systems and may pose a significant threat to both reproductive performance and welfare in the longer term. At present, many potential mechanisms of action for individual chemicals have been identified but knowledge of factors affecting the rate of tissue exposure and of the effects of combinations of chemicals on physiological systems is poor. Nevertheless, both are vital for the identification of risks to animal productivity and welfare.

The present study was designed to investigate the relationship between pre-mating nutrition and the relative amounts of a panel of developmentally relevant genes in ovine oocytes and granulosa cells. Cast age ewes were fed a ration providing 0.5× (0.5 M) or 1.5× (1.5 M) live weight maintenance requirements for 2 weeks before slaughter. The ewes were synchronized and superovulated with FSH and pregnant mares serum gonadotropin. At slaughter, oocytes and granulosa cells were aspirated from follicles >2 mm in diameter and the relative abundance of 8 and 17 transcripts in oocytes and granulosa cells respectively were analyzed by semi-quantitative RT-PCR. In the oocytes, no differences between groups were observed for five transcripts (GDF9,BMP15, c-kit, glucose transporter 1 (SLC2A1), and hexokinase 1), but a lower amount of glucose transporter 3 (SLC2A3), sodium/glucose cotransporter 1 (SLC5A1), and Na+/K+ATPase mRNAs was detected in the 0.5 M group. Increased expression ofPTGS2,HAS2, and the leptin receptor long form was observed in granulosa cells from the 0.5 M group. No differences between groups were observed for the other transcripts (early growth response factor-1, estrogen receptor-α, LH and FSH receptors, gremlin 1, pentraxin 3, KIT ligand, glucose transporters 1, 3, and 8,IGF1, IGF1 receptor, leptin receptor, and tumor necrosis factor-stimulated gene 6). Expression of leptin and sodium/glucose cotransporter 1 was not detected in both groups. The present data indicate that pre-mating nutrition is associated with alteration in the mRNA content in oocytes and surrounding follicle cells in ewes, which may account for the reduced reproductive performance typical of ewes that are fed a restricted ration for a short period of time before mating.

It is becoming increasingly evident that cancer constitutes a group of diseases involving altered stem-cell maturation/differentiation and the disturbance of regenerative processes. The observed malignant transformation is merely a symptom of normal differentiation processes gone astray rather than the primary event. This review focuses on the role of cancer stem cells (CSCs) in three common but also relatively under-investigated cancers: head and neck, ovarian, and testicular cancer. For didactic purpose, the physiology of stem cells is first introduced using hematopoietic and mesenchymal stem cells as examples. This is followed by a discussion of the (possible) role of CSCs in head and neck, ovarian, and testicular cancer. Aside from basic information about the pathophysiology of these cancers, current research results focused on the discovery of molecular markers specific to these cancers are also discussed. The last part of the review is largely dedicated to signaling pathways active within various normal and CSC types (e.g. Nanog, Nestin, Notch1, Notch2, Oct3 and 4, Wnt). Different elements of these pathways are also discussed in the context of therapeutic opportunities for the development of targeted therapies aimed at CSCs. Finally, alternative targeted anticancer therapies arising from recently identified molecules with cancer--(semi-)selective capabilities (e.g. apoptin, Brevinin-2R) are considered.

Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time. Normal stem cells in the adult organism are responsible for renewal and repair of aged or damaged tissue. Adult stem cells are present in virtually all tissues and during most stages of development. In this review, we introduce the reader to the basic information about the field. We describe selected stem cell isolation techniques and stem cell markers for various stem cell populations. These include makers for endothelial progenitor cells (CD146/MCAM/MUC18/S-endo-1, CD34, CD133/prominin, Tie-2, Flk1/KD/VEGFR2), hematopoietic stem cells (CD34, CD117/c-Kit, Sca1), mesenchymal stem cells (CD146/MCAM/MUC18/S-endo-1, STRO-1, Thy-1), neural stem cells (CD133/prominin, nestin, NCAM), mammary stem cells (CD24, CD29, Sca1), and intestinal stem cells (NCAM, CD34, Thy-1, CD117/c-Kit, Flt-3). Separate section provides a concise summary of recent clinical trials involving stem cells directed towards improvement of a damaged myocardium. In the last part of the review, we reflect on the field and on future developments.

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocytein vitromaturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs duringin vitromaturation has been investigated. Results indicate that exposure of cumulus–oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.

The dioxin/aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsive to both natural and man-made environmental compounds. AhR and its nuclear partner ARNT are expressed in the female reproductive tract in a variety of species and several indications suggest that the AhR might play a pivotal role in the physiology of reproduction. Furthermore, it appears to be the mediator of most, if not all, the adverse effects on reproduction of a group of highly potent environmental pollutants collectively called aryl hydrocarbons (AHs), including the highly toxic compound 2,3,7,8-tetrachlor-odibenzo-p-dioxin (TCDD). Although a large body of recent literature has implicated AhR in multiple signal transduction pathways, the mechanisms of action resulting in a wide spectrum of effects on female reproduction are largely unknown. Here we summarize the major types of molecular cross-talks that have been identified for the AhR and linked cell signaling pathways and that are relevant for the understanding of the role of this transcription factor in female reproduction.

The arylhydrocarbon receptor (AhR) is known to mediate toxic responses to dioxin (2,3,7,8-tetrachlorodibenzo-p- dioxin) and related compounds and has been extensively characterized from a toxicological viewpoint. However, it has recently been reported that the AhR may have a central role in ovarian physiology. To investigate the role of AhR during oocyte maturation, we analyzed the expression of AhR, its nuclear partner AhR nuclear translocator, and the major target gene CYP1A1, in bovine cumulus-oocyte complexes (COCs) by semiquantitative RT-PCR and Western blot. Coexpression of AhR and AhR nuclear translocator was observed in both oocytes and surrounding cumulus cells before and after in vitro maturation (IVM). Furthermore, after IVM, both cell types showed a clear up-regulation of AhR mRNA compared with the expression at 0 h. Constitutive expression of CYP1A1 mRNA was observed in immature oocytes at the background level, whereas no expression was observed in the surrounding cumulus cells. Interestingly, a significant increase in CYP1A1 expression level was observed in both oocytes and cumulus cells after IVM. To further investigate the role of AhR in CYP1A1 up-regulation and oocyte maturation, COCs were treated throughout IVM with the AhR antagonists, alpha-naphthoflavone and resveratrol. Both antagonists decreased the level of CYP1A1 in COCs compared with controls. Furthermore, CYP1A1 down-regulation was accompanied by a reduced ability of oocytes to complete in vitro maturation until metaphase II stage. These results suggest that CYP1A1 induction in COCs is necessary for correct proceeding of in vitro oocyte maturation in bovine and suggest a physiological role of AhR during resumption of meiosis.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 microg/ml. Groups treated withor =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.

Polychlorinated biphenyls (PCBs) can interfere with normal reproductive functions acting as endocrine disruptors. Aroclor-1254 (A-1254), is a pool of more than 60 congeners used for in vitro studies because its composition is representative of PCBs environmental pollution. We previously demonstrated that the exposure of bovine oocytes to A-1254 during in vitro maturation (IVM) was detrimental not only to the maturation process but also induced a significant increase of polyspermy and a reduction of developmental competence. Therefore, we investigated whether A-1254 acts on two processes that occur during IVM and may be related with its negative effects: maternal mRNA polyadenylation and cortical granules (CGs) migration and exocytosis. Bovine cumulus-oocyte complexes (COCs) were exposed to 0.1 microg/ml of A-1254 during IVM, a level of exposure known to affect oocyte maturation, fertilization, and developmental competence. Oocyte exposure to A-1254 altered the poly(A) tail length of 5 out of 10 genes examined. PCBs effect on mRNA polyadenylation was different depending on the gene considered and resulted either in a shorter or in a longer poly(A) tail. At the end of maturation, Aroclor treated oocytes presented clustered CG in a significantly higher percentage than the control group. In addition, CG exocytosis after 8 hr of fertilization occurred at significantly lower extent in zygotes derived from the exposed group compared to control. Our results indicated that the lower developmental competence of oocytes exposed to PCBs during IVM can be related to the interaction of these contaminants with mechanisms regulating maternal mRNA storage in the ooplasm and normal CGs function.

Glucose is readily been taken up and utilized by preimplantation embryos from different species. However, a comprehensive analysis of the glucose transporter expression throughout preimplantation development is still missing. Here, we have investigated the expression of facilitative glucose transporters (Glut1-5 and 8) and sodium-dependent-glucose transporter (SGLT-I) in bovine oocytes and preimplantation embryos up to d16 of development, using RT-PCR and immunohistochemistry. The embryos were produced in vitro by IVM-IVF. Glut1, Glut3, Glut8, and SGLT-I were expressed in all stages studied. Glut4 transcripts were first detected at the blastocyst stage. Glut2 expression was restricted to the period of blastocyst elongation at d14 and d16. Transcription of the fructose transporter Glut5 started at the 8-/16-cell stage. Our results show a distinct expression pattern for glucose transporters during bovine embryo development in vitro indicating specialized functions for these isoforms at different developmental stages in bovine embryos. Mol. Reprod. Dev. 60:370-376

Previous studies on sheep have demonstrated effects of maternal nutrition on fetal development with effects being observed on reproductive function (Rae et al. 2002 Anim. Reprod. Sci. 72, 63–71), blood pressure (Gopalakrishnan et al. 2004 Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R12–R20), and behavior (Erhard et al. 2004 Behav. Brain Res. 151, 25–35) in the adult offspring. It is known that gene expression in developing oocytes and very early embryos is influenced by their environment and some of the effects of nutrition on reproductive and other systems are likely to be expressed through such early changes (Hyttel 2000 Anim. Reprod. Sci. 60–61, 49–60). Effects of nutrition on oocyte function (i.e. before ovulation and fertilization) and resultant embryo quality have been observed (Gonzalez-Bulnes et al. 2004 Reprod. Fertil. Dev. 16, 421–435), but effects on oocyte gene expression have not been investigated. The aim of the present study was to investigate the effects of premating nutrition on the expression of selected genes in sheep cumulus cells and oocytes. For this purpose, 16 cast age ewes of proven fertility were used. At the time of manipulation and study, all ewes had a condition score of approximately 2.5. During the 2 weeks before slaughter, ewes of one group were fed a ration providing 0.5-live weight (n = 10) maintenance requirements and the other group 1.5-live weight (n = 6) maintenence requirements. This treatment ensured that all animals had similar numbers of potentially ovulatory follicles but different patterns of ovarian follicle maturation and, accordingly, different numbers of follicles and oocytes undergoing the final stages of maturation. The ewes's reproductive cycles were synchronized using intravaginal pessaries inserted 2 weeks before slaughter. At slaughter, ovaries were recovered and oocytes and associated cumulus cells were aspirated from all follicles >2 mm in diameter. For each ovary, oocytes derived from follicles with a diameter 4 mm were pooled separately, and stored for further analysis. Associated cumulus masses were collected and stored singly for subsequent investigations. As expected from previous observations, the number of visible follicles, the ratio of small-to-large antral follicles, and the total numbers of oocytes per ewe were similar for control and underfed ewes. Furthermore, the expression levels of the markers for oocyte growth, GDF-9, BMP-15, and c-kit, were investigated. Data were analyzed by ANOVA followed by Duncan test. Results showed no major differences between groups, independent of follicle diameter. These data suggest that the nutritional status of donor ewes did not significantly affect the expression profiles of these genes. However, further analyses on a greater panel of genes is required to rule out premating nutrition effects on oocyte gene expression. Furthermore, effects on the cumulus cell gene expression profile remain to be investigated. This work was supported by the University of Milan, FIRST 2006; Scottish Executive Environment and Rural Affairs Department.

The arylhydrocarbon receptor (AhR), a ligand activated transcription factor, has been extensively characterized from a toxicological point of view due to its ability to mediate the adverse effects of a variety of halogenated aromatic hydrocarbons. Recent reports on AhR knockout mice suggest that the AhR may play a role in ovarian physiology. We have previously demonstrated that AhR activity (as indicated by the up-regulation of the target gene cytochrome p450 1A1: CYP1A1) is stimulated during bovine oocyte IVM in the absence of exogenous ligands. Furthermore, exposure to specific AhR antagonists, besides down-regulating the expression of CYP1A1, significantly impairs the ability of the oocyte to complete maturation until the metaphase II stage (Pocar et al. 2004 Endocrinology 145, 1594–1601). The aim of the present study was to further investigate the mechanisms underlying the AhR activation during IVM. Several reports point to a critical role of phosphorylation in the regulation of the AhR-complex. Furthermore, the mitogen-activated protein kinase (MAPK, extracellular regulated kinase (ERK 1 and 2)) cascade has been shown to play a crucial role in regulating meiotic cell cycles during bovine oocyte maturation. A total of 572 bovine cumulus-oocyte complexes were used to investigate the potential role of the MAPK in modulating the activity of the AhR during IVM. The effect of the broad-spectrum serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the induction of CYP1A1 during oocyte maturation was investigated. As expected, exposure to 6-DMAP induced meiotic arrest (at the stage of germinal vesicle/germinal vesicle breakdown) and down-regulated the expression level of phosphorylated ERK 1 and 2. Interestingly, a significant down-regulation of the target genes CYP1A1 and CYP1B1 (9.5% and 26.8% of control, respectively) and an up-regulation of the AhR (199.4% of control) were observed at the mRNA level. This phenomenon was partially reversible after a period of further 24 h of culture in the absence of 6-DMAP. In this condition, besides a recovery of oocyte maturation and phosphorylation status of ERK 1 and 2 to levels comparable to control, a significant up-regulation of CYP1A1 mRNA was observed (68.3% of control). Finally, to confirm the role of serine/threonine kinases in modulating the activity of AhR during resumption of meiosis, we exposed the oocytes to cycloheximide, a protein synthesis inhibitor, also known to arrest oocyte maturation. Furthermore, although cycloheximide exposure induced meiotic arrest, no significant differences in the expression levels of AhR or its target genes compared to control were observed. Each experiment was replicated at least three times. Data were assessed using ANOVA followed by Duncans multiple rance test. The criterion for significance was set at P < 0.05. In conclusion, our results strongly suggest that 6-DMAP-sensitive kinase(s) is (are) involved in the regulation of AhR during bovine oocyte maturation. Further analyses are necessary to understand the biological significance of these observations.