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Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

MicroRNAs are promising biological markers for prenatal diagnosis. They regulate placental development and are present in maternal plasma. Maternal metabolic diseases are major risk factors for placental deterioration. We analysed the influence of a maternal insulin-dependent diabetes mellitus on microRNA expression in maternal plasma and in blastocysts employing an in vivo rabbit diabetic pregnancy model and an in vitro embryo culture in hyperglycaemic and hypoinsulinaemic medium. Maternal diabetes led to a marked downregulation of Dicer protein in embryoblast cells and Drosha protein in trophoblast cells. MiR-27b, miR-141 and miR-191 were decreased in trophoblast cells and in maternal plasma of diabetic rabbits. In vitro studies indicate, that maternal hyperglycaemia and hypoinsulinaemia partially contribute to the downregulation of trophoblastic microRNAs. As the altered microRNA expression was detectable in maternal plasma, too, the plasma microRNA signature could serve as an early biological marker for the prediction of trophoblast function during a diabetic pregnancy.

The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets inin vitrocultured preimplantation rabbit blastocysts andin vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1;CCND1), translation initiation (eukaryotic translation initiation factor 4E;EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: geneSLC7A8) and proliferation (proliferating cell nuclear antigen;PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns ofCCND1andEIF4Ewere increased in embryos from diabetic rabbits, while the expression of proliferation markerPCNAwas decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy.

Title of the paper: Level estimation of metabolic parameters in the glucose tolerance test (GTT) of young bulls The metabolic parameters of the glucose tolerance test are suitable for an additional recommendation for the valuation of the breeding bulls before the start of the offspring’s test could be given. Very important is the level of glucose half live to the begin of lactation of the cows. The other limit is the hyperglycemias greater than 10 mmol/l (or 180 mg/dl) in the glucose level in the blood. The glucose half live measured nearly 48 minutes is comparable in the 8th week post partum in lactation of cows with the young bulls in the age one year. Their combination with the pedigree breeding value is leading to increase of information, that could be utilised to the selection of improper bulls if the present results were confirmed.

Maternal metabolic diseases such as diabetes mellitus with diabetogenic hypoinsulinemia and hyperglycemia change periconceptional developmental conditions in utero. In preimplantation rabbit embryos, all major metabolic pathways are affected. Alterations in protein, lipid and glucose metabolism, adipokines, advanced glycation end products (AGEs) and reactive oxygen species (ROS) are described in this review. The embryonic metabolism is characterized by a high plasticity which enables survival of most preimplantation embryos under the non-physiological developmental conditions in diabetic mothers. Adiponectin, for example, compensates for the missing insulin-driven glucose supply and stimulates intracellular lipid accumulation in embryonic cells. AGEs and ROS are clear indicators of metabolic stress. The price paid for survival, however, needs to be taken into consideration. It is an increase in lipogenesis and proteinogenesis, leading to metabolic stress and with potentially negative long-term health effects.

Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml–1) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes. Copyright © 2014 John Wiley & Sons, Ltd.

Diabetes mellitus (DM) during pregnancy is one of the leading causes of perinatal morbidity and birth defects. The mechanism by which maternal hyperglycemia, the major teratogenic factor, induces embryonic malformations remains unclear. Advanced glycation end products (AGEs) are known to accumulate during the course of DM and contribute to the development of diabetic complications. Employing a diabetic rabbit model, we investigated the influence of maternal hyperglycemia during the preimplantation period on AGE formation (pentosidine, argpyrimidine, andNε-carboxymethyllysine (CML)) in the reproductive tract and the embryo itself. As a consequence of type 1 DM, the AGE levels in blood plasma increased up to 50%, correlating closely with an AGE accumulation in the endometrium of diabetic females. Embryos from diabetic mothers had increased protein-bound CML levels and showed enhanced fluorescent signals for AGE-specific fluorescence in the blastocyst cavity fluid (BCF). The quantification of CML by HPLC–mass spectrometry (MS/MS) showed a higher amount of soluble CML in the BCF of blastocysts from diabetic rabbits (0.26±0.05 μmol/l) compared with controls (0.18±0.02 μmol/l). The high amount of AGEs in blastocysts from diabetic mothers correlates positively with an increasedAGER(receptor for AGE (RAGE)) mRNA expression. Our study gives alarming insights into the consequences of poorly controlled maternal diabetes for AGE formation in the embryo. Maternal hyperglycemia during the preimplantation period is correlated with an increase in AGE formation in the uterine environment and the embryo itself. This may influence the development of the embryo through increased AGE-mediated cellular stress by RAGEs.

According to the “developmental origin of health and disease” hypothesis, the metabolic set points of glucose and lipid metabolism are determined prenatally. In the case of a diabetic pregnancy, the embryo is exposed to higher glucose and lipid concentrations as early as during preimplantation development. We used the rabbit to study the effect of maternal diabetes type 1 on lipid accumulation and expression of lipogenic markers in preimplantation blastocysts. Accompanied by elevated triglyceride and glucose levels in the maternal blood, embryos from diabetic rabbits showed a massive intracellular lipid accumulation and increased expression of fatty acid transporter 4, fatty acid–binding protein 4, perilipin/adipophilin, and maturation of sterol-regulated element binding protein. However, expression of fatty acid synthase, a key enzyme for de novo synthesis of fatty acids, was not altered in vivo. During a short time in vitro culture of rabbit blastocysts, the accumulation of lipid droplets and expression of lipogenic markers were directly correlated with increasing glucose concentration, indicating that hyperglycemia leads to increased lipogenesis in the preimplantation embryo. Our study shows the decisive effect of glucose as the determining factor for fatty acid metabolism and intracellular lipid accumulation in preimplantation embryos.

Objective: The food contaminants bisphenol A (BPA), diethylhexylphthalate (DEHP), and tributyltin (TBT) are potent endocrine-disrupting compounds (EDC) known to interfere with adipogenesis. EDC usually act in mixtures and not as single compounds. The aim of this study was to investigate the effects of a simultaneous exposure of BPA, DEHP, and TBT on mesenchymal stem cell differentiation into adipocytes. Methods: Multipotent murine mesenchymal stem cells (C3H10T1/2) were exposed to EDC mixtures in high concentrations, i.e. MIX-high (10 µmol/l BPA, 100 µmol/l DEHP, 100 nmol/l TBT), and in environmentally relevant concentrations, i.e. MIX-low (10 nmol/l BPA, 100 nmol/l DEHP, 1 nmol/l TBT). The exposure was performed either for the entire culture time (0-12 days) or at distinct stages of adipogenic differentiation. At day 12 of cell culture, the amount of adipocytes, triglyceride content (TG), and adipogenic marker gene expression were analyzed. Results: MIX-high increased the development of adipocytes and the expression of adipogenic marker genes independently of the exposure window. The total TG amount was not increased. The low-concentrated EDC mixture had no obvious impact on adipogenesis. Conclusion: In EDC mixtures, the adipogenic effect of TBT and DEHP predominates single effects of BPA. Mixture effects of EDC are not deducible from single compound experiments.

STUDY QUESTION How does a maternal diabetic hyperadiponectineamia affect signal transduction and lipid metabolism in rabbit preimplantation blastocysts? SUMMARY ANSWER In a diabetic pregnancy increased levels of adiponectin led to a switch in embryonic metabolism towards a fatty acid-dependent energy metabolism, mainly affecting genes that are responsible for fatty acid uptake and turnover. WHAT IS KNOWN ALREADY Although studies in cell culture experiments have shown that adiponectin is able to regulate lipid metabolism via 5′-AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα), data on the effects of adiponectin on embryonic lipid metabolism are not available. In a diabetic pregnancy in rabbits, maternal adiponectin levels are elevated fourfold and are accompanied by an increase in intracellular lipid droplets in blastocysts, implying consequences for the embryonic hormonal and metabolic environment. STUDY DESIGN, SIZE, DURATION Rabbit blastocysts were cultured in vitro with adiponectin (1 μg/ml) and with the specific AMPK-inhibitor Compound C for 15 min, 1 h and 4 h (N ≥ 3 independent experiments: for RNA analysis, n ≥ 4 blastocysts per treatment group; for protein analysis three blastocysts pooled per sample and three samples used per experiment). Adiponectin signalling was verified in blastocysts grown in vivo from diabetic rabbits with a hyperadiponectinaemia (N ≥ 3 independent experiments, n ≥ 4 samples per treatment group, eight blastocysts pooled per sample). PARTICIPANTS/MATERIALS, SETTING, METHODS In these blastocysts, expression of molecules involved in adiponectin signalling [adaptor protein 1 (APPL1), AMPK, acetyl-CoA carboxylase (ACC), p38 mitogen-activated protein kinases (p38 MAPK)], lipid metabolism [PPARα, cluster of differentiation 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid binding protein (FABP4), carnitine palmityl transferase 1 (CPT1), hormone-senstive lipase (HSL), lipoprotein lipase (LPL)] and members of the insulin/insulin-like growth factor (IGF)-system [IGF1, IGF2, insulin receptor (InsR), IGF1 receptor (IGF1R)] were analyzed by quantitative RT-PCR and western blot. Analyses were performed in both models, i.e. adiponectin stimulated blastocysts (in vitro) and in blastocysts grown in vivo under increased adiponectin levels caused by a maternal diabetes mellitus. MAIN RESULTS AND THE ROLE OF CHANCE In both in vitro and in vivo models adiponectin increased AMPK and ACC phosphorylation, followed by an activation of the transcription factor PPARα, and CPT1, the key enzyme of β-oxidation (all P < 0.05 versus control). Moreover, mRNA levels of the fatty acid transporters CD36, FATP4 and FABP4, and HSL were upregulated by adiponectin/AMPK signalling (all P < 0.05 versus control). Under diabetic developmental conditions the amount of p38 MAPK was upregulated (P < 0.01 versus non-diabetic), which was not observed in blastocysts cultured in vitro with adiponectin, indicating that the elevated p38 MAPK was not related to adiponectin. However, a second effect of adiponectin has to be noted: its intensification of insulin sensitivity, by regulating IGF availability and InsR/IGF1R expression. LARGE SCALE DATA Not applicable. LIMITATIONS REASONS FOR CAUTION There are two main limitations for our study. First, human and rabbit embryogenesis can only be compared during blastocyst development. Therefore, the inferences from our findings are limited to the embryonic stages investigated here. Second, the increased adiponectin levels and lack of maternal insulin is only typical for a diabetes mellitus type one model. WIDER IMPLICATIONS OF THE FINDINGS This is the first mechanistic study demonstrating a direct influence of adiponectin on lipid metabolism in preimplantation embryos. The numbers of young women with a diabetes mellitus type one are increasing steadily. We have shown that preimplantation embryos are able to adapt to changes in the uterine milieu, which is mediated by the adiponectin/AMPK signalling. A tightly hormonal control during pregnancy is essential for survival and proper development. In this control process, adiponectin plays a more important role than known so far. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the German Research Council (DFG RTG ProMoAge 2155), the EU (FP7 Epihealth No. 278418, FP7-EpiHealthNet N°317146), COST Action EpiConcept FA 1201 and SALAAM BM 1308. The authors have no conflict(s) of interest to disclose.

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.

Background: Recent studies have disclosed a close relationship between maternal obesity, fetal metabolism and pre- and postnatal development. The lipid metabolism in preimplantation embryos is a possible target of metabolic programming. Methods: 31 genes of beta-oxidation and fatty acid and cholesterol uptake, synthesis and regulation were analyzed in day 3.5 blastocysts from NZO (obese) and C57Bl/6 (normal weight) mice by RT-PCR and semiquantitative PCR. Results: The most obvious difference between both strains was the lack of the RXR gamma transcript in NZO blastocysts. In adult NZO mice, RXR gamma is detectable in most tissues. In a semiquantitative analysis, a higher transcription rate of fatty acid transport protein 4 (p = 0.004) and a reduced transcript number of fatty acid synthase (p = 0.049) was found in NZO blastocysts. Cholesterol synthesis regulation was modified in NZO blastocysts, as indicated by the ratio of sterol regulatory element-binding protein (SREBP) 2 / insulin-induced gene 1 (Insig 1) (p = 0.001). Conclusion: In mouse blastocysts enzymes and signal molecules of fatty acid and cholesterol metabolism resemble those expressed postnatally. Distinct differences in transcription rates of genes between blastocysts from obese and non-obese mothers indicate that preimplantation embryo development is an early target for metabolic programming.

In the rabbit reproductive model, maternal experimentally induced insulin-dependent diabetes mellitus (expIDD) leads to accumulation of lipid droplets in blastocysts. Cholesterol metabolism is a likely candidate to explain such metabolic changes. Therefore, in the present study we analysed maternal and embryonic cholesterol concentrations and expression of related genes in vivo (diabetic model) and in vitro (embryo culture in hyperglycaemic medium). In pregnant expIDD rabbits, the serum composition of lipoprotein subfractions was changed, with a decrease in high-density lipoprotein cholesterol and an increase in very low-density lipoprotein cholesterol; in uterine fluid, total cholesterol concentrations were elevated. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), very low-density lipoprotein receptor (VLDLR), sterol regulatory element binding transcription factor 2 (SREBF2), insulin-induced gene-1 (INSIG1) and cholesterol 7 alpha-hydroxylase (CYP7A1) mRNA was decreased in the liver and low-density lipoprotein receptor (LDLR) mRNA expression was decreased in the adipose tissue of diabetic rabbits. In embryos from diabetic rabbits, the mean (+/- s.e.m.) ratio of cholesterol concentrations in trophoblasts to embryoblasts was changed from 1.27 +/- 2.34 (control) to 0.88 +/- 3.85 (expIDD). Rabbit blastocysts expressed HMGCR, LDLR, VLDLR, SREBF2 and INSIG1 but not CYP7A1, without any impairment of expression as a result of maternal diabetes. In vitro hyperglycaemia decreased embryonic HMGCR and SREBF2 transcription in rabbit blastocysts. The findings of the present study show that a diabetic pregnancy leads to distinct changes in maternal cholesterol metabolism with a minor effect on embryo cholesterol metabolism.

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.

Since the discovery of adipokines, the adipose tissue is no longer considered to be an inactive fat storage. It secretes a variety of bioactive molecules, which regulate body metabolism and energy homeostasis. One of these molecules is the adipokine adiponectin. In different tissues, adiponectin triggers metabolic effects through the adenosine monophosphate-activated protein kinase (PRKA), which is a master regulator in glucose and lipid metabolism. Recent studies point to a role for adiponectin in reproduction. Adiponectin and its receptors are present in female reproductive tract during pregnancy, and the preimplantation embryo is fully equipped with adiponectin. Here, we show that both receptor isoforms, ADIPOR1 and ADIPOR2, are expressed in 6-day-old rabbit blastocysts. To investigate the signaling pathway of adiponectin in preimplantation embryos, rabbit blastocysts were cultured in vitro and stimulated with adiponectin. Supplementation of adiponectin (1 μg/ml) enhanced PRKA alpha 1/2 (PRKAA1/2) phosphorylation and decreased expression of phosphoenolpyruvate carboxykinase 2 (PCK2), a key regulator of gluconeogenesis. Inhibition of PRKAA1/2 by Compound C (10 μM) restored PCK2 transcription. Adiponectin enhanced embryonic glucose uptake and led to a translocation of solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4), previously known as GLUT4. We conclude that adiponectin influences the glucose metabolism of rabbit blastocysts via the phosphorylation of PRKAA1/2, which in turn results in a decrease of gluconeogenesis and an increase in glycolysis. The regulatory influence of adiponectin on glucose metabolism of blastocysts may be of specific interest in pathophysiological situations, such as obesity during pregnancy.

Because of the increasing incidence of worldwide obesity, cell culture models which enable the study of adipose tissue development are of particular importance. The murine embryonic stem cell (ESC) line CGR8 differentiates into adipocytes with a differentiation efficiency of up to 15%. A critical step for the analysis of stem cell-derived adipogenesis is the reliable separation of adipocytes. Here we report on how to (i) gently separate the cells of embryoid bodies (EBs) and (ii) identify and sort adipocytes from the rest of the heterogeneous cell mixture. Up to the present, no adipocyte specific surface marker is known for fluorescence activated cell sorting (FACS). After separation we employed two independently existing FACS methods for adipocyte cell sorting. These methods are based on Nile red staining and granularity. For stem cell-derived adipocytes only the combination of both methods led to a reliable, efficient, and highly reproducible FACS analysis, as shown by the presence and absence of adipocyte specific markers in positively and negatively sorted cells. ' 2010 International Society for Advancement of Cytometry

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

The mammalian embryo is encased in a glycoproteinaceous coat, the zona pellucida (ZP) during preimplantation development. Prior to implantation, the blastocyst must undergo 'hatching' or ZP escape. In hamsters, there is a thinning of the ZP followed by a focal lysis and a complete dissolution of the ZP during blastocyst hatching. Earlier studies from our laboratory have indicated a role for cysteine proteases in the hatching phenomenon. In this study, we tested the effect of specific inhibitors of the three classes of cysteine protease on blastocyst hatching. Cystatin, an endogenous cathepsin inhibitor, blocked blastocyst hatching. Similarly, Fmoc-Tyr-Ala-diazomethane, a synthetic cathepsin inhibitor, blocked hatching. Both showed dose-dependent and temporal inhibition of hatching. However, Z-Val-Ala-Asp-fluoromethylketone, a synthetic caspase inhibitor, and calpastatin, an endogenous calpain inhibitor, had no effect on hatching. The cathepsins were localized to blastocyst cells. Exogenous addition of cathepsins L, P or B to cultured 8-cell embryos caused a complete ZP dissolution. The expression of mRNA and protein of cathepsins L and P was observed in peri-hatching blastocysts. Cathepsins L and P were detected in trophectodermal projections and in the ZP of peri-hatching blastocysts. These data provide the first evidence that blastocyst-derived cathepsins are functionally involved as zonalytic factors in the hatching of blastocysts in the golden hamster.

Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations, and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR), the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells. Undifferentiated P19 cells were exposed to 1–100 nm TCDD and characterized for AhR signaling. For studying cell differentiation, P19 cells were exposed to 10 nm TCDD at stage of embryoid body formation, and analyzed on glucose metabolism and cardiac differentiation during the next 3 wk. TCDD treatment activated the AhR-signaling cascade within 1 h, confirmed by AhR translocation, induction of cytochrome P450 1A1 expression, and activation of the xenobiotic response element. Although cell viability and transcription of the cardiac marker protein α-myosin heavy chain were affected, TCDD did not inhibit the differentiation of P19 cells to pulsating cardiomyocytes. TCDD significantly down-regulated the expression levels of the glucose transporter (GLUT) isoforms 1 and 3. After 24-h TCDD treatment, GLUT1 was no longer localized in the plasma membrane of P19 cells. The impaired GLUT expression correlated with a lower glucose uptake in 5-d-old embryoid bodies. The TCDD effects were mediated by AhR, as shown by preculture with the AhR antagonist α-naphthoflavone. Our data demonstrate that an AhR-mediated disturbance in GLUT expression and insufficient glucose uptake may be major mechanisms in TCDD embryo toxicity.

The insulin/IGF system plays a critical role in embryo growth and development. We have investigated the expression of insulin receptor (IR) and IGF-I receptor (IGF-IR) and the activation of their downstream pathways in rabbit 6-d-old blastocysts. IR was expressed in embryoblast (Em, inner cell mass) and trophoblast (Tr) cells, whereas IGF-IR was localized mainly in Em. Isoform A (IR-A) represents the main insulin isoform in blastocysts and was found in Em and Tr cells. IR-B was detectable only in Tr. IR/IGF-IR signaling pathways were analyzed after stimulation with insulin (17 nm) or IGF-I (1.3 nm) in cultured blastocysts. Insulin stimulated Erk1/2 in Em and Tr and Akt in Tr but not in Em. IGF-I activated both kinases exclusively in Em. The target genes c-fos (for MAPK kinase-1/Erk signaling) and phosphoenolpyruvate carboxykinase (PEPCK, for PI3K/Akt signaling) were also specifically regulated. Insulin down-regulated PEPCK RNA amounts in Tr by activation of the phosphatidylinositol 3-kinase/Akt pathway. Expression of c-fos by insulin and IGF-I was different with respect to time and fortitude of expression, mirroring again the specific IR and IGF-IR expression patterns in Em and Tr. Taken together, we show that IGF-I acts primarily mitogenic, an effect that is cell lineage-specifically restricted to the Em. By contrast, insulin is the growth factor of the Tr stimulating mitogenesis and down-regulating metabolic responses. As soon as blastocyst differentiation in Em and Tr has been accomplished, insulin and IGF-I signaling is different in both cell lineages, implying a different developmental impact of both growth factors.

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.

Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions. Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis.

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.

Environmental contaminants binding to transcription factors, such as the aryl hydrocarbon receptor (AhR) and the alpha and gamma peroxisome proliferator-activated receptors (PPARs), contribute to adverse effects on the reproductive system. Expressing both the AhR and PPARs, the human granulosa cell line KGN offers the opportunity to investigate the regulatory mechanisms involved in receptor crosstalk, independent of overriding hormonal control. The aim of the present study was to investigate the impact of two environmental contaminants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an AhR ligand) and di-(2-ethylhexyl) phthalate (DEHP, a PPAR ligand), on gonadotrophin sensitivity and estrogen synthesis in KGN cells. Accumulation of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in DEHP-exposed cells was measured by high-performance liquid chromatography mass spectrometry, thereby demonstrating DEHP metabolism to MEHP by KGN cells. By employing TCDD ( an AhR agonist), rosiglitazone (a PPARgamma agonist) or bezafibrate (a PPARalpha agonist), the presence of a functional AhR and PPAR cascade was confirmed in KGN cells. Cytotoxicity testing revealed no effect on KGN cell proliferation for the concentrations of TCDD and DEHP used in the current study. FSH-stimulated cells were exposed to TCDD, DEHP or a mix of both and estradiol synthesis was measured by enzyme-linked immunosorbent assay and gene expression by quantitative RT-PCR. Exposure decreased estradiol synthesis (TCDD, DEHP, mix) and reduced the mRNA expression of CYP19 aromatase (DEHP, mix) and FSHR (DEHP). DEHP induced the expression of the alpha and gamma PPARs and AhR, an effect which was inhibited by selective PPAR antagonists. Studies in the human granulosa cell line KGN show that the action of endocrine-disrupting chemicals may be due to a direct activation of AhR, for example by TCDD, and by a transactivation via PPARs, for example by DEHP, inducing subsequent transcriptional changes with a broad range of effects on granulosa cell function.

Maternal insulin and insulin-like growth factors (IGFs) promote development and growth of preimplantation embryos. They regulate differentiation-specific gene expression and balance embryonic energy and glucose metabolism. Changes in maternal metabolism affect the developmental conditions within the uterus and, among others, the availability of insulin and IGFs. We used the rabbit model to study the insulin/IGF and cAMP-responsive-element binding protein (CREB)/adiponectin networks in early embryonic development in a diabetic pregnancy. Our results demonstrate that uterine hypoinsulinaemia elevates adiponectin levels and expression of the adiponectin receptor (adipoR) 1 in the endometrium and the embryo. This effect is mediated by a paracrine IGF adjustment and cell-lineage specific regulation of the transcription factor CREB in the blastocyst. The up-regulation of adiponectin and adipoR1 expression by IGFs is part of an embryonic adaptation process, compensating for the lack of maternal insulin to maintain embryonic glucose metabolism. Embryonic CREB/ATFs act as insulin/IGF sensors. This view is supported by our data demonstrating no difference in glucose uptake in blastocysts from diabetic rabbits. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which in turn maintains glucose uptake in blastocysts grown in diabetic mothers. In conclusion, CREB-regulated embryonic adiponectin expression is a functional connecting link between maternal insulin supply and embryonic metabolic adaptation. In diabetes, this failsafe system may compensate for the loss of insulin and helps to maintain embryo development. A conversion of the embryonic metabolism from insulin- to adiponectin-dependent glucose metabolism allows the embryo to adapt to a diabetic environment. However, its long-term consequences for offspring health need to be carefully considered. Supported by DFG Na418, EU FP7-EpiHealth 278418 and the Wilhelm Roux Programme, MLU

Molecular cloning of the partial cDNA coding sequences of the four erbB receptors and the epidermal growth factor (EGF)like ligands EGF, transforming growth factor a (TGF), and heparin-binding EGF (HB-EGF) has provided the basis for a comprehensive analysis of the spatiotemporal expression pattern of the EGF receptor/ligand system during the peri-implantation period in the rabbit. Employing nonradioactive in situ hybridization and immunolocalization, we observed differential expression of erbB1‐erbB3 within the trophectoderm of the blastocyst. ErbB1 was strongly expressed in the cytotrophoblast but was downregulated upon syncytium formation. ErbB3 was a product of both the cyto- and syncytiotrophoblast. Despite the expression of erbB2 mRNA, the trophectoderm was devoid of immunoreactive ErbB2. ErbB4 gene activity was exclusively detected in the trophoblast at midpregnancy. The luminal and glandular epithelium and stroma of the nonpregnant, pseudopregnant, and pregnant rabbit uterus at Day 6 of gestation also expressed ErbB1‐ErbB3. In the peri-implantation period, gene activities of erbB1‐erbB3 were upregulated upon decidualization. At the site of implantation, uterine luminal epithelial cells apposing the preimplantation blastocyst displayed a distinct membrane immunolocalization of ErbB2, identifying the uterine epithelium as target for EGF, TGFa, and HB-EGF derived from both the embryonic trophectoderm and the uterine epithelium. In the luminal epithelium at the antimesometrial uterine site, HB-EGF gene activity was upregulated at the time of blastocyst attachment, but this upregulation was not reflected in an increase in immunoreactive HB-EGF. The detection of tyrosine phosphorylated ErbB2 in the rabbit placenta indicated the presence of a functional ErbB/EGF-like system in the pregnant rabbit uterus. This study provides strong evidence for a role of the ErbB/EGFlike system in embryo/maternal interactions during the peri-implantation period in the rabbit.

Employing postpubertal testicular tissue, we determined the cDNA coding sequence of a truncated canine relaxin-like factor (RLF) consisting of a signal peptide of 28 amino acids (aa), a B-domain of 23 aa, a truncated C-domain of 34 aa, and an A domain of 26 aa, respectively. Within the B-domain of canine RLF, the putative relaxin receptor binding motif contained a single substitution with the C-terminal arginine replaced by a serine residue, and the putative RLF receptor binding motif was truncated. Leydig cells specifically expressed RLF in the normal postpubertal and cryptochid testis as well as in testicular Leydig cell adenoma. The epididymis was an additional source of RLF in the dog. In the female reproductive tract, expression of immunoreactive RLF and relaxin were compared. Within the ovary, RLF, but not relaxin, was detected in follicular theca interna and granulosa cells and the corpus luteum. In the nonpregnant uterus, luminal and glandular epithelium coexpressed RLF and relaxin. Uteroplacental tissue at early stages of gestation revealed RLF expression in the proliferative fetal villous cytotrophoblast and in maternal uterine cells. In the mature canine placenta, the trophoblast surrounding the maternal blood vessels and the hemophagous cytotrophoblast of the paraplacental zone expressed RLF. Canine relaxin was absent in the paraplacental areas. Western analysis of placental tissue extracts revealed the presence of specific immunoreactive bands likely resembling unprocessed and enzymatically cleaved RLF. Differential expression of RLF and relaxin appears to reflect distinct autocrine and paracrine functions of RLF in canine reproductive tissues.

We have determined the cDNA sequence of preprorelaxin in the pregnant one-humped camel by employing reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Camel preprorelaxin consisted of 600 base pairs (bp) encoding a protein of 199 amino acids (aa) with a signal peptide of 25 aa (75 bp), a B domain of 28 aa (84 bp), a C domain of 121 aa (366 bp), and an A domain of 24 aa (72 bp). The N terminus of the C domain of camel prorelaxin contained the unique proline-rich repetitive sequence (-RPAP)3-(-K/RPAL-)2, and within the B domain the classical -GRELVR- receptor binding motif was found. Camel preprorelaxin showed highest homology with porcine (74.6%) and equine (65.4%) relaxin. The ovary and the uteroplacental unit were a dual source of relaxin in the pregnant dromedary. Within the ovary, weak expression of relaxin was detected in large luteal cells of the mature corpus luteum. In the ovarian follicles, immunoreactive relaxin, but not relaxin mRNA, was detected in the granulosa and theca interna cell layer. Beginning at around Day 93 of gestation and coinciding with increasing interdigitation of the fetal villus with the underlying maternal endometrium, uterine luminal epithelial cells in the uteroplacental tissue expressed relaxin. Weak expression of immunoreactive relaxin, but not relaxin mRNA, was observed in villous trophoblast cells. Pseudostratified trophoblast cells at the base of the placental villi and multinucleate giant cells did not express relaxin.

The arylhydrocarbon receptor (AhR) and the arylhydrocarbon receptor nuclear translocator (ARNT) are members of the PAS gene family mediating toxic effects of xenobiotics such as dioxin and polychlorinated biphenyls. We have analyzed the expression and cellular distribution of rabbit AhR and ARNT mRNA and protein level in the nonpregnant uterus and the pregnant and pseudopregnant uterus at Days 6 to 12 of gestation. In the preimplantation uterus at Day 6 of gestation and in the interimplantation and pseudopregnant uterus at Days 7, 8, 9, and 12 of gestation, low levels of AhR transcripts were detected in the glandular uterine epithelium. Upon attachment of the blastocyst at Day 7 of gestation, a strong expression of AhR and ARNT mRNA was observed in the luminal and glandular epithelium of the antimesometrial uterine compartment. In contrast, AhR and ARNT expression was low in the luminal epithelium of the paraplacental and the mesometrial placental fold. AhR mRNA was also detected in the trophoblast cells. During early placentation at Day 9 of gestation, expression of AhR and ARNT was first observed in the perivascular decidualized stromal cells and, at Day 12, extended to the decidualized stromal cells of the placental bed. Within the placenta, the syncytiotrophoblast expressed only low levels of AhR and ARNT mRNA and no protein. The specific expression patterns of AhR and ARNT during early gestation suggest functional roles for both transcription factors during feto-maternal interactions in the rabbit.

The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor and mediates carcinogenic, teratogenic, and toxic effects of xenobiotics such as dioxin and coplanar polychlorinated biphenyls. The AhR nuclear translocator (ARNT) is involved in AhR signal transduction. We have analyzed the expression of AhR and ARNT mRNA and AhR protein in Day 3 pc (postcoitum) rabbit morulae and Days 4 and 6 pc blastocysts using RT-PCR, nested PCR, whole mount in situ hybridization, and whole mount immunohistochemistry with subsequent confocal laser scanning analysis. AhR and ARNT transcripts were detected in all stages investigated, indicating coexpression of both transcription factors. AhR protein was localized in the cytoplasm. It was detected in Day 3 pc morulae and in blastocysts. In Day 4 pc blastocysts, only trophoblast cells but not embryoblast cells were immunopositive. However, at Day 6 pc, the embryoblast cells also expressed AhR protein and this expression was correlated with the degeneration of Rauber's trophoblast layer.

Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E--D and L--Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta.

The cat placenta is known to secrete large amounts of relaxin. We employed uteroplacental tissue at approximately Day 35 of gestation to determine the nucleic acid sequence of feline preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Feline preprorelaxin cDNA was found to consist of 540 base pairs encoding a protein of 180 amino acids (aa). We identified a signal peptide of 25 aa, a B domain of 33 aa, a C domain of 98 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the B domain contained one substitution from the classical GRELVR motif (L→F). Feline preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a specific 1-kilobase transcript present in total RNA of feline uteroplacental tissue but not of liver tissue. Nonradioactive in situ hybridization was used to localize relaxin mRNA, and immunohistochemistry was used to localize the relaxin hormone and cytokeratin, in tissues of the feto-maternal interface recovered from two queens at Day 35 of gestation. Specific hybridization signals for relaxin mRNA were exclusively detected in cells located in the lamellar placental labyrinth but were absent from other placental and non-placental uterine parts. The cells expressing relaxin mRNA also displayed immunoreactivity for cytokeratin and were, therefore, identified as trophoblast cells. Immunoreactive relaxin colocalized in those placental areas expressing relaxin mRNA. Trophoblast cells located at the villous chorioallantoic tips invading the endometrium and extravillous trophoblast cells in the junctional placental zone were devoid of relaxin.

Objective: To determine the expression and localization of the arylhydrocarbon (dioxin) receptor in human endometrium throughout the normal menstrual cycle. Design: Retrospective immunohistochemical and in situ hybridization study. Setting: Academic research unit. Patient(s): Premenopausal women (n = 86), aged 25 to 45 years, with histologically normal endometrium undergoing curettage or hysterectomy. Intervention(s): Endometrial samples were collected from days 3 to 26 of the cycle by superficial scrapings of the uterine cavity or by hysterectomy. Main Outcome Measure(s): Expression of arylhydrocarbon receptor mRNA and protein. Result(s): Arylhydrocarbon receptor was expressed in 43% of the endometria studied and was correlated with the day of the cycle. The maximum of immunopositive endometria was found around the time of ovulation. Immunostaining decreased with increasing age of the patients. The receptor protein was localized exclusively in the apical part of the cytoplasm in the epithelial cells of the endometrial glands. In women positive for arylhydrocarbon receptor, arylhydrocarbon receptor mRNA was expressed in the cytoplasm of endometrial epithelial cells. Conclusion(s): Our results describe the expression of the arylhydrocarbon receptor in human endometrium and indicate a possible involvement of this transcription factor in endometrial function in women during the reproductive phase.

Preimplantation development depends on multiple interactions between mother and embryo. The Epidermal Growth Factor Receptor (EGF-R) and its ligands are potential components of the embryo-maternal cross-talk. Employing RT-PCR, in situ hybridization, and immunohistochemistry, we investigated on mRNA and protein level the expression of EGF-R, Epidermal Growth Factor (EGF), Transforming Growth Factor alpha (TGF-α), and Heparin-binding EGF-like Growth Factor (HB-EGF) in spherical and elongating bovine blastocysts between day 13 and day 16 of gestation, and in endometrium at day 13 of gestation. EGF-R mRNA and protein were detected in trophoblast and endoderm cells of all blastocyst stages that were studied, and in luminal and some glandular epithelial cells of the endometrium at day 13. EGF protein was detected in both blastocysts and endometrial epithelium. TGF-α transcripts and protein were present in blastocysts prior to and after elongation and in uterine glandular and luminal epithelium at day 13 of gestation. HB-EGF mRNA and protein was shown in the endoderm, and the protein also was detected immunohistochemically in about 45% of the blastocysts. This presence of the EGF receptor-ligand system in the endometrium and the preimplantation embryo at the time of blastocyst elongation suggests an important role for these growth factors during bovine preimplantation development. Mol. Reprod. Dev. 51:402–412, 1998. © 1998 Wiley-Liss, Inc.

The epidermal growth factor receptor (EGF-R) is a 170-kDa transmembrane protein, of which a truncated 100-kDa form (trEGF-R) lacking the cytoplasmic and the transmembrane domains, and an oncogenic 68-kDa form (v-erb) lacking the extracellular domain have been described. The trEGF-R is secreted and not able to transmit signals into the cell. Growth factors of the EGF family have been shown in porcine uterine fluids and blastocyst. Employing differential immunohistochemistry, we found the extracellular domain, but not the cytoplasmic domain, of the EGF-R in porcine endometrium on Days 9-11 of pregnancy. Blastocysts from Days 9 to 11 post coitum (p.c.) were positive for both antibodies, indicating the presence of the full-size receptor. Western Blotting of endometrial protein resulted in a 100-kDa band, but no 170-kDa band. No evidence for a coexpression of the 170- or 68-kDa forms of the receptor was found. ELISA analysis demonstrated trEGF-R in porcine uterine flushings. We conclude that 1) the trEGF-R is the only EGF-R form present in porcine endometrium, and 2) growth factors of the EGF family in porcine uterine fluids can exert their function via the EGF-R only on blastocysts, not on the endometrium.

The food contaminant tributyltin (TBT) is an endocrine disrupting compound (EDC) promoting adipogenic differentiation in vitro and in vivo. Although prenatal TBT exposure has been shown to induce obesity, the underlying mechanisms and the role of the transcription factor PPARγ are not clarified yet. At different stages of adipogenesis, multipotent murine mesenchymal stem cells (MSC), C3H10T1/2, were exposed to TBT and analyzed for adipogenic differentiation, PPARγ promoter activation and PPARγ1, PPARγ2, Pref-1 and SOX9 expression. Depending on the exposure window, TBT promoted subsequent adipogenesis independently and dependently from PPARγ. In undifferentiated MSC, TBT exposure induced a transcriptional PPARγ-independent repression of Pref-1 and SOX9, which are both suppressors of adipogenic cell fate commitment. During hormonal induction TBT additionally enhanced adipogenic differentiation by PPARγ signaling. The impact of TBT on early cell fate development documents a novel mechanistic insight in the development of adipocytes derived from MSC and its susceptibility to EDC.

The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

In a previous study oral administration of a commercial PCB mixture (Aroclor 1,260) to female rabbits (4 mg/kg b.wt. for 14 weeks) resulted in a significant accumulation of PCBs in 6-d-old blastocysts and increased preimplantation embryo mortality (1). In the present study, the direct toxicity of PCBs on preimplantation rabbit embryos was investigated during in vitro culture. One-day-old cleavage stages and 3-d-old rabbit morulae were cultured in BSM II medium supplemented with 1.5% BSA in a reduced oxygen concentration of 5% O2. They were exposed to 50, 5, or 0.5 microgram Aroclor 1,260/mL culture medium for 24 h. PCB (50 micrograms) led to a complete degeneration of the exposed embryos. Following exposure to 5 micrograms only 16% of the morulae developed into blastocysts. The others were either arrested in the morulae stage or were degenerated. Cell proliferation of the nondegenerated embryos was approximately 20% of that of corresponding control embryos. Compared with nonexposed controls, addition of 0.5 microgram PCB/mL showed either no or only a slight impairment of development. Preliminary embryo transfer experiments showed that morulae exposed to 5 micrograms PCB with clear signs of degeneration after 24 h in vitro culture were able to implant. Aroclor 1,260 is embryotoxic in a dose-dependent manner in cultured rabbit preimplantation embryos.

The renaissance of the laboratory rabbit as a reproductive model for human health is closely related to the growing evidence of periconceptional metabolic programming and its determining effects on offspring and adult health. Advantages of rabbit reproduction are the exact timing of fertilization and pregnancy stages, high cell numbers and yield in blastocysts, relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structured similarly to the human placenta. To understand, for example, the mechanisms of periconceptional programming and its effects on metabolic health in adulthood, these advantages help to elucidate even subtle changes in metabolism and development during the pre- and peri-implantation period and during gastrulation in individual embryos. Gastrulation represents a central turning point in ontogenesis in which a limited number of cells program the development of the three germ layers and, hence, the embryo proper. Newly developed transgenic and molecular tools offer promising chances for further scientific progress to be attained with this reproductive model species.

The commercial polychlorinated biphenyl (PCB) formulation Aroclor 1260 (4 mg/kg body weight), technical grade dichlorodiphenyltrichloroethane (DDT; 3 mg) and Lindane (gamma-hexachlorocyclohexane; 0.8 mg) were administered orally, either separately or in combination, to sexually mature female rabbits three times per week for 12-15 weeks. Oviductal and uterine luminal fluid, cleavage stage embryos (day 1 post coitum), blastocysts (day 6), fetuses, exocoelic fluid and placentae (day 11) were analysed, firstly for chlorinated hydrocarbon residues, and secondly for embryonic and fetal development. The doses applied were well tolerated by the treated animals. PCB and DDT accumulated in uterine secretions (day 6) but not in oviductal luminal fluid (day 1). Both chlorinated hydrocarbons were found in preimplantation blastocysts. Residues in day 11 fetuses were 16- (DDT) or 18-fold (PCB) higher than in day 6 blastocysts. Significant amounts were also detected in placental tissue and in exocoelic fluid. A specific accumulation of the highly chlorinated biphenyl congener no. 180 was noted in fetuses, placentae and exocoelic fluid. The clear accumulation of the chlorinated hydrocarbon compounds in luminal fluid and embryonic tissue is contrasted by rather weak effects on fertility. No statistically significant differences between treated animals and controls were observed for fertilization rate and pre- and post-implantation (up to day 11 post coitum) losses. However, in females exposed to PCB, a 20% higher loss of blastocysts was noticed, as compared with controls (P > 0.05). This effect was shown on day 6 of embryonic development and may be due to the embryotoxic activities of PCB.

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.

Insulin-like growth factors (IGFs) are well-known regulators of embryonic growth and differentiation. IGF function is closely related to insulin action. IGFs are available to the preimplantation embryo through maternal blood (endocrine action), uterine secretions (paracrine action) and by the embryo itself (autocrine action). In rabbit blastocysts, embryonic IGF1 and IGF2 are specifically strong in the embryoblast (ICM). Signalling of IGFs and insulin in blastocysts follows the classical pathway with Erk1/2 and Akt kinase activation. The aim of this study was to analyse signalling of IGFs in experimental insulin dependent diabetes (exp IDD) in pregnancy, employing a diabetic rabbit model with uterine hypoinsulinemia and hyperglycaemia. Exp IDD was induced in female rabbits by alloxan treatment prior to mating. At 6 days p.c., the maternal and embryonic IGFs were quantified by RT-PCR and ELISA. In pregnant females, hepatic IGF1 expression and IGF1 serum levels were decreased while IGF1 and IGF2 were increased in endometrium. In blastocysts, IGF1 RNA and protein was approx. 7.5-fold and 2-fold higher, respectively, than in controls from normoglycemic females. In cultured control blastocysts supplemented with IGF1 or insulin in vitro for 1 or 12 h, IGF1 and insulin receptors as well as IGF1 and IGF2 were downregulated. In cultured T1D blastocysts activation of Akt and Erk1/2 was impaired with lower amounts of total Akt and Erk1/2 protein and a reduced phosphorylation capacity after IGF1 supplementation. Our data show that the IGF axis is severely altered in embryo-maternal interactions in exp IDD pregnancy. Both, the endometrium and the blastocyst produce more IGF1 and IGF2. The increased endogenous IGF1 and IGF2 expression by the blastocyst compensates for the loss of systemic insulin and IGF. However, this counterbalance does not fill the gap of the reduced insulin/IGF sensitivity, leading to a developmental delay of blastocysts in exp IDD pregnancy.

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.

Insulin and insulin-like-growth-factor 1 (IGF1) are components of the uterine secretions. As potent growth factors they influence early embryo development. The underlying molecular mechanisms are largely unknown. Here we report on the effects of insulin and IGF1 on early gastrulation in rabbit blastocysts. We have studied blastocysts grown in vivo in metabolically healthy rabbits, in rabbits with type 1 diabetes and in vitro in the presence or absence of insulin or IGF1. Embryonic disc morphology and expression of Brachyury , Wnt3a and Wnt4 were analysed by qPCR and IHC. Pre-gastrulated blastocysts (stage 0/1) cultured with insulin or IGF1 showed a significantly higher capacity to form the posterior mesoderm and primitive streak (stage 2 and 3) than blastocysts cultured without growth factors. In gastrulating blastocysts the levels of the mesoderm-specific transcription factor Brachyury and the Wnt signalling molecules Wnt3a and Wnt4 showed a stage-specific expression pattern with Brachyury transcripts increasing from stage 0/1 to 3. Wnt4 protein was found spread over the whole embryoblast. Insulin induced Wnt3a, Wnt4 and Brachyury expression in a temporal- and stage-specific pattern. Only blastocysts cultured with insulin reached the Wnt3a, Wnt4 and Brachyury expression levels of stage 2 in vivo blastocysts, indicating that insulin is required for Wnt3a, Wnt4 and Brachyury expression during gastrulation. Insulin-induced Wnt3a and Wnt4 expression preceded Brachyury . Wnt3a-induced expression could be depleted by MEK1 inhibition (PD98059). Involvement of insulin in embryonic Wnt3a expression was further shown in vivo with Wnt3a expression being notably down regulated in stage 2 blastocysts from rabbits with type 1 diabetes. Blastocysts grown in diabetic rabbits are retarded in development, a finding which supports our current results that insulin is highly likely required for early mesoderm formation in rabbit blastocysts by inducing a distinct spatiotemporal expression profile of Wnt3a, Wnt4 and Brachyury .

A well-balanced activity of the aryl hydrocarbon receptor (AhR) is necessary for normal ovarian function. As known from murine AhR knock-out (KO) models, the AhR is involved in folliculogenesis, gonadotrophin receptor expression, proliferation of granulosa cells and intraovarian estrogen signalling. Highly potent, non-physiological ligands such as the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lead to a blockade of ovulation, estrogen receptor degradation and reduction of estrogen levels. Estrogen synthesis is a typical function of granulosa cells and essential for normal cyclicity and fertility. We employed the human granulosa cell line KGN to further characterize AhR signalling and AhR function in granulosa cell physiology. Real-time PCR quantification of the target genes Cyp1a1 and Cyp1b1 and reporter gene assays after stimulation with TCDD or beta-naphthoflavone (BNF) or inhibition with alpha-naphthoflavone (ANF) or 3'-methoxy-4'-nitroflavone (3,4-MNF) of the AhR demonstrated constitutive activity and functionality of AhR pathway in KGN granulosa cells. In untreated KGN cells, AhR protein was exclusively detected in the nuclear fraction. TCDD stimulation affected the gonadotrophin receptor but not estrogen receptor β (ERβ) protein expression. Additionally, the constitutively activated AhR suppressed aromatase expression and estrogen synthesis (enzyme-linked immunoassay, ELISA) and enhanced proliferation [Bromodeoxyuridine (BrdU) ELISA] of KGN cells. Activation of the AhR by BNF did not override this inhibitory effect on estrogen synthesis or proliferation. In conclusion, the AhR pathway is constitutively activated and functional in human KGN granulosa cells. It is a potential target for endocrine disruption by exogenous ligands and subsequent dysfunction of granulosa cells.