Influence of Von Willebrand Disease (VWD) and pregnancy on the expression of angiogenic factors in the porcine female reproductive tract.

Von Willebrand Disease (VWD) is a heritable disorder caused by defects of the Von Willebrand Factor (VWF), leading to deficiencies in coagulation and also angiogenesis. Women affected by VWD frequently show bleeding concerning the reproductive tract and may present with increased rates of miscarriages. We used a porcine model representing VWD type 1 and type 3 as well as the wildtype. Samples were obtained from the reproductive tract of non-pregnant sows and sows pregnant at time of placentation. Relative expression of the genes CALR, CCN2, CXCL8, ECE1, EDN1, F8, IGFBP7, and LGALS3 was analyzed. CCN2 and FVIII proteins were additionally analyzed using immunohistochemistry. In uterus and ovary significant upregulation of CCN2 was seen in non-pregnant pigs affected by VWD. This might be caused by the higher VEGFA-levels in these pigs and could have an influence angiogenesis. During pregnancy, CCN2 expression increased in wildtype pig uteri but hardly changed in those of pregnant pigs affected by VWD, presumably because the expression level in the latter pigs already was significantly increased before pregnancy. F8 expression was significantly reduced in uterus and ovary of VWD-affected pigs. VWF is known to protect FVIII from decomposition and a lack of VWF leads to lower levels of FVIII. Our results suggest that a reduced F8 expression primarily might contribute to those reduced FVIII levels in VWD-affected pigs. Additional significant results involving the pregnant pigs were detected for CALR, EDN1, and LGALS3. These genes are promising candidates for more detailed future studies.

Estradiol-17ß Is Influenced by Age, Housing System, and Laying Performance in Genetically Divergent Laying Hens (Gallus gallus f.d.).

The estrogen estradiol-17ß is known as one of the major gonadal steroid hormones with different functions in reproduction. In this study we analyzed estradiol-17ß concentration in laying hens of four pure bred chicken laying lines at four different time intervals of the laying period (17th-19th week of age, 33rd-35th week of age, 49th-51st week of age, and 72nd week of age). The high performing white egg (WLA) and brown egg (BLA) layer lines as well as the low performing white (R11) and brown (L68) layer lines were kept in both single cages and a floor housing system. We investigated whether there were differences in estradiol -17ß concentrations between lines at different ages that could be related to selection for high egg production or phylogenetic origin of the animals, and whether there was an influence of housing conditions on estradiol-17ß. Estradiol-17ß concentrations differed between high and low performing layer lines at all time intervals studied. High performing hens showed higher estradiol-17ß concentrations compared to low performing hens. In all lines, highest estradiol-17ß concentration was measured at their 49th to their 51st week of age, whereas the peak of laying intensity was observed at their 33rd to their 35th week of age. Additionally, hens with fewer opportunities for activity housed in cages showed higher estradiol-17ß concentrations than hens kept in a floor housing system with more movement possibilities. We could show that laying performance is strongly linked with estradiol -17ß concentration. This concentration changes during laying period and is also influenced by the housing system.

Melatonin improves rate of monospermic fertilization and early embryo development in a bovine IVF system.

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10-11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode's Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10-11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

Identification and Functional Annotation of Genes Related to Bone Stability in Laying Hens Using Random Forests.

Skeletal disorders, including fractures and osteoporosis, in laying hens cause major welfare and economic problems. Although genetics have been shown to play a key role in bone integrity, little is yet known about the underlying genetic architecture of the traits. This study aimed to identify genes associated with bone breaking strength and bone mineral density of the tibiotarsus and the humerus in laying hens. Potentially informative single nucleotide polymorphisms (SNP) were identified using Random Forests classification. We then searched for genes known to be related to bone stability in close proximity to the SNPs and identified 16 potential candidates. Some of them had human orthologues. Based on our findings, we can support the assumption that multiple genes determine bone strength, with each of them having a rather small effect, as illustrated by our SNP effect estimates. Furthermore, the enrichment analysis showed that some of these candidates are involved in metabolic pathways critical for bone integrity. In conclusion, the identified candidates represent genes that may play a role in the bone integrity of chickens. Although further studies are needed to determine causality, the genes reported here are promising in terms of alleviating bone disorders in laying hens.

Keel Bone Damage in Laying Hens-Its Relation to Bone Mineral Density, Body Growth Rate and Laying Performance.

Keel bone damage is an important animal welfare problem in laying hens. Two generations of four layer lines, differing in phylogenetic background and performance level and kept in single cages or floor pens were weighed and scored for keel bone deformities (KBD) during the laying period. KBD, keel bone fractures (KBF) and the bone mineral density (BMD) of the keels were assessed post mortem. For BMD, relationships to laying performance and body growth were estimated. Caged hens showed more deformities, but fewer fractures and a lower BMD of the keel bone than floor-housed hens. White-egg layers had a lower BMD (0.140-0.165 g/cm2) and more KBD than brown-egg layers (0.179-0.184 g/cm2). KBF occurred more often in the high-performing lines than the moderate-performing ones. However, in the high-performing lines, BMD was positively related to total egg number from 18 to 29 weeks of age. The adult body weight derived from fitted growth curves (Gompertz function) had a significant effect (p < 0.001) on keels' BMD. The study contributes to the understanding of predisposing factors for keel bone damage in laying hens. It showed that the growth rate has a rather subordinate effect on keels' BMD, while the BMD itself greatly affects KBD.

Influence of Age and Phylogenetic Background on Blood Parameters Associated With Bone Metabolism in Laying Hens.

The high laying performance of today's laying hens places enormous demands on their mineral metabolism. While up-to-date data are rare, the present study aimed to describe blood parameters associated with egg laying and bone metabolism during the pre-laying period, in the course of the laying period and the daily egg laying cycle. Ten to 15 laying hens of two high-performing, phylogenetically divergent lines (BLA: brown-egg layer; WLA: white-egg layer), kept in single cages were blood sampled at 17, 25, 29, 49, and 69 weeks of age. Sampling was made at 6 a.m., 10 a.m., 2 p.m. and, with the exception of week 17, 6 p.m. Blood samples were analyzed for concentrations of total and ionized calcium, inorganic phosphate (PO4), markers of bone formation (osteocalcin) and resorption [carboxyterminal crosslinked telopeptide of type I collagen (CTX-I)], 25-hydroxycholecalciferol (25(OH)D3) and estradiol-17ß. In the pre-laying period (17 week), the estradiol-17ß level calculated for WLA was more than twice as high as the level calculated for BLA, while no significant difference could be observed in the laying period (25 to 69 weeks). BLA hens had significantly higher total calcium concentrations at 49 weeks of age as well as up to twice as high levels of osteocalcin and 25(OH)D3 than WLA at any time of the day from 25 to 69 weeks of age. While osteocalcin, CTX-I and 25(OH)D3 concentrations were significantly higher before the onset of lay, total calcium and estradiol-17ß levels significantly increased from 17 to 69 weeks of age. In contrast, PO4 values varied only slightly during the experimental period and ionized calcium was highest at 17 and 49 weeks of age and lowest around peak production (29 week). In the course of the daily egg laying cycle blood concentrations clearly reflected the stage of egg formation. Our results provide up-to-date data of bone- and egg laying-associated blood parameters of two contemporary purebred layer lines over the course of the pre- and egg-laying period and the daily egg laying cycle. Differences between brown- and white-egg layers raise questions, whether phylogenetic background determines their efficiency to cope with high calcium demands relating to egg production.

EMR + with the AWC improves endoscopic resection speed compared to ESD: a porcine ex-vivo pilot study.

INTRODUCTION: To improve resection speed and to reach higher en bloc resection rates in lesions ≥ 2 cm, a novel grasp and snare EMR technique termed "EMR+", accomplished by an additional working channel (AWC), was developed. Its use compared to endoscopic submucosal dissection (ESD) is evaluated for the first time. MATERIAL AND METHODS: We prospectively conducted a randomized pre-clinical ex-vivo pilot study in explanted porcine stomachs for the comparison of EMR + with classical ESD of mucosal-based lesions. Prior to intervention, we set flat lesions with a standardized size of 3 × 3 cm. RESULTS: The median time of procedure was significantly shorter in the EMR + group (median 10.5 min, range 4.4-24 min) than in the ESD group (median 32 min, range 14-61.6 min, p < .0001). The rate of en bloc resection was significantly lower in the EMR + group (38 % vs. 95 %) (p < .0001). Nevertheless, an improvement in the learning curve for EMR + was achieved after the first 12 procedures, with a subsequent en bloc resection rate of 100 %. CONCLUSIONS: EMR + could improve the efficiency of mucosal resection procedures. Initial experience demonstrates a higher and satisfactory en bloc resection rate after going through the learning curve of EMR+.

Correction: Jansen, S., et al. Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines. Animals 2020, 10, 850.

The authors wish to make the following corrections to this paper [...].

Bone Health or Performance? Adaptation Response of Genetically Divergent Chicken Layer Lines to a Nutritive Calcium Depletion.

In modern laying hybrids, calcium (Ca) homeostasis is immensely challenged by daily eggshell calcification. However, excessive mobilization of Ca from bones may lead to osteoporosis, which then manifests in a high incidence of poor bone quality. The aim of this study was to characterize the hens' adaptation response to an alternating dietary Ca restriction. The animal model consisted of four purebred layer lines, differing in laying performance (high vs. moderately performing lines) and phylogenetic origin (white- vs. brown-egg lines). According to the resource allocation theory, hens selected for high egg production were assumed to show a different response pattern to cope with this nutritive challenge compared to moderately performing lines. Data collected included egg number, egg quality traits, body weight and bone characteristics. The Ca depletion led to a temporary drop in egg production and shell quality and a loss of bone stability due to Ca mobilization. The white-egg lines response was more pronounced, whereas the brown-egg lines were less sensitive towards reduced Ca supply. Our study shows that the hens' responsiveness to coping with a nutritive Ca depletion is not ultimately linked to genetic selection for increased egg production but rather to phylogenetic origin.

Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines.

Impaired animal welfare due to skeletal disorders is likely one of the greatest issues currently facing the egg production industry. Reduced bone stability in laying hens is frequently attributed to long-term selection for increased egg production. The present study sought to analyse the relationship between bone stability traits and egg production. The study comprised four purebred layer lines, differing in their phylogenetic origin and performance level, providing extended insight into the phenotypic variability in bone characteristics in laying hens. Data collection included basic production parameters, bone morphometry, bone mineral density (BMD) and bone breaking strength (BBS) of the tibiotarsus and humerus. Using a multifactorial model and regression analyses, BMD proved to be of outstanding importance for bone stability. Only for the tibiotarsus were morphometric parameters and the bone weight associated with BBS. Within the chicken lines, no effect of total eggshell production on BBS or BMD could be detected, suggesting that a high egg yield itself is not necessarily a risk for poor bone health. Considering the complexity of osteoporosis, the estimated genetic parameters confirmed the importance of genetics in addressing the challenge of improving bone strength in layers.

In Vitro and In Vivo Interspecies Chimera Assay Using Early Pig Embryos.

Chimeric pigs harboring organs derived from human stem cells are promising for patient-specific regenerative therapies. Induced pluripotent stem cells (iPSCs) can contribute to all cell types of the fetus, including germline after injection into embryos. However, ethical concerns prohibit testing human iPSCs in chimera assays. Here, we evaluated porcine embryos as hosts for an interspecies chimera assay using iPSCs from either cynomolgus monkeys (cyiPSCs) or mouse (miPSCs). To establish an in vitro culture system compatible for cyiPSCs and porcine embryos, we determined blastocyst development in eight different stem cell media. The highest developmental rates of blastocysts were achieved in Knockout Dulbecco's modified Eagle's medium with 20% knockout serum replacement. We found that cyiPSCs injected into porcine embryos survived in vitro and were mostly located in the trophectoderm (TE). Instead, when miPSCs were injected into porcine embryos, the cells rapidly proliferated. The behavior of chimeras developed in vitro was recapitulated in vivo; cyiPSCs were observed in the TE, but not in the porcine epiblast. However, when miPSCs were injected into in vivo derived porcine embryos, mouse cells were found in both, the epiblast and TE. These results demonstrate that porcine embryos could be useful for evaluating the interspecies chimera-forming ability of iPSCs from different species.

Radiographic examination of keel bone damage in living laying hens of different strains kept in two housing systems.

A high prevalence of deviations and fractures of the keel bone is a widespread welfare problem in laying hens. The aim of this study was to experimentally investigate this multifactorial problem throughout the laying period and to compare the prevalence and severity in different layer lines and different housing systems. High performing white (WLA) and brown (BLA) pure bred layer lines and low performing white (R11, G11) and brown layer lines (L68) were kept in both single cages and a floor housing system. A total of 97 hens (19 or 20 from each line, respectively) were repeatedly radiographed in the 35th, 51st and 72nd week of age. Fracture prevalence increased with age (p<0.001). The proportion of deviated keel bone area increased only for caged BLA, WLA and R11 hens (p<0.05) and was significantly higher for caged WLA and R11 hens compared to floor-housed WLA and R11 hens in the 72nd week of age (p<0.05). In the 72nd week of age hens in the floor housing system showed significantly more fractures than hens kept in cages (p<0.05). Prevalence of keel bone deviations was significantly higher in the white layer line R11 but significantly lower in the white layer line G11 compared to both brown layer lines and WLA (p<0.05). Brown layers showed significantly more fractures than white layers (p<0.05) in the 51st and 72nd week of age. Within the brown layers there was a significantly lower prevalence of deviations (p<0.05) and fractures (p<0.05) in the low performing (L68) compared to the high performing line (BLA). Our results show a different development of keel bone damage in caged compared to floor-housed hens under experimental conditions. Additionally, they indicate genetic effects on keel bone damage.

Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification.

Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle.

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24 h IVM/control), cAMP30 (2 h pre-IVM (forskolin-IBMX), 30 h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

Effects of long-term dietary supplementation with conjugated linoleic acid on bovine oocyte lipid profile.

Nutritional and environmental conditions around conception and during early embryonic development may have significant effects on health and well-being in adult life. Here, a bovine heifer model was used to investigate the effects of rumen-protected fat supplementation on oocyte quality and embryo development. Holstein-Friesian heifers (n=84) received a dietary supplement consisting of rumen-protected conjugated linoleic acid (CLA) or stearic acid (SA), each on top of an isocaloric basic diet. Oocytes were collected via ultrasound-guided follicular aspiration and subjected to in vitro maturation followed by either desorption electrospray ionisation mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in vitro fertilisation and embryo culture. The type of supplement significantly affected lipid profiles of in vitro-matured oocytes. Palmitic acid and plasmalogen species were more abundant in the mass spectra of in vitro-matured oocytes after rumen-protected SA supplementation when compared with those collected from animals supplemented with CLA. Lipid concentrations in blood and follicular fluid were significantly affected by both supplements. Results show that rumen-protected fatty-acid supplementation affects oocyte lipid content and may pave the way for the establishment of a large-animal model for studies towards a better understanding of reproductive disorders associated with nutritional impairments.

Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality.

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development.

Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag(+)-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

Gold nanoparticles interfere with sperm functionality by membrane adsorption without penetration.

Abstract To examine gold nanoparticle reprotoxicity, bovine spermatozoa were challenged with ligand-free or oligonucleotide-conjugated gold nanoparticles synthesized purely without any surfactants by laser ablation. Sperm motility declined at nanoparticle mass dose of 10 µg/ml (corresponding to ∼14 000 nanoparticles per sperm cell) regardless of surface modification. Sperm morphology and viability remained unimpaired at all concentrations. Transmission electron microscopy showed an modification dependant attachment of nanoparticles to the cell membrane of spermatozoa, but provided no evidence for nanoparticle entrance into sperm cells. A molecular examination revealed a reduction of free thiol residues on the cell membrane after nanoparticle exposure, which could explain the decrease in sperm motility. Sperm fertilising ability decreased after exposure to 10 µg/ml of ligand-free nanoparticles indicating that agglomerated ligand-free nanoparticles interfere with membrane properties necessary for fertilisation. In conclusion, nanoparticles may impair key sperm functions solely by interacting with the sperm surface membrane.

Ectopic expression of human telomerase RNA component results in increased telomerase activity and elongated telomeres in bovine blastocysts.

Telomeres play an important role in aging, and are critical for the regenerative capacity of mammalian cells. The holoenzyme telomerase rebuilds telomeres and is composed of two components, the catalytic protein telomerase reverse transcriptase (TERT) and the telomerase RNA (TERC). TERC is ubiquitously expressed in somatic cells and is thought to have no regulatory effects on telomerase activity. Transgenic expression of human TERT (hTERT) in bovine somatic and embryonic cells extends telomere length and enhances telomerase activity. To obtain further insight into the regulatory capacity of the two telomerase components, we have studied the ability of hTERC and hTERT to increase telomerase activity and telomere length in bovine embryos. Expression plasmids for the human RNA component (hTERC) and/or the catalytic subunit of human telomerase (hTERT), respectively, were injected into the cytoplasm of in vitro-produced bovine zygotes. Ectopic expression of hTERC increased telomerase activity and telomere length in bovine blastocysts. Coexpression of hTERT and hTERC did not result in further telomere elongation when compared to the hTERC group. These data indicate that TERC is one of the limiting factors of telomerase activity in bovine blastocysts, and further establish bovine preimplantation embryos as a useful model to modulate telomere length with impact for basic embryology and derivation of pluripotent cells.

DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors.

The developmental capacity of oocytes from prepubertal cattle is reduced compared with their adult counterparts, and epigenetic mechanisms are thought to be involved herein. Here, we analyzed DNA methylation in three developmentally important, nonimprinted genes (SLC2A1, PRDX1, ZAR1) and two satellite sequences, i.e. 'bovine testis satellite I' (BTS) and 'Bos taurus alpha satellite I' (BTαS). In parallel, mRNA expression of the genes was determined by quantitative real-time PCR. Oocytes were retrieved from prepubertal calves and adult cows twice per week over a 3-week period by ultrasound-guided follicular aspiration after treatment with FSH and/or IGF1. Both immature and in vitro matured prepubertal and adult oocytes showed a distinct hypomethylation profile of the three genes without differences between the two types of donors. The methylation status of the BTS sequence changed according to the age and treatment while the methylation status of BTαS sequence remained largely unchanged across the different age and treatment groups. Relative transcript abundance of the selected genes was significantly different in immature and in vitro matured oocytes; only minor changes related to origin and treatment were observed. In conclusion, methylation levels of the investigated satellite sequences were high (>50%) in all groups and showed significant variation depending on the age, treatment, or in vitro maturation. To what extent this is involved in the acquisition of developmental competence of bovine oocytes needs further study.