H. Kliem

Expression and localisation of extracellular matrix degrading proteases and their inhibitors during the oestrous cycle and after induced luteolysis in the bovine corpus luteum.

The corpus luteum (CL) offers the opportunity to study high proliferative processes during its development and degradation processes during its regression. We examined the mRNA expression of matrix metalloproteases (MMP)-1, MMP-2, MMP-9, MMP-14, MMP-19, tissue inhibitor of MMP (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), uPA-receptor (uPAR), PA-inhibitors (PAI)-1, PAI-2 in follicles 20 h after GnRH application, CLs during days 1-2, 3-4, 5-7 and 8-12 of the oestrous cycle as well as after induced luteolysis. Cows in the mid-luteal phase were injected with Cloprostenol and the CLs were collected at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR determined mRNA expressions. Expression from 20 h after GnRH to day 12: MMP-1, MMP-2, MMP-14 and tPA showed a clear expression, but no regulation. TIMP-1 and uPAR mRNA increased when compared with the follicular phase. TIMP-2, MMP-9, MMP-19 and uPA increased from the follicular phase to days 8-12. PAI-1 and PAI-2 expression increased from days 1-7 and decreased to days 8-12. Induced luteolysis: MMP-1, MMP-2, MMP-9, MMP-14, MMP-19 and TIMP-1 all increased at different time points and intensities, whereas TIMP-2 was constantly decreased from 24 to 64 h. The plasminogen activator system and their inhibitors were up-regulated from 2 to 64 h, tPA was already increased after 0.5 h. Immunohistochemistry for MMP-1, MMP-2, MMP-14: an increased staining for MMP-1 and MMP-14 was seen in large luteal cells beginning 24 h after PGF2alpha application. MMP-2 showed a strong increase in staining in endothelial cells at 48 h.

A differentially methylated single CpG-site is correlated with estrogen receptor alpha transcription.

DNA methylation of the promoter region of estrogen receptor alpha (ESR1) is recognized as an epigenetic mechanism that regulates its mRNA abundance. We questioned whether tissues in male growing piglets were influenced in terms of DNA methylation by the developmentally occurring distinct plasma estradiol-17β (E2) concentrations. Additionally, we aimed at broadening the currently limited understanding of the epigenetic regulation of ESR1 in physiological settings. Three distinct genetic regions of ESR1 were analyzed using a combination of methylation-sensitive high resolution melting (MS-HRM) and pyrosequencing. Unexpectedly, major E2 concentration differences were only marginally associated with minor variations in DNA methylation and mRNA abundance. However, by analyzing two tissues showing the greatest differences in transcript abundance, we were able to find one single CpG site in the +1kb intragenic region of ESR1 strikingly differently methylated between heart vs. epididymis. Interestingly, this single CpG-site was identified as a putative binding site for the transcriptional repressor TG-interacting factor 1 (TGIF) which can recruit histone deacetylase 1 (HDAC1) leading to chromatin condensation. Indeed, chromatin immunoprecipitation confirmed a reduced histone H3 presence at the specific ESR1 location in case of higher DNA methylation. We therefore hypothesize that ESR1 expression may be manifested by a single-CpG-site based methylation difference impairing transcription factor binding.

Regulatory changes of apoptotic factors in the bovine corpus luteum after induced luteolysis.

The corpus luteum (CL) offers the opportunity to study not only proliferative, but also regressive processes. During luteolysis of the CL a sudden death of luteal and endothelial cells seems to be involved (apoptosis). The aim of this study was to examen the mRNA expression of factors known to be involved in apoptotic processes: monocyte chemoattractant protein‐1 (MCP‐1), factors of the extrinsic and intrinsic apoptotic pathways, caspase3, ‐6, ‐7 and interferone gamma (IFNγ). Luteolysis was induced by injection of 500 µg Cloprostenol during mid‐luteal phase. The CLs were collected at 0.5, 2, 4, 12, 24, 48, and 64 hr after PGF2α‐injection. Control CLs (Days 8–12) were collected at the slaugtherhouse. Real‐time RT‐PCR determined the mRNA expressions. Western blot analysis of poly(ADP‐ribose) polymerase (PARP‐1) and IFNγ as well as protein measurement of tumor necrosis factor α (TNFα) by EIA were performed. The mRNA levels of MCP‐1, IFNγ and most factors of the extrinsic pathway were significantly increased between 0.5 and 2 hr. The factors of the intrinsic pathway were mostly later up‐regulated at 24–48 hr after PGF2α. Caspase6 and 3 revealed a significant increase from 2 and 12 hr, respectively, whereas caspase7 was significantly up‐regulated after 24 hr. The protein level of TNFα increased significantly to a maximum level at 12 hr. The Western blot revealed an increasing level of an 89 kDa fragment of PARP‐1 from 12 to 24 hr, which is specific for apoptosis. We assume that the extrinsic pathway is more important for the onset of luteolysis, because of its earlier and higher increase during induced luteolysis. Mol. Reprod. Dev. 76: 220–230, 2009.

Effect of the luteinising hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degrading proteinases and their inhibitors in bovine follicles

The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1-2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF(121), VEGF(165), VEGF(189)) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).

Determination of oxytocin in milk of cows administered oxytocin

To address peoples concerns of exogenous oxytocin (OT) administration to lactating bovines, a study was undertaken to (a) establish an enzyme immunoassay (EIA) for OT determination in milk, (b) quantify OT in milk of cows administered OT, and (c) study influence of pasteurization on OT stability in milk. A sensitive EIA validated according to the criteria of European Union-Decision 2002/657/EC was developed for OT in skim milk in an analytical range of 10-250pgmL(-1) with a decision limit (CCalpha) of 30pgmL(-1) and detection capability (CCbeta) of 41.5pgmL(-1). Milk samples collected from cows (n=38) administered either 25 or 50IU OT prior to milking were investigated for the presence of OT. There was no significant difference among both groups with the mean concentrations of OT being 15.8 and 14.9pgmL(-1) for cows subjected to 25 and 50IU OT administration, respectively. The OT levels in skim milk of control cows (n=30; untreated) were basal (around 10pgmL(-1)). All the analyzed milk samples were below the CCalpha value of 30pgmL(-1). Pasteurization of OT spiked milk samples at different temperature and sample holding conditions reduced the immunological activity of OT to 43% at 110 degrees C. However, no further decline occurred in the immunological activity with increased pasteurization temperature and time. It was concluded that the milk OT concentrations after OT administrations were minimal and below the assay decision limit. However, OT was quite stable to pasteurization in OT spiked milk.

Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue

Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

Expression and Localization of Gap Junctional Connexins 26 and 43 in Bovine Periovulatory Follicles and in Corpus Luteum During Different Functional Stages of Oestrous Cycle and Pregnancy

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1-3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8-12 were injected with PGF2alpha analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.

HOXA10 mRNA expression and promoter DNA methylation in female pig offspring after in utero estradiol-17β exposure

Early exposure to environmental estrogens may exert lasting impacts on health. In rodents, homeobox A10 (HOXA10) was demonstrated to be a target of early endocrine disruption, as indicated by persistent changes in uterine HOXA10 expression and promoter DNA methylation in the offspring. This study aimed at analyzing long-term effects of estradiol-17β on porcine uterine HOXA10. Therefore, offspring were exposed in utero to low (0.05 and 10μg/kg body weight/day) and high (1000μg/kg body weight/day) doses, respectively. We, furthermore, investigated whether promoter DNA methylation was generally involved in regulating HOXA10 expression. Unexpectedly, the maternal estrogen exposure did not distinctly impact HOXA10 expression and promoter DNA methylation in either pre- or postpubertal offspring. Although differential HOXA10 expression was observed in endometrial tissue during the estrous cycle and the pre-implantation period, no concurrent substantial changes occurred regarding promoter DNA methylation. However, by comparing several tissues displaying larger differences in transcriptional abundance, HOXA10 expression correlated with promoter DNA methylation in prepubertal, but not postpubertal, gilts. Thus, promoter DNA methylation could affect gene expression in pigs, depending on their stage of development. Clearly, early estrogen exposure exerted other effects in pigs as known from studies in rodents. This may be due to endocrine differences as well as to species-specific peculiarities of tissue sensitivity to estradiol-17β during critical windows of development.

Maternal low-dose estradiol-17β exposure during pregnancy impairs postnatal progeny weight development and body composition

Endocrine disrupting chemicals with estrogenic activity play an important role as obesogens. However, studies investigating the most potent natural estrogen, estradiol-17β (E2), at low dose are lacking. We examined endocrine and physiological parameters in gilts receiving distinct concentrations of E2 during pregnancy. We then investigated whether adverse effects prevail in progeny due to a potential endocrine disruption. E2 was orally applied to gilts during the entire period of pregnancy. The concentrations represented a daily consumption at the recommended ADI level (0.05 μg/kg body weight/day), at the NOEL (10 μg/kg body weight/day) and at a high dosage (1000 μg/kg body weight/day). Plasma hormone concentrations were determined using enzyme immuno assays. Offspring body fat was assessed by dual-energy X-ray absorptiometry scanning. In treated gilts receiving 1000μg E2/kg body weight/day we found significantly elevated plasma E2 levels during pregnancy, paralleled by an increased weight gain. While offspring showed similar weight at birth, piglets exhibited a significant reduction in weight at weaning even though their mothers had only received 0.05 μg E2/kg body weight/day. At 8 weeks of age, specifically males showed a significant increase in overall body fat percentage. In conclusion, prenatal exposure to low doses of E2 affected pig offspring development in terms of body weight and composition. In line with findings from other obesogens, our data suggest a programming effect during pregnancy for E2 causative for the depicted phenotypes. Therefore, E2 exposure may imply a possible contribution to childhood obesity.

Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens.

Bovine mastitis, the inflammation of the udder, is a major problem for the dairy industry and for the welfare of the animals. To better understand this disease, and to implement two special techniques for studying mammary gland immunity in vitro, we measured the innate immune response of primary bovine mammary epithelial cells (pbMEC) from six Brown Swiss cows after stimulation with the heat-inactivated mastitis pathogens, Escherichia coli 1303 and Staphylococcus aureus 1027. The cells were extracted and cultivated from milk instead of udder tissue, which is usually done. The advantages of this technique are non-invasiveness and less contamination by fibroblasts. For the first time, pbMEC gene expression (GE) was measured with a microfluidic high-throughput real-time reverse transcription-quantitative PCR platform, the BioMark HD™ system from Fluidigm. In addition to the physiological analysis, the precision and suitability of this method was evaluated in a large data set. The mean coefficient of variance (± s.e.) between repeated chips was 4.3 ± 0.4% for highly expressed and 3.3 ± 0.4% for lowly expressed genes. Quantitative PCR (qPCR) replicate deviations were smaller than the cell culture replicate deviations, indicating that biological and cell culture differences could be distinguished from the background noise. Twenty-two genes (complement system, chemokines, inflammatory cytokines, antimicrobial peptides, acute phase response and toll-like receptor signalling) were differentially expressed (P < 0.05) with E. coli. The most upregulated gene was the acute phase protein serum amyloid A3 with 618-time fold. S. aureus slightly induced CCL5, IL10, TLR4 and S100A12 expression and failed to elicit a distinct overall innate immune response. We showed that, with this milk-derived pbMEC culture and the high-throughput qPCR technique, it is possible to obtain similar results in pbMEC expression as with conventional PCR and with satisfactory precision so that it can be applied in future GE studies in pbMEC.