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Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge., Journal of reproduction and development, 68 (1), ISSN:0916-8818, ISSN:1348-4400

Progesterone (P4) plays a pivotal role in maintenance of pregnancy in many mammalian species. Species-specific P4 metabolites have been shown to function as primary acting progestogen and the receptor binding capacity varies between species. The European roe deer (Capreolus capreolus) displays a 4-5 month period of embryonic diapause, which decouples fertilization from implantation. The majority of roe deer have two corpora lutea that secrete P4. No changes in P4 concentrations have been observed during pre-implantation embryo development. As 5α-DHP is known to play a major role during pregnancy in elephants and horses, we hypothesized that 5α-DHP functions as additional progestogen facilitating embryo reactivation. The profile of 11 progestogens was quantified in roe deer plasma over the course of diapause and resumption of embryo development including P4, 3α- and 3β-DHP, 20α- and 20β-DHP, 5α- and 5β-DHP, 3α,5α- and 3α,5β-THP, as well as 3β,5α- and 3β,5β-THP. While P4 was most abundant during diapause and resumption of development, 20α-DHP was the most abundant P4 metabolite. This is different than in pregnant elephants, where 5α-DHP was most abundant, and the luteal phase in cattle, where 3α,5α-THP was most abundant. With the exception of a weak correlation of 3β,5α-THP, none of the progestogens significantly correlated with embryonic development in the roe deer. Thus, plasma 5α-DHP does not seem to play a role in embryo reactivation. We propose that progestogens might contribute to priming the endometrium for supporting embryo development and preparation for implantation.

The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. ‘Cellular detoxification’ in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and ‘cortical cytoskeleton’, ‘regulation of substrate adhesion-dependent cell spreading’ and ‘melanosome’ were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.

The phenomenon of embryonic diapause has been reported to possibly affect ovarian follicular development. The aim of the current study was to identify the impact of the embryonic diapause period on follicle number and oocyte quality in the European roe deer (Capreolus capreolus). In this species, the embryo undergoes five months of developmental delay. We have determined the antral follicle count (AFC), oocyte morphological quality, oocyte meiotic resumption capacity and the transcriptome profile in immature (GV) and in vitro matured (MII) oocytes obtained during embryonic diapause and after embryo reactivation. A drop in AFC, number of oocytes suitable for in vitro maturation and oocytes reaching the MII stage was observed after embryonic reactivation. A large impact of the diapause period was evident when MII oocytes were compared against GV oocytes using an RNA-seq approach. A total of 1028 genes significantly differed between MII and GV oocytes during diapause and 1795 genes after reactivation. These genes included PDE3A and SOD2, known for being involved in oocyte meiotic arrest and oxidative stress. In addition, tumour-related genes included NDRG3, NCSTN and UGDH, which have been associated with cell proliferation, migration and metabolism, were found. Comparing diapause versus reactivation, differential transcript abundance in both GV and MII oocytes was observed (44 and 32 differentially expressed genes, respectively). Our results suggest that the embryonic diapause and reactivation periods modulate follicular dynamics, influencing the oocyte towards lower quality after reactivation. This is may be related to increased apoptosis, abnormal mitochondria function/structure and metabolic dysfunction in the maturing oocyte. © 2020 Third International Symposium on Embryonic Diapause.

Estrogen metabolism results in the formation of inactive estrogen sulphates and glucuronides. Despite the lack of receptor binding, circulating conjugated estrogens might serve as a reservoir for the active form through the involvement of specific cleaving enzymes. In order to elucidate the potential role that estrogen conjugates play in the regulation of the estrous cycle, we determined the concentration of progesterone, estrogen and estrogen conjugates in serum and endometrial homogenates of cycling gilts. In addition, we determined the mRNA expression changes of enzymes (UDP glucuronosyltransferase (UGT), β-glucuronidase (GUSB), sulphotransferases (SULT) and steroid sulphatase (STS)) and transporters (multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATPs)) involved in the estrogen metabolism in the endometrium across the estrous cycle. GUSB displayed highest expression at estrous (day 0), decreasing expression during metestrus (day 3 and 6), minimal expression on day 10 and 12, and increasing expression towards proestrus (day 18), suggesting either a stimulation by estrogens or a negative impact of progesterone. The mRNA expression of the influx-transporter OATP1A2 significantly increased from day 0 to 6 and decreased again by day 10, while the efflux-transporters (MRP1, MRP2, and MDR1) displayed minimal expression at day 3 and 6. The mRNA expression of the UDP-glucuronsyltransferases followed a similar pattern, with minimal expression found at day 6. The analyses of the concentration of local and circulating steroid hormones points towards an interaction of the analyzed transporters and enzymes with steroid hormones, thereby possibly regulating the reservoir of active steroids contributing to the endometrial function.

During early pregnancy, the secretome of both oviduct and uterus serves as exchange medium for signaling factors between embryo and mother and provides the embryo with nutrients. The preimplantation embryo can utilize the fatty acids (FA) therein via direct incorporation into cell membrane lipid bilayers and for energy production via β-oxidation. The FA concentration and composition of the oviduct (OF) and uterine fluid (UF) might be regulated by ovarian hormones to meet the changing needs of the growing embryo. In our study, we analyzed the FA profile of blood plasma (BP) and reproductive fluid samples obtained post mortem from slaughtered mares by gas chromatography mass spectrometry. Cycle stage was determined by visual evaluation of the ovary and measurement of plasma progesterone. No major effect of cycle stage on the FA profile was observed. However, the composition of FA was different between BP and both OF and UF. While linoleic, stearic, oleic and palmitic acid were the four most prevalent FA in both BP and reproductive fluids, the latter contained higher concentrations of arachidonic, eicosapentaenoic and dihomo-γ-linolenic acid. The finding suggests selective endometrial transport mechanisms from plasma into the reproductive fluids or increased endometrial synthesis of selected FA.

Porcine conceptuses synthesize estrogens between Day 11 and 12 as signal for maternal recognition of pregnancy. A preimplantational estrogen exposure to pregnant gilts has been associated with embryonic losses and changes in endometrial mRNA expression. MicroRNAs (miRNAs) play a key role in the mRNA regulation by modulating the expression. Effects of estrogens on endometrial miRNAs have not been investigated in this context so far. Thus, we studied the endometrial expression profile of miRNAs in the pig at gestational Day 10 after daily estradiol-17β (E2) application starting at fertilization using either 0, 0.05 (ADI-acceptable daily intake), 10 (NOEL-no-observed-effect level) and 1,000 (high dose) µg E2/kg body weight/day, respectively. In endometrial homogenates, E2 (p < 0.001) and total estrogen concentrations (p < 0.001) were significantly increased, namely 28- and 160-fold, respectively, in the high dose group as compared to the control. Additionally, total estrogens were sixfold elevated in the NOEL group. Interestingly, high-throughput sequencing of small non-coding RNA libraries did not indicate any differentially expressed miRNAs between the treatment groups and the control group. The expression of 12 potential E2 target miRNAs investigated by RT-qPCR were equally unaffected. Thus, preimplantational E2 exposure resulted in significantly higher endometrial estrogen concentrations, but did not perturb the expression profile of endometrial miRNAs.

In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABAA-r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20β-dihydroprogesterone (20β-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3β-dihydroprogesterone (3β-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5β-reduced progestogens (5β-dihydroprogesterone (5β-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20β-DHP. Further studies will need to focus particularly on the period around luteolysis., Theriogenology, 142, ISSN:1879-3231, ISSN:0093-691x

This study aimed to identify interactions between state of lactation (dry or early lactating) and immune responder group (low, medium, or high) for energy metabolism traits as well as metabolic and immunological traits in dairy cows. In early lactation, when the energy priority of cows shifts toward the mammary gland, the energy available to be partitioned toward the immune system may differ among individuals. The equilibrium between energy supply from feed, digestion, and body reserve mobilization and energy expenditure with milk, immune system, methane, and heat production is delicate in this stage. Seventeen Holstein cows entering their second to fifth lactation were kept under comparable feeding, housing, and management conditions and were studied from 14 ± 6 d before calving to 11 ± 3 d after calving. Feed intake, milk yield, body condition, blood metabolites, and cortisol as well as gaseous exchange in respiration chambers were measured. The latter was used to quantify methane emission and to calculate resting metabolic rate and heat production. Subsets of blood leukocytes and peripheral blood mononuclear cells (PBMC) were monitored. Activation and proliferation of the PBMC in response to the mitogen phytohemagglutinin ante- and postpartum were assessed using the oxygen consumption rate (24-h cell culture assay) and the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (72-h cell culture assay). Cows were classified based on the in vitro proliferative response of the PBMC measured postpartum in low (n = 6), medium (n = 5), and high (n = 6) responders. We found no interaction of state of lactation with responder group for feed intake, milk yield, efficiency, metabolic traits, and immune cell activation ante- and postpartum. However, after calving, low-responder cows produced less methane per unit of body weight and per unit of energy-corrected milk compared with the other cows. This might be indicative of a low rumen fermentation intensity. Low responders might therefore suffer from a lower availability of digestible energy in early lactation and not be able to sustain the shift from immune cell activation to proliferation. If so, the selection of environmentally friendly low-methane emitters could promote phenotypes with a compromised immune response in the critical early lactation.

Numerous intrauterine changes take place across species during embryo development. Following fertilization in July/August, the European roe deer (Capreolus capreolus) embryo undergoes diapause until embryonic elongation in December/January. Embryonic elongation prior to implantation is a common feature among ungulates. Unlike many other ruminants, the roe deer embryo does not secrete interferon-tau (IFNτ). This provides the unique opportunity to unravel IFNτ-independent signaling pathways associated with maternal recognition of pregnancy (MRP). This study aimed at identifying the cell-type–specific endometrial gene expression changes associated with the MRP at the time of embryo elongation that are independent of IFNτ in roe deer. The messenger RNA (mRNA) expression of genes known to be involved in embryo–maternal communication in cattle, pig, sheep, and mice was analyzed in laser capture microdissected (LMD) endometrial luminal, glandular epithelial, as well as stromal cells. The mRNA transcript abundances of the estrogen (ESR1), progesterone receptor (PGR), and IFNτ-stimulated genes were lower in the luminal epithelium in the presence of an elongated embryo compared to diapause. Retinol Binding Protein-4 (RBP4), a key factor involved in placentation, was more abundant in the luminal epithelium in the presence of an elongated embryo. The progesterone receptor localization was visualized by immunohistochemistry, showing an absence in the luminal epithelium and an overall lower abundance with time and thus prolonged progesterone exposure. Our data show a developmental stage-specific mRNA expression pattern in the luminal epithelium, indicating that these cells sense the presence of an elongated embryo in an IFNτ-independent manner., Biology of Reproduction, 101 (5), ISSN:0006-3363, ISSN:1529-7268

Maternal exposure to estrogens can induce long-term adverse effects in the offspring. The epigenetic programming may start as early as the period of preimplantation development. We analyzed the effects of gestational estradiol-17β (E2) exposure with two distinct low doses, corresponding to the acceptable daily intake “ADI” and close to the no-observed-effect level “NOEL”, and a high dose (0.05, 10, and 1000 µg E2/kg body weight daily, respectively). The E2 doses were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls leading to a significant increase in E2 in plasma, bile and selected somatic tissues including the endometrium in the high-dose group. Conjugated and unconjugated E2 metabolites were as well elevated in the NOEL group. Although RNA-sequencing revealed a dose-dependent effect of 14, 17, and 27 differentially expressed genes (DEG) in the endometrium, single embryos were much more affected with 982 DEG in female blastocysts of the high-dose group, while none were present in the corresponding male embryos. Moreover, the NOEL treatment caused 62 and 3 DEG in female andmale embryos, respectively. Thus, we detected a perturbed sex-specific gene expression profile leading to a leveling of the transcriptome profiles of female and male embryos. The preimplantation period therefore demonstrates a vulnerable time window for estrogen exposure, potentially constituting the cause for lasting consequences. The molecular fingerprint of low-dose estrogen exposure on developing embryos warrants a careful revisit of effect level thresholds.Summary SentenceMaternal oral low-dose estrogen exposure during the preimplantation period specifically targeted female embryos by inducing a male-like gene expression profile.

Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage and genome activation. However, the composition and regulation of this critical environment remain rather poorly defined. This study uses anin vitropreparation of the bovine oviduct epithelium to investigate the formation and composition ofin vitro-derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct-specific glycoprotein 1 inivDOF and show that the amino acid and carbohydrate content resembles that of previously reportedin vivodata. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes and the corresponding flux of amino acids withinivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully thein vivoenvironment and impacts onivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on thein vitrooviduct preparation was evaluated. These experiments help clarify the dynamic function of the oviductin vitroand suggest a number of future research avenues, such as investigating epithelial–fibroblast interactions, probing the molecular aetiologies of subfertility and optimising embryo culture media.

Data from various studies indicate that the ovarian function in dairy cows can be compromised during intramammary infections. Therefore, in this study, we investigated if an experimentally induced mastitis has an effect on corpus luteum (CL) function in 14 lactating cows. On d 9 of the estrous cycle (d 1=ovulation), cows received a single dose of 200 μg of Escherichia coli lipopolysaccharide (LPS; dissolved in 10 mL of NaCL; n=8) or 10 mL of saline (control; n=6) into one quarter of the mammary gland. Measurements included plasma cortisol, haptoglobin, and progesterone (P4) concentrations, as well as luteal size (LTA) and relative luteal blood flow (rLBF). Sampling was performed on d 1, 4, and 8. On d 9, the main examination day, sampling was performed immediately before (0 h), every 1h (or at 3-h intervals for LTA and rLBF) until 9 h, as well as 12 and 24 h after treatment. Thereafter, measurements were taken on d 12, 15, 18, and then every 2 d until ovulation. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment. Quantitative real-time PCR was applied to assess mRNA expression of steroidogenic factors (STAR, HSD3B), caspase 3, toll-like receptors (TLR2, -4), tumor necrosis factor α (TNFA), and prostaglandin-related factors (PGES, PGFS, PTGFR). Intramammary LPS infusion caused considerable inflammatory responses in the treated udder quarters. No decrease in plasma P4 concentrations was noted after LPS-challenge, and P4 levels did not differ between LPS-treated and control cows. Furthermore, LTA and rLBF values were not decreased after LPS challenge compared with the values obtained immediately before treatment. However, LPS infusion increased plasma levels of cortisol and haptoglobin compared with the control group. In the CL, mRNA abundance of TLR2 and TNFA was increased in cows after LPS-challenge (but not in control cows), whereas TLR4, steroidogenic, and prostaglandin-related factors remained similar to the mRNA abundance before treatment. In conclusion, intramammary LPS challenge induces systemic inflammatory reactions which alter the luteal mRNA abundance of TLR2 and TNFA but does not induce lysis of the CL.

The objective was to characterize effects of Escherichia coli LPS (given i.v.) on corpus luteum (CL) and embryonic viability in early pregnant cattle. Eight non-lactating German Holstein cows were given 0.5 µg/kg LPS on 35 ± 3 day (mean ± s.e.m.) of pregnancy, whereas seven heifers, 41 ± 6 day pregnant, were given 10 mL saline (control group). Transrectal B-mode examinations of the CL were done at -1, 3, 6, 12, 24, 48, 72 and 96 h relative to treatment. Blood samples were collected at -1, 0.5, 1, 2, 3, 4, 6, 9, 12, 24, 48, 72 and 96 h. At 12 and 48 h, the CL was biopsied. None of the cows still in the experiment 10 day after LPS (n = 7) had embryonic loss. In LPS-treated cows, luteal area decreased (from 4.1 to 3.1 cm2; P ≤ 0.05) within 6 h and until 48 h. Luteal blood flow decreased by 39% (P ≤ 0.05) within the first 6 h after LPS, but returned to pre-treatment values by 48 h. Plasma P4 decreased by 62% (P ≤ 0.05), reached a nadir (2.7 ± 0.6 ng/mL) at 12 h after LPS and was not restored to pre-treatment (P ≤ 0.05). In luteal tissue, mRNAs for STAR and for FGF1 were lower (P ≤ 0.05) in LPS than in saline-treated cattle at 12 h, with no difference between groups at 48 h. Levels of mRNAs for CASP3 and FGF2 were not different between groups (P > 0.05) at 12 or 48 h after treatment. In conclusion, LPS transiently suppressed CL function, but did not induce embryonic mortality.

This in vitro study examined the ability of important immune modulators [β-hydroxybutyrate (BHB), cortisol, prolactin, isoproterenol and insulin] to influence the responsiveness of peripheral blood mononuclear cells (PBMC) from multiparous dairy cows 29 ± 2 days before and 14 ± 3 days after calving. The activation and proliferation of PBMC in response to the mitogen phytohemagglutinin was estimated by the oxygen consumption rate after 24 hr and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) method after 72 hr respectively. In early lactation, the presence of 2 compared to 0.5 mmol/L BHB reduced PBMC activation (p

Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal Interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational, and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 hours. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific Enzyme-linked-immunosorbent Assay (ELISA). The biological activity was assessed by quantifying the response of Interferon stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold, and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

Background: Along with trophoblast elongation (Days 10 to 12), estradiol is secreted in increasing amounts for recognition of pregnancy. Endometrial secretions driven by ovarian progesterone and conceptus signals are essential for conceptus growth and development. Results of transcriptome analyses of whole endometrial tissue samples in the pig indicated the need for cell type-specific endometrial gene expression analysis for a better understanding of transcriptome changes associated with establishment of pregnancy. Results: The most distinct transcriptome profile and the majority of differentially expressed genes (DEGs) were identified in luminal epithelium (LE). Many DEGs were found only in the cell type-specific analysis. The functional classification of DEGs identified in specific endometrial cell types revealed various distinct functions and pathways. Genes related to immune activation, estrogen signaling pathway, embryo development, and cell proliferation were upregulated in LE of pregnant gilts. Genes involved in sterol biosynthetic and metabolic processes and extracellular matrix were upregulated in stroma. Genes associated with cell communication such as via exosomes and vesicles were found as differential in LE, glandular epithelium (GE), and stroma (S). Conclusions: This study revealed that conceptus signals induce different transcriptomic regulations in the endometrial compartments/cell types related to their specific function during recognition and establishment of pregnancy., BMC Genomics, 19

Endocrine disrupting chemicals (EDC) interfere with the natural hormone balance and may induce epigenetic changes through exposure during sensitive periods of development. In this study, the effects of short-term estradiol-17β (E2) exposure on various tissues of pregnant sows (F0) and on day 10 blastocysts (F1) were assessed. Intergenerational effects were investigated in the liver of 1-year old female offspring (F1). During gestation, sows were orally exposed to two low doses and a high dose of E2 (0.05, 10, and 1000 µg/kg body weight/day). In F0, perturbed tissue specific mRNA expression of cell cycle regulation and tumour suppressor genes was found at low and high dose exposure, being most pronounced in the endometrium and corpus luteum. The liver showed the most significant DNA hypomethylation in three target genes; CDKN2D, PSAT1, and RASSF1. For CDKN2D and PSAT1, differential methylation in blastocysts was similar as observed in the F0 liver. Whereas blastocysts showed hypomethylation, the liver of 1-year old offspring showed subtle, but significant hypermethylation. We show that the level of effect of estrogenic EDC, with the periconceptual period as a sensitive time window, is at much lower concentration than currently presumed and propose epigenetics as a sensitive novel risk assessment parameter.

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, has detrimental effects on the structure and function of bovine corpus luteum (CL)in vivo. The objective was to investigate whether these effects were mediated directly by LPS orviaLPS-induced release of PGF2α. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and isolated perfused for 240 min. After 60 min of equilibration, LPS (0.5 μg/ml) was added to the medium of five ovaries, whereas an additional six ovaries were not treated with LPS (control). After 210 min of perfusion, all ovaries were treated with 500 iu of hCG. In the effluent perfusate, concentrations of progesterone (P4) and PGF2αwere measured every 10 and 30 min, respectively. Punch biopsies of the CL were collected every 60 min and used for RT-qPCR to evaluate mRNA expression of receptors for LPS (TLR2,-4) and LH (LHCGR); the cytokineTNFA; steroidogenic (STAR,HSD3B), angiogenic (VEGFA121,FGF2), and vasoactive (EDN1) factors; and factors of prostaglandin synthesis (PGES,PGFS,PTGFR) and apoptosis (CASP3,-8,-9). Treatment with LPS abolished the hCG-induced increase in P4(P≤0.05); however, there was a tendency (P=0.10) for increased release of PGF2αat 70 min after LPS challenge. Furthermore, mRNA abundance ofTLR2,TNFA,CASP3,CASP8,PGES,PGFS, andVEGFA121increased (P≤0.05) after LPS treatment, whereas all other factors remained unchanged (P>0.05). In conclusion, reduced P4responsiveness to hCG in LPS-treated ovariesin vitrowas not due to reduced steroidogenesis, but was attributed to enhanced apoptosis. However, an impact of luteal PGF2αcould not be excluded.

The pre-implantation period is prone to embryonic losses in bovine. Embryo–maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acidwas reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome.Summary SentenceThe pre-implantation embryo elongation in bovine is affected by dietary omega 3/6 fatty acids likely due to an effect on the ovary rather than a change of the uterine secretome.

Endometrial cell co-culture (ECC) with single embryo may reflect endometrium responses in vivo. Bovine Day-6 in vitro-produced morulae were cultured until Day-8 in modified synthetic oviductal fluid (mSOF), or on the epithelial side of ECC. Expression of epithelial- and stromal-cell transcripts was analyzed by RT-PCR in ECC with one male (ME) or female embryo (FE). Concentrations of ARTEMIN (ARTN) and total protein were determined in epithelial cell-conditioned medium. ECCs yielded embryos with more cells in the inner cell mass than embryos cultured in mSOF. Embryos altered transcript expression only in epithelial cells, not in stromal ones. Thus, ME induced larger reductions than FE and controls (i.e., no embryos cultured) in hexose transporter solute carrier family 2 member 1 (SLC2A1) and member 5 (SLC2A5), connective tissue growth factor (CTGF), artemin (ARTN), and interferon alpha and beta receptors subunit IFNAR1 and IFNAR2. FE reduced SLC2A1 and SLC2A5, and increased ARTN expression with respect to controls. ME tended to reduce total protein concentration (P < 0.082) in ECC-conditioned medium, while ARTN protein and gene expressions strongly correlated (R > 0.90; P < 0.05) in the group of ME or FE, but not in controls (without embryo). Isolated male and female embryos may differentially release signaling factors that induce sexually dimorphic responses in endometrial cells.

This study aimed to investigate the in vitro contractility of the myometrium and its relationship to the blood concentrations of estradiol-17β (E2β), progesterone (P4), 15-keto-13,14-dihydro-PGF2α (PGFM) and ionised calcium (Ca2+) prior to tissue harvest in 12 healthy Holstein-Friesian cows in late pregnancy. Three circular (CM) and 3 longitudinal myometrial (LM) strips were dissected during a caesarean section and mounted in an organ bath containing modified Krebs solution (KS). The spontaneous contractility was recorded during five 30-min time periods (T1 to T5), after which the strips were exposed to increasing concentrations of oxytocin (OT; 10-10-10-7M), a natural PGF2α-analogue (PGF; 10-7-10-4M) and KS (Cont) for four 30-min time periods (T6 to T9). The variables area under the curve (AUC), mean (MA) and maximal amplitude (maxA) were calculated for each T. The blood P4, E2s, Ca2+ and PGFM values averaged 4.0±1.7ng/mL, 482.3±63.7 pg/mL, 0.8±0.3 mmol/L and 125.3±63.7pg/mL. The LM strips had greater AUC, MA, and maxA than CM, and OT caused greater AUC and MA in both muscle layers than PGF or control treatment (OT>PGF>Cont). Estradiol-17β correlated with AUC and MA of LM at T1 to T5 (r=0.69; P≤0.05). In conclusion, LM and CM strips have different contractile performance but show enhanced activity when stimulated with OT and less activity after PGF stimulation if compared with Cont. Blood concentrations of E2β may be useful as an indicator of uterine contractile performance in late pregnant cattle.

Interferon-tau (IFNτ) is the conceptus derived specific early pregnancy signal in bovidae. Locally, IFNτ induces an IFNτ specific gene expression (ISG) in endometrial cells and by this it averts luteolysis of the corpus luteum (CL) by suppressing prostaglandin production. Moreover, it was shown that IFNτ also induces ISG in the liver in pregnant sheep and in liver biopsies from Holstein Friesian heifers on Day 18 of pregnancy. The objective of the present study was to confirm increased hepatic ISG in vivo on Day 18 of pregnancy and to prove if hepatocytes and not non-parenchymal cells react to IFNτ by using immunohistochemistry and primary bovine hepatocytes stimulated in vitro with recombinant bovine IFNτ. For the animal experiment, Angus heifers (n = 12) were cycle synchronized and the Day of ovulation (Day 0) was defined by ovarian ultrasonography and verified by progesterone

In the equine industry, horse breeders aim to produce one foal per mare per year. Thus, mares are bred while nursing. In high-yielding dairy cattle, concurrent lactation and conception may affect embryo quality, but effects on uterine fluid (UF) quality are unknown. No data are available in horses. The aim of our study was to analyse the effect of nursing on protein, metabolite and fatty acid (FA) compositions of UF at ~7.5 days post-ovulation. Anglo-Arab mares (multiparous (2.7 ± 0.9 foals), 10.8 ± 2.5 years old) that were either nursing (N; 105 ± 12 days of lactation) or barren (B) were inseminated with the semen of the same stallion at induction of ovulation with human chorionic gonadotrophin. Ovulation was confirmed by ultrasound within 48 h. At 7.4 ± 0.7 days post-ovulation, UF was collected with a human tampon that was left for 10 min in the uterus before embryo collection. Only mares for which no embryo was collected were selected (n = 5 in both groups). Trypsin digestion followed by liquid chromatography-tandem mass spectrometry analysis was used for proteomic analysis, with subsequent characterisation with the PANTHER platform using an overrepresentation test (Fisher's exact type with false discovery rate correction) with the PANTHER pathway database. An untargeted approach based on liquid chromatography-tandem mass spectrometry was performed for metabolomics (normalisation to 50 µg of protein), and data were analysed with the MS Peaks to Pathways tool of the MetaboAnalyst platform, using both the GSEA and mummichog algorithms (cut-off = 0.05). Fatty acid composition (% of total FA) was analysed using gas chromatography. Differences between groups were analysed using a linear model with permutation using R software with the pgirmess package (P = 0.05 for significance). Altogether, 2706 proteins with at least two peptides were identified in mares’ UF, with 164 being differentially expressed. Ubiquitin proteasome, involved in embryo-endometrium interactions, was the most enriched pathway in N mares (fold enrichment = 15.12; P

The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1±0.8 years old (mean±s.e.m.), were given 10 ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 μg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm2, P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P4) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P4 concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.

Eosinophilic cells accumulate in the capillaries of the bovine Graafian follicle shortly before ovulation and in the early developing corpus luteum (CL). Suppressing the migration of these eosinophilic cells by dexamethasone allowed us to evaluate their possible function in the CL development. Brown Swiss cows (n = 10) were randomly subdivided into two groups (n = 5). Every group was used once as control group and once as experimental group with two oestrous cycles between each treatment. Eighteen hours (h) after oestrus synchronization, dexamethasone or saline was given. Ovulation was induced 24 h later with gonadotropin-releasing hormone. Another injection of dexamethasone or saline was given 12 h later. Eosinophilic cells in the blood were counted daily until day 7 after the first dexamethasone injection. The collection of ovaries took place at days 1, 2 and 5. Gene expression, protein concentration and location of angiogenic factors, chemokines, insulin-like growth factor 1 (IGF1) and eosinophilic cells were studied. No eosinophilic cells were found in the CL of the treatment group. Blood progesterone decreased significantly in the dexamethasone group from day 8 to 17. The protein concentration of FGF2 increased significantly in CL tissue at day 2 and VEGFA decreased. Local IGF1 gene expression in the CL was not regulated. We assume from our data that the migration of eosinophilic cells into the early CL is not an essential, but an important stimulus for angiogenesis during early CL development in cattle.

Epigenetic events controlling the transcriptional regulation of genes involved in endometrial function during the estrous cycle and early pregnancy have only sparsely been investigated. We analyzed the gene expression of DNA methyltransferases and the most prominent endocrine transcriptional mediator estrogen receptor alpha (ESR1) in the bovine endometrium of heifers at 0, 12 and 18 days following estrous and at day 18 after insemination. The luminometric methylation assay for the investigation of global DNA methylation and an elegant combination of methylation-sensitive high resolution melting and pyrosequencing for local methylation levels of ESR1 were deployed. In spite of differential gene expression of ESR1 among groups, no differences in endometrial ESR1 DNA methylation during neither estrous cycle nor early pregnancy were determined. Global DNA methylation prevailed at similar low levels in endometrium, likely controlled by the observed moderate DNMT3b expression. Thus, the epigenetic contribution of DNA methylation influencing endometrial function seems rather limited. However, because a control tissue expressing only minute amounts of ESR1 transcripts was locally significantly higher methylated, DNA methylation might contribute to an appropriate tissue-specific expression status underlying further specific control mechanisms of gene transcription.

Embryonic developmental arrest, known as diapause, has been reported in more than 130 species. However, its mechanisms are still not completely understood. In the roe deer, the only known ungulate that exhibits this phenomenon, diapause lasts for approximately 5 months, starting after the rut period in mid-July to early August and ending with embryo elongation and implantation in December/January. Little is known regarding oocyte characteristics during this period. Here, we analysed the roe deer oocyte transcriptome as a model to understand diapause effects on oocyte features during embryonic developmental arrest and reactivation. During regular hunting, immature oocytes were obtained by ovary slicing from diapause and nondiapause stages, and classified according to morphological characteristics. Only oocytes with >2 layers of compact cumulus cells and cytoplasm from 30 hunted females were used for analyses. Immature oocytes were denuded and snap frozen. Additional oocytes were cultured in maturation medium for 20-24h. Matured oocytes with a present polar body were snap frozen. Two pools of 10 immature and mature oocytes for both diapause and nondiapause stages were included (at least 4 donors/pool). Oocyte pools were processed using the Smart-seq 2 single-cell protocol (Illumina Inc., San Diego, CA, USA) for full-length cDNA and library preparation. We performed RNA-seq on an Illumina sequencer. The obtained Fastq files were clipped and analysed with a locally installed version of the Galaxy platform. Sequences were mapped against the roe deer transcriptome (unpublished data) and annotated against human and bovine transcripts. Differentially expressed genes (DEG; false discovery rate

Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

Uterine secretions have a dominant impact on the environment in which embryo development takes place. The uterine serpins (SERPINA14, previously known as UTMP) are found most abundantly during pregnancy in the uterus of ruminants. Although progesterone is currently assumed to be the major regulator of SERPINA14 expression, our recent study of transcriptome changes in bovine endometrium during the estrous cycle unexpectedly detected a marked upregulation of SERPINA14 mRNA levels at estrus. The present study describes the full-length mRNA sequence, genomic organization, and putative promoter elements of the SERPINA14 gene. The SERPINA14 mRNA abundance was quantified by real-time RT-PCR in intercaruncular endometrium at several time points during the estrous cycle and early pregnancy. Highest levels were found at estrus, followed by a dramatic decrease and a moderate expression during the luteal phase. Transcript levels were higher in pregnant endometrium compared with controls at Day 18. At estrus, immunoreactive protein was localized in deep glandular epithelium, and Western blotting concomitantly showed the 52-kDa form in uterine flushings. SERPINA14 mRNA was significantly upregulated in glandular endometrial cells in vitro after stimulation with estradiol-17beta and progesterone, but not after interferon-tau treatment. Our results clearly demonstrate that SERPINA14 appears distinctly in bovine endometrium during the estrus phase. A supporting role toward providing a well-prepared endometrial environment for passing gametes, especially sperm, is assumed.

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography–tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF1α (stable metabolite of PGI2) was predominant followed by PGF2α>PGE2>PGD2≈TXB2 (stable metabolite of TXA2). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF2α/PGE2 ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF1α (6.4 ng/ml) followed by PGF2α (1.1 ng/ml) and PGE2 (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI2 and PGF2α receptors were abundantly expressed by the trophoblast, abundant amounts of PGI2 and PGF2α in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE2 in the preimplantation uterus may be essential rather than detrimental for successful reproduction.

The aim of this study was to examine the hypothesis that follicular and uterine perfusion as well as endometrial gene expression during the ovulatory period differs after induction of luteolysis during the 1(st) follicular wave compared with the 2(nd) wave or in intact cycle. Nine healthy non-lactating Holstein-Friesian cows were examined during three estrous cycles. A prostaglandin F(2alpha) analogue (PGF) was administered randomly either on Day 7 (1(st) wave cycle) or Day 11 (2(nd) wave cycle) after detection of ovulation (=Day 1). No hormonal treatment was used in the intact cycle with spontaneous ovulation. Transrectal Doppler sonography was conducted daily after PGF injection and in the intact cycle beginning on Day 18 of the estrous cycle until ovulation. Follicular blood flow (FBF) was determined by measuring the maximum area of colour pixels on digitalized images of the follicles. Uterine blood flow was quantified by the time-averaged maximum velocity (UTAMV) and pulsatility index (PI) in both uterine arteries. Blood flow measurements were carried out on Days -1 and 0. Endometrial biopsy specimens were taken on Day 1 and analyzed for the gene expressions of estrogen, progesterone, oxytocin and VEGF receptors and eNOS and iNOS using RT-PCR. The interval from PGF injection to ovulation was shorter (P0.05) in 1(st) wave cycles than in 2(nd) wave cycles. On Days 0 and -1, FBF was greater (P0.05) in 1(st) wave cycles than in intact and 2(nd) wave cycles. On Day -1, UTAMV was greater (P0.05) in 1(st) wave cycles than in intact and 2(nd) wave cycles. There were no differences (P0.05) in FBF and UTAMV values between 2(nd) wave and intact cycles. No differences (P0.05) were detected in the gene expressions of endometrial receptors and enzymes between intact, 1(st) and 2(nd) wave cycles. The results show that follicular and uterine perfusion during the ovulatory phase are higher after induction of luteolysis during the 1(st) follicular wave compared with the 2(nd) wave or intact cycle. There were no effects on endometrial gene expression.

The endometrium undergoes marked functional changes during estrous cycle and pregnancy. As the adjacent environment of the conceptus, it represents the maternal interface for embryo-maternal communication, which is essential to maintain pregnancy. Transcriptome studies provide the unique opportunity to assess molecular profiles changing in response to endocrine or metabolic stimuli or to embryonic pregnancy recognition signals. Here we review the current state of transcriptome profiling techniques and the results of a series of transciptome studies comparing bovine endometrium samples during the estrous cycle or endometrium samples from pregnant vs. non-pregnant animals. These studies revealed specific mRNA profiles which are characteristic for the functional status of the endometrium. Transcriptome studies of endometrial samples recovered during the pre-attachment period identified many interferon-stimulated genes, genes that are possibly involved in embryo-maternal immune modulation ( C1S, C1R, C4, SERPING1, UTMP, CD81, IFITM1, BST2), as well as genes affecting cell adhesion ( AGRN, CD81, LGALS3BP, LGALS9, GPLD1, MFGE8, and TGM2) and remodeling of the endometrium ( CLDN4, MEP1B, LGMN, MMP19, TIMP2, TGM2, MET, and EPSTI1). The results of these transcriptome studies were compared to those of similar microarray analyses in human, mouse and Rhesus monkey to identify similarities in endometrial biology between mammalian species and species-specific differences. Future studies will cover dynamic transcriptome changes between different stages of early pregnancy, the relationship between metabolic problems in dairy cows and the functionality of reproductive tissues as well as endometrium transcriptome profiles in recipients of somatic cell nuclear transfer embryos.

Inflammation of the uterus is associated with disturbed ovarian function and reduced reproductive performance in dairy cows. To investigate the influence of endometritis on the bovine corpus luteum, 8 heifers received intrauterine infusions with either phosphate-buffered saline (PBS; 9mL) or Escherichia coli lipopolysaccharide (LPS; 3µg/kg of body weight diluted in 9mL of PBS) at 6-h intervals from 12h before and until 9d after ovulation during 2 cycles in a random order (ovulation=d 1). An untreated cycle was examined before and after PBS and LPS cycles, and the mean values from both untreated cycles were used as control. In all cycles, blood sampling and ultrasonography of the ovaries were performed on d 0, 1, 2, 4, 6, 8, 9, 10, 12, 15, 18, and then every 2d until ovulation. Endometrial cells were collected for cytology and quantitative real-time reverse transcriptase PCR on d 0, 6, and 9, and on d 0 and 6, respectively, and luteal tissue was collected for quantitative real-time reverse transcriptase PCR on d 6 and 9. Both, PBS and LPS infusions induced subclinical endometritis, which was accompanied by increased endometrial mRNA abundance of proinflammatory cytokines IL1β, IL8, and tumor necrosis factor α. Additionally, LPS challenge induced premature luteolysis, which was characterized by increased plasma concentrations of PGF2α metabolite, decreased plasma progesterone concentrations, and reduced luteal size and blood flow compared with the control. The luteal mRNA expression of the LPS receptor TLR4, PGE synthase, and the apoptosis-related factor CASP3 were higher, and those of steroidogenic factors STAR and HSD3B, the PGF receptor, and the angiogenic factor VEGFA121 were lower after LPS challenge compared with the control. In conclusion, repeated intrauterine LPS infusions during the first 9d of the estrous cycle alter gene expression and shorten the lifespan of the bovine corpus luteum.

The aim of this study was to investigate the effect of puerperal uterine disease on histopathologic findings and gene expression of proinflammatory cytokines in the endometrium of postpuerperal dairy cows; 49 lactating Holstein–Friesian cows were divided into two groups, one without (UD–; n = 29) and one with uterine disease (UD+; n = 21), defined as retained fetal membranes and/or clinical metritis. General clinical examination, vaginoscopy, transrectal palpation, and transrectal B-mode sonography were conducted on days 8, 11, 18, and 25 and then every 10 days until Day 65 (Day 0 = day of calving). The first endometrial sampling (ES1; swab and biopsy) was done during estrus around Day 42 and the second endometrial sampling (ES2) during the estrus after synchronization (cloprostenol between days 55 and 60 and GnRH 2 days later). The prevalence of histopathologic evidence of endometritis, according to the categories used here, and positive bacteriologic cultures was not affected by group (P > 0.05), but cows with uterine disease had a higher prevalence of chronic purulent endometritis (ES1; P = 0.07) and angiosclerosis (ES2; P ≤ 0.05) than healthy cows. Endometrial gene expression of IL1α (ES2), IL1β (ES2), and TNFα (ES1 and ES2) was higher (P ≤ 0.05) in the UD+ group than in the UD– group. In conclusion, puerperal uterine disease had an effect on histopathologic parameters and on gene expression of proinflammatory cytokines in the endometrium of postpuerperal cows, indicating impaired clearance of uterine inflammation in cows with puerperal uterine disease.

The endometrium plays a central role among the reproductive tissues in the context of early embryo–maternal communication and pregnancy. This study investigated transcriptome profiles of endometrium samples from day 18 pregnant vs non-pregnant heifers to get insight into the molecular mechanisms involved in conditioning the endometrium for embryo attachment and implantation. Using a combination of subtracted cDNA libraries and cDNA array hybridisation, 109 mRNAs with at least twofold higher abundance in endometrium of pregnant animals and 70 mRNAs with higher levels in the control group were identified. Among the mRNAs with higher abundance in pregnant animals, at least 41 are already described as induced by interferons. In addition, transcript levels of many new candidate genes involved in the regulation of transcription, cell adhesion, modulation of the maternal immune system and endometrial remodelling were found to be increased. The different expression level was confirmed with real-time PCR for nine genes. Localisation of mRNA expression in the endometrium was shown byin situhybridisation forAGRN,LGALS3BP,LGALS9,USP18,PARP12andBST2. A comparison with similar studies in humans, mice, and revealed species-specific and common molecular markers of uterine receptivity.

Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15 and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.

The extracellular matrix (ECM) component hyaluronan (HA) is considered to contribute to the optimal development of the gametes and the embryo. In this study, the regulation and localization of HA, its synthases (HAS 1-3) and the receptors CD44, RHAMM, and putative HARE were investigated in bovine oviducts during the estrous cycle. HA could be demonstrated in the entire lamina propria of the oviduct. Immunoreactive CD44 was found in the muscle layer additionally to the lamina propria. As for HA the luminal epithelium was devoid of CD44. Introducing quantitative real-time RT-PCR, a remarkable cyclic change of CD44 occurred. In addition, CD44 expression was much higher in the isthmus than in the ampulla. RHAMM transcripts were elevated particularly in the ampulla during estrus. A major part of the bovine putative HARE coding sequence was characterized. An analysis of the tissue distribution revealed transcripts not restricted to liver, spleen, and lymph node, providing evidence for a local putative HARE-mediated turnover of HA. These results suggest that in the bovine oviduct the cell surface receptor CD44 in particular might inversely regulate HA during the estrous cycle. The local turnover of HA through synthases and receptors may indicate its participation in the bovine oviduct supporting gamete maturation and early embryonic development.

Although many efforts have been made to improve the sperm normal penetration ability for in vitro embryo production (IVP), sperm capacitation in bovine remains a bottleneck for in vitro fertilization (IVF). The variable responses of sperm to the different supplements in IVF medium makes the process suboptimal for every bull. Theophylline, a methylxanthine, has recently been reported to enhance and maintain in vitro blastocyst production due to prolonged motility and longevity of bovine sperm (Kang et al. 2015 J. Reprod. Dev. 61, 99-105). Here, we evaluated the effects of theophylline supplementation during IVF on developmental rates of bulls with known low and average blastocyst production under standard conditions. A total of 1498 cumulus–oocyte complexes were obtained by slicing from bovine ovaries. Cumulus-oocyte complexes were submitted to in vitro maturation (IVM) for 24 h. In vitro fertilization was performed for 19 h with (2.5 mM) or without theophylline using frozen–thawed sperm from 5 different bulls at a final sperm concentration of to 2 × 106 cells mL−1. Two of the bulls had a previous report of low blastocyst rates (A, B) and 3 showed an average blastocyst production (C, D, E). After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. R software (https://www.r-project.org/) was used to evaluate cleavage and blastocyst rates using a two-sample t-test. The supplementation of theophylline significantly increased blastocyst rates for bulls A, B, and E (Table 1; P 0.05). These preliminary results show that in vitro blastocyst production for low performance bulls can be improved by theophylline supplementation during IVF. The underlying mechanism probably involves phosphodiesterase (PDE) inhibition to increase intra-sperm/oocyte cAMP levels improving oocyte-sperm interaction. Table 1.Total oocytes and mean (± SEM) cleavage and blastocyst rates of bull sperm treated or not with theophylline The authors thank Susanne Meese and Ulrich Witschi from Swissgenetics for the cooperation and donation of frozen bull sperm.

The objective of this study was to investigate the effect of time of first postpartum ovulation on endometrial inflammation in dairy cows with and without uterine disease during the early puerperal period. Transvaginal follicular puncture (FP) was carried out to suppress postpartum ovulation and formation of a CL until Day 42. Fifty-three lactating Holstein Friesian cows were divided into four groups on the basis of presence (UD+) or absence (UD-) of uterine disease, which was defined as retained fetal membranes and/or metritis, and whether FP had (FP+) or had not been (FP-) carried out. This resulted in the following groups: UD-FP- (n = 15), UD-FP+ (n = 13), UD+FP- (n = 13), and UD+FP+ (n = 12). Cloprostenol was given on Days 55 to 60 postpartum, and GnRH was administered 2 days later for synchronization of ovulation. In the FP- groups, endometrial swab and biopsy samples were collected during the second estrus (approximately Day 40) and during the estrus after synchronization. In the FP+ groups, the same samples were collected during the first estrus (approximately Day 49) and during the estrus after synchronization. The prevalence of positive bacteriologic cultures of the endometrium was not affected by FP (P > 0.05). Histologic signs of endometritis were more severe in UD+FP- cows at second sampling than in UD+FP+ cows (P ≤ 0.05). Endometrial expression of IL1α (in UD- after first sampling and in UD+ after second sampling) and IL1β (in UD- and UD+ after first sampling) was higher (P ≤ 0.05) in FP- cows than in FP+ cows. Regardless of group, cows with histopathologic evidence of endometritis had higher expression (P ≤ 0.05) of IL1α, IL1β, IL6, and TNFα than cows without endometritis. In conclusion, suppression of early ovulation by transvaginal FP enhances clearance of uterine inflammation in postpartum cows.

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.

Ongoing detailed investigations into embryo–maternal communication before implantation reveal that during early embryonic development a plethora of events are taking place. During the sexual cycle, remodelling and differentiation processes in the endometrium are controlled by ovarian hormones, mainly progesterone, to provide a suitable environment for establishment of pregnancy. In addition, embryonic signalling molecules initiate further sequences of events; of these molecules, prostaglandins are discussed herein as specifically important. Inadequate receptivity may impede preimplantation development and implantation, leading to embryonic losses. Because there are multiple factors affecting fertility, receptivity is difficult to comprehend. This review addresses different models and methods that are currently used and discusses their respective potentials and limitations in distinguishing key messages out of molecular twitter. Transcriptome, proteome and metabolome analyses generate comprehensive information and provide starting points for hypotheses, which need to be substantiated using further confirmatory methods. Appropriate in vivo and in vitro models are needed to disentangle the effects of participating factors in the embryo–maternal dialogue and to help distinguish associations from causalities. One interesting model is the study of somatic cell nuclear transfer embryos in normal recipient heifers. A multidisciplinary approach is needed to properly assess the importance of the uterine milieu for embryonic development and to use the large number of new findings to solve long-standing issues regarding fertility.

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.

Amino acids (AAs) are crucial for the developing conceptus prior to implantation. To provide insights into the requirements of the bovine embryo, we determined the AA composition of the uterine fluid. At days 12, 15, and 18 post-estrus, the uteri of synchronized pregnant and non-pregnant Simmental heifers were flushed for the analysis of 41 AAs and their derivatives by liquid chromatography–tandem mass spectrometry. The ipsilateral endometrium was sampled for quantitative PCR. In addition to a pregnancy-dependent increase of the essential AAs (PSLC15A3) were significantly increased at day 18 of pregnancyin vivo. In addition,SLC15A3was predominantly stimulated by trophoblast-derived interferon-τ in stroma cells of anin vitroco-culture model of endometrial cells. Our results show an increased concentration of AAs most likely to optimally provide the elongating pre-attachment conceptus with nutrients.

Early embryonic development is critically dependent on both maternal preparation and embryonic signalling of pregnancy. Matrix metallopeptidases (MMP) contribute to spatial and temporal matrix remodeling in the bovine endometrium. In this study we observed distinct changes in expression of MMP2, MMP14, and the metallopeptidase inhibitor TIMP2 between different phases of the estrous cycle indicating an endocrine regulation. An increase of TIMP2 protein abundance was ascertained in the uterine lumen during the time of embryo elongation. The expression pattern and cellular localization correlate well with the assumed effects of MMPs on release and activation of cytokines and growth factors directing cell migration, differentiation, and vascularization during this pivotal period of development. Specifically, active MMP2 in the endometrium may determine the allocation of growth factors supporting conceptus development. The presence of a day 18 conceptus in vivo and day 8 blastoysts in vitro induced endometrial TIMP2 mRNA expression. The results imply that TIMP2 is involved in very early local maternal recognition of pregnancy. Matrix metallopeptidases are likely to participate in remodeling processes preparing a receptive endometrium for a timely and precise regulation of embryo development.

A well-balanced immunological interaction between mother and the semi-allogenic embryo is of particular importance. The objective of the present study was to analyse mechanisms of immune tolerance in bovine pregnancy during peri-implantation. Simmental heifers inseminated with either cryopreserved spermatozoa or seminal plasma were killed 12, 15 or 18 days after oestrus. Uteri were flushed for the recovery of conceptuses and the ipsilateral intercaruncular endometrium was sampled for gene expression analysis. Indoleamine 2,3-dioxygenase (IDO) mRNA, coding for the initial enzyme of the kynurenine pathway, was 18-fold (P

Bovine trophoblast cells release interferon-τ (IFNT), a type I IFN, as the pregnancy recognition signal. Since type I IFNs exert growth inhibitory and proapoptotic actions, the effect of the conceptus on components of the apoptosis pathways was determined in the bovine endometrium during the periimplantation period. Uteri of Simmental heifers were flushed post mortem at days 12, 15, and 18 of cycle or pregnancy for the recovery of conceptuses and the sampling of ipsilateral endometrial tissue at slaughter for quantitative RT-PCR, immunohistochemistry, caspase activity and TUNEL assays. Endometrium samples of pregnant animals revealed increased transcript levels for the proapoptotic genes XAF1 (day 15: 2.9-fold; day 18: 15.1-fold; p = 0.005) and CASP8 (day 18: 2.4-fold; p = 0.007). The mRNA expression increased significantly with the day of the cycle for the proapoptotic genes FASLG, TNFSF10, TNF and TNFSF1A (p = 0.004, p = 0.006, p = 0.001 and p = 0.007) and the antiapoptotic gene BIRC4 (p = 0.03). We detected high amounts of FASLG transcripts in day 18 conceptuses (16-fold higher than day 18 endometria). This finding was validated at the protein level by immunohistochemistry. To further analyse the endometrial activation of the caspase cascade, the activities of initiator caspase 8 and effector caspases 3/7 were determined luminometrically. No difference between pregnant and cyclic animals was found for either caspase activity. Additionally, a TUNEL assay showed no increase of apoptotic cells in the pregnant endometrium. In conclusion, although the bovine conceptus induces the expression of proapoptotic genes, neither an activation of a caspase cascade nor an increase of apoptotic cells was noticed. These results suggest inhibitory mechanisms preventing endometrial cells from programmed cell death.

Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo–maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo–maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.

During the oestrous cycle, the bovine endometrium exhibits characteristic morphological and functional changes, which are mainly induced by progesterone (P4), oestrogens and oxytocin. We studied the response of the endometrium to this changing hormonal environment at the transcriptome level using a custom-made cDNA microarray. Endometrium samples were recovered from Simmental heifers on days 0 (oestrus), 3.5 (metoestrus), 12 (dioestrus) and 18. The latter group was divided into animals with high (late dioestrus) and low P4levels (preoestrus). Significance analysis of microarrays revealed 269 genes exhibiting significant changes in their transcript levels during the oestrous cycle in distinct temporal patterns. Two major types of expression profiles were observed, which showed the highest mRNA levels during the oestrus phase or the highest levels during the luteal phase respectively. A minor group of genes exhibited the highest mRNA levels on day 3.5. Gene ontology (GO) analyses revealed GO categories related to extracellular matrix remodelling, transport, and cell growth and morphogenesis enriched at oestrus, whereas immune response and particular metabolic pathways were overrepresented at dioestrus. Generation of gene interaction networks uncovered the genes possibly involved in endometrial remodelling (e.g. collagen genes,TNC,SPARC,MMP2,MEP1B,TIMP1,TIMP2,HTRA1), regulation of angiogenesis (e.g.ANGPTL2,TEK,NPY,AGT,EPAS1,KLF5 ), regulation of invasive growth (e.g.PCSK5, tight junction proteins,GRP,LGALS1,ANXA2,NOV,PLAT,MET,TDGF1,CST6,ITGB4), cell adhesion (e.g.MUC16,LGALS3BP) and embryo feeding (e.g.SLC1A1,SLC11A2,SLC16A1,SEPP1,ENPP1). Localisation of mRNA expression in the endometrium was analysed forCLDN4,CLDN10,TJP1,PCSK5,MAGED1, andLGALS1.