Generation of a novel three-dimensional scaffold-based model of the bovine endometrium.

Bovine in vitro endometrial models that resemble tissue function in vivo are needed to study infertility, long-term uterine alterations induced by pathogens and impact of endocrine disruptor chemicals on reproductive function and other reproductive system complications that cause high economic losses in livestock species. The present study aimed to generate an innovative, reproducible, and functional 3D scaffold-based model of the bovine endometrium structurally robust for long term-culture. We developed a multicellular model containing both endometrial epithelial and stromal cells. Epithelial cells organized to form a luminal-like epithelial layer on the surface of the scaffold. Stromal cells produced their own extracellular matrix forming a stable subepithelial compartment that physiologically resembles the normal endometrium. Both cell types released prostaglandin E2 and prostaglandin F2α following a treatment with oxytocin and arachidonic acid. Additionally signal pathways mediating oxytocin and arachidonic acid stimulation of prostaglandin synthesis were analyzed by real time PCR (RT-PCR). Oxytocin receptor (OXTR), prostaglandin E2 receptor 2 (EP2), prostaglandin E2 receptor 4 (EP4), prostaglandin F receptor (PTGFR), prostaglandin E synthase (PTGES), PGF-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (COX-2) expression was detected in both control and treatment groups, however, only significant changes in abundance of OXTR mRNA transcripts were found. The results obtained by this study are a step forward in bovine in vitro culture technology. This 3D scaffold-based model provides a platform to study regulatory mechanisms involved in endometrial physiology and can set the basis for a broader tool for designing and testing novel therapeutic strategies for recurrent uterine pathologies.

Metabolomic Profiling of Bos taurus Beef, Dairy, and Crossbred Cattle: A Between-Breeds Meta-Analysis.

Cattle breeds may differ substantially in their metabolism. However, the metabolomes of dairy and beef cattle are not well-known. Knowledge of breed-specific metabolic features is essential for biomarker identification and to adopt specific nutritional strategies. The muscle hypertrophy (mh), a beef cattle phenotype present in Asturiana de los Valles (AV) but absent in Asturiana de la Montaña (AM) and Holsteins, may underlie such differences. We compared the plasma metabolomes of Holstein, AV, AM, and crossbred cattle recipients selected for meta-analysis within an embryo transfer (ET) program. Blood samples were collected on day 0 (oestrus) and day 7 (prior to ET) (N = 234 samples × 2 days). Nuclear magnetic resonance quantified N = 36 metabolites in plasma, and more metabolic differences between breeds were found on day 0 (N = 19 regulated metabolites) than on day 7 (N = 5). AV and AM largely differed from Holstein cattle (N = 55 and 35 enriched metabolic pathways, respectively); however, AV and AM differed in N = 6 enriched pathways. Metabolic activity was higher in AV than in Holstein cattle, as explained in part by the mh phenotype. The metabolomic characterization of breeds facilitates biomarker research and helps to define the healthy ranges of metabolite concentrations.

Nuclear magnetic resonance analysis of female and male pre-hatching embryo metabolites at the embryo-maternal interface.

INTRODUCTION: Bovine female and male embryos differentially release metabolites with signalling effects to culture media. However, it is unknown if the embryo-maternal interface (EMI) metabolome is modified by embryonic sex. OBJECTIVE: To analyse using a combination of 1H NMR and a co-culture of endometrial cells the EMI. RESULTS: Twenty-six metabolites were identified and quantified in the EMI, nine metabolites reflected the sex of the embryo rather than their presence. CONCLUSIONS: 1H NMR is sensitive enough to perform quantitative analysis of sex-induced differences in the EMI. These results may help to understand the embryo-maternal dialogue on the basis of embryonic sex.

Differential release of cell-signaling metabolites by male and female bovine embryos cultured in vitro.

Male and female early bovine embryos show dimorphic transcription that impacts metabolism. Individual release of metabolites was examined in a 24h single culture medium from Day-6 male and female morulae that developed to Day-7 expanded blastocysts. Embryos were produced in vitro, fertilized with a single bull and cultured in SOFaaci+6  g/L BSA. The embryonic sex was identified (amelogenin gene amplification). Embryos (N = 10 males and N = 10 females) and N = 6 blank samples (i.e. SOFaaci+6  g/L BSA incubated with no embryos) were collected from 3 replicates. Metabolome was analyzed by UHPLC-TOF-MS in spent culture medium. After tentative identification, N = 13 metabolites significantly (P < 0.05; ANOVA) differed in their concentrations between male and female embryos, although N = 10 of these metabolites showed heterogeneity (Levene's test; P > 0.05). LysoPC(15:0) was the only metabolite found at higher concentration in females (fold change [FC] male to female = 0.766). FC of metabolites more abundant in male culture medium (N = 12) varied from 1.069 to 1.604. Chemical taxonomy grouped metabolites as amino-acids and related compounds (DL-2 aminooctanoic acid, arginine, 5-hydroxy-l-tryptophan, and palmitoylglycine); lipids (2-hexenoylcarnitine; Lauroyl diethanolamide; 5,6 dihydroxyprostaglandin F1a; LysoPC(15:0); DG(14:0/14:1(9Z)/0:0) and triterpenoid); endogenous amine ((S)-N-Methylsalsolinol/(R)-N-Methylsalsolinol); n-acyl-alpha-hexosamine (N-acetyl-alpha-d-galactosamine 1-phosphate); and dUMP, a product of pyrimidine metabolism. Among the compounds originally contained in CM, female embryos significantly depleted more arginine than males and blank controls (P < 0.001). Male and female embryos induce different concentrations of metabolites with potential signaling effects. The increased abundance of metabolites released from males is consistent with the higher metabolic activity attributed to such blastocysts.

In vitro cultured bovine endometrial cells recognize embryonic sex.

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.

Low serum concentration in bovine embryo culture enhances early blastocyst rates on Day-6 with quality traits in the expanded blastocyst stage similar to BSA-cultured embryos.

In bovine, single in vitro embryo culture in protein-free medium from Day-6 to Day-7 leads to expanded blastocyst (XB) with improved pregnancy and birth rates after cryopreservation. Under these conditions, early blastocysts (EB) progress to the XB stage at higher rates than morulae (M). However, embryo production with BSA in culture prior to Day-6 leads to low EB rates. We investigated whether a very low FCS concentration (0.1%) in culture from Day-1 to Day-6 would improve EB rates and, subsequently, increase XB rates on Day-7 after single culture in protein-free medium. The quality of embryos produced was evaluated in terms of survival to cryopreservation, apoptosis percentage, lipid accumulation and transfer to recipients. On Day-6, EB rates from embryos cultured with FCS were higher than with BSA (P=0.022). On Day-7, XB rates were higher in embryos from Day-6 EB than from Day-6M, both with and without FCS (P<0.005). After vitrification/warming of Day-7 XB, 100% embryos survived at 24h in all treatments, and total cell number and apoptosis percentage were not affected by the presence of FCS or embryonic stage on Day-6. Cryopreserved and fresh embryos produced with FCS until Day-6, and then deprived of protein and cultured individually, led to pregnancies after ET. In conclusion, minute FCS concentration improves EB rates on Day-6 leading, after one-day single culture without protein, to more XBs. The quality of XB produced with FCS compares well with XB produced with BSA in terms of apoptosis, lipid accumulation and pregnancy.

Hepatoma-derived growth factor: Protein quantification in uterine fluid, gene expression in endometrial-cell culture and effects on in vitro embryo development, pregnancy and birth.

Hepatoma-derived growth factor (HDGF) is present in the endometrium of cows and other mammals. Recombinant HDGF (rHDGF) improves bovine blastocyst development in vitro. However, specific culture conditions and essential aspects of HDGF uterine physiology are yet unknown. In this work we quantified total HDGF protein in uterine fluid (UF) by multiple reaction monitoring (MRM), and analyzed effects of rHDGF on specific embryonic stages with Day-6 bovine embryos cultured in vitro with and without BSA, and on pregnancy viability and calf phenotypes after embryo transfer to recipients. In addition, mRNA abundance of HDGF in endometrial cells co-cultured with one male or one female embryo was quantified. In the presence of BSA, rHDGF had no effect on blastocyst development; however, in BSA-free culture rHDGF mainly promoted development of early blastocysts in contrast with morulae. As the presence of HDGF contained in commercial BSA replacements was suspected, western blot confirmed HDGF identification in BSA both with and without fatty acids. Total HDGF quantified by MRM tended to increase in UF without vs. UF with embryos (P = 0.083). Pregnancy and birth rates, birth weight and calf measurements did not differ between embryos cultured with rHDGF and controls without rHDGF. However, HDGF abundance in cultured epithelial, endometrial cells tended to increase (P < 0.08) in culture with one male embryo. rHDGF acts selectively on specific embryonic stages, but care should be taken with specific macromolecular supplements in culture. The endometrial expression of HDGF can be regulated by the embryonic sex. The use of rHDGF is compatible with pregnancy and birth of normal calves.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Expression and localization of ARTEMIN in the bovine uterus and embryos.

Artemin a member of the glial cell line-derived neurotrophic factor (GDNF) family is present in mice and human preimplantation embryos, and reproductive tract, during early pregnancy promoting embryo development in vitro. The presence of artemin in cattle embryos and reproductive tract, however, is unknown. In the present work we identified for first time artemin in bovine uterine fluid (UF) (Western blot), endometrium (RT-PCR, Western blot and immunohistochemistry) and embryos (RT-PCR and immunohistochemistry) during early preimplantation development. In addition, GFRalpha3, a component of the artemin receptor was localized in blastocysts produced in vitro. Individually developing embryos released ARTEMIN in culture medium and triggered ARTEMIN mRNA down-regulation in epithelial cells from endometrial cell cultures. Our results suggest that ARTEMIN derived from early embryos and maternal reproductive tract may exert important roles during early development in cattle.

Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro.

In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24h in culture (P=0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P<0.06) mostly as a result of a higher number of miscarriages (P<0.013), reflecting lower viability. Birthweight, gestation length, height and thorax circumference did not differ between embryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (P<0.05) of insulin-like growth factor 2 receptor (IGF2R; imprinting) and activating transcription factor 4 (ATF4) and DNA-damage-inducible transcript 3 (DDIT3; endoplasmic reticulum stress) transcripts, with DNA methyltransferase 3A (DNMT3A; imprinting) tending to increase (P=0.062). However, in hatched blastocysts that survived cryopreservation, glucose-6-phosphate dehydrogenase (G6PD) was overexpressed in embryos cultured without protein (P<0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation.

Protein in culture and endogenous lipid interact with embryonic stages in vitro to alter calf birthweight after embryo vitrification and warming.

Short-term protein removal in vitro improves long-term blastocyst competence to survive vitrification. We investigated the mechanisms and effects underlying protein removal. Day-6 morulae and early blastocysts were cultured individually with and without protein for 24h. Development and lipid content were analysed in expanded blastocysts derived from morulae (M-XB) and from early blastocysts (EB-XB). Expression of genes involved in lipid metabolism, stress responses and apoptosis was analysed in fresh and vitrified-warmed M-XB produced with and without protein. Pregnancy rates, birth rates and birthweight (BW) were recorded after transfer of embryos. Day-7 EB-XB production rates (with, 66.9±6.2 and without, 68.8±6.0 protein) were higher than M-XB rates (with, 21.4±4.6 and without, 9.4±4.6 protein; P<0.005). EB-XB showed fewer lipids than M-XB (P=0.03). In fresh M-XB, expression of sterol regulatory element binding protein (SREBP1) was lower with (4.1±2.2) than without (13.6±2.2) protein, contrary to results obtained for Patatin-like phospholipase domain containing 2, Hormone-sensitive lipase and Bcl-2-associated X protein (P<0.05). Protein did not affect pregnancy rates and birth phenotypes (P>0.05). However, BW was higher (P<0.01) in calves born from vitrified M-XB (48.6±3.4kg) than from EB-XB (39.8±2.9kg). Such effects were more pronounced in females (P<0.001). Calves from fresh embryos did not show BW differences. These results indicate that embryonic kinetics and vitrification impact birth phenotypes, at least in females. Alterations might involve exogenous protein and mobilisation of lipid stocks.

Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium.

Metabolic differences between early male and female embryos can be reflected in culture medium (CM). We used a single bovine embryo culture step (24h) supporting improved birth rates under chemically defined conditions (CDC) to investigate biomarker detection of embryonic sex in contrast to classical BSA-containing medium. In vitro matured slaughterhouse oocytes were fertilized in vitro with a single bull. Embryos were initially cultured in synthetic oviduct fluid with BSA. On day-6, morulae were cultured individually in droplets with (BSA) or without protein (CDC). On day-7, expanded blastocysts were sexed (amelogenin gene amplification) and CM was stored at -145°C until metabolomic analysis by UHPLC-TOF MS. N=10 embryos per group (i.e. male-protein; female-protein; male-non-protein; female-non-protein) were produced. Statistical analysis revealed N=6 metabolites with different concentrations in CM, N=5 in male embryos (methionine, tryptophan, N-stearoyl-valine, biotin and pipecolic acid), N=1 in female embryos (threonine) (P<0.05 in BSA; P<10-7 in CDC). Only the clear threshold between males and females in CDC allowed correct classification of 100% males and 91% females within 5 out of 6 biomarkers (one female outlier showing the male biomarker profile). The use of CDC represents a critical aspect in the efficient detection of embryonic sex biomarkers by metabolomics.

Developmental kinetics of in vitro-produced bovine embryos: An aid for making decisions.

Embryo developmental kinetics and embryo survival after cryopreservation have been correlated with embryo quality and viability. The main objectives of this work were to analyze developmental ability and quality of in vitro-produced bovine embryos in relation to their kinetics and to establish a criterion of quality to predict further viability. Embryos were classified and grouped by their specific stage of development (2, 3-4, or ≥ 5 cells) at 44 hours post insemination (hpi) and cultured separately up to Day 8. On Days 7 and 8, good quality expanded blastocysts were vitrified or frozen. Cryopreserved surviving hatched embryos were stained for cell counts. Embryos at a more advanced stage (3-4 cells, and ≥5 cells) developed to morulae (P < 0.001) and blastocysts (P < 0.01) at higher rates than those embryos that had cleaved once by 44 hpi. Vitrification improved the hatching rates of blastocysts at 48 hours (P < 0.001) when compared with slow-rate freezing within each group of embryos (3-4 cells and ≥5 cells). After vitrification/warming, blastocysts coming from 3- to 4-cell embryos had higher hatching rates at 48 hours than those that came from ≥5-cell embryos. With regard to differential cell counts, no effect of the initial developmental stage was observed after warming/thawing. However, trophectoderm and total cells were higher in vitrified/warmed than in the frozen/thawed embryos (P < 0.001). These data show that selecting IVF embryos at 44 hpi, after the evaluation of their in vitro embryo development, could be used as noninvasive markers of embryo developmental competence and may help to select IVF embryos that would be more suitable for cryopreservation.

PGF2α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus.

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo-maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (ß-actin, NFkB, clusterin and immunoproteosome 20S ß5i subunit-i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F2 α and PGE2 concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF2 α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE2. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo-maternal interactions in vivo in monotocous species.

Early embryonic and endometrial regulation of tumor necrosis factor and tumor necrosis factor receptor 2 in the cattle uterus.

Tumor necrosis factor (TNF) alpha likely mediates embryomaternal communication in mammals. In bovine, we have previously found that the uterine fluid of heifers that carried early embryos shows downregulation in the TNF and nuclear factor κB system. In this work, we assessed the expression of TNF and its receptor TNFR2 in the bovine endometrium and embryos during blastocyst development. Moreover, to explore the endometrial immune response to early embryos, we analyzed the number of CD45 leukocytes in the bovine endometrium. Day 8 endometrium and blastocyst recovered from animals after transfer of Day 5 embryos showed TNF and TNFR2 mRNA transcription and protein colocalization. The presence of embryos increased endometrial TNF and TNFR2 protein, whereas endometrial leukocytes decreased. Blastocysts exposed to the uterine tract had undetectable levels of TNF and lower levels of TNFR2 mRNA. These results suggest that the endometrium might lower the TNF concentration in the blastocyst by (1) regulating TNF secretion into the uterine fluid and (2) inducing decreased TNF and TNFR2 mRNA transcription in the embryo. Thus, TNF and TNFR2 might participate in early embryomaternal communication.

Survival of vitrified in vitro-produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure.

Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro-produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro-produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a modified fiber plug designed to allow direct ET after in-straw cryoprotectant (CP) dilution. They were warmed using the one-step process (0.25 M sucrose, 5 minutes) in a 0.25 mL French straw. Embryo recovery associated with the modified fibreplug system was less reliable than with the conventional system. However, no differences were seen between the systems in terms of in vitro embryo survival among those finally recovered. Finally, IVP blastocysts were vitrified using conventional fibreplugs to maintain a high embryo recovery rate, and then warmed using the one-step warming in-straw CP dilution procedure, but using an adapter with a wider opening coupled to the French straw and a heated metal chamber to protect and keep the straw at 41 °C (experiment 4). No differences were seen in embryo survival rates between the two groups. The CVM combined with this new one-step warming in-straw CP dilution procedure could be used for direct ET under field conditions.

Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate=52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80±0.053; plasma: 0.89±0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607±0.038 (CM, expanded blastocysts) and 0.672±0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.