Modelling aspects of oviduct fluid formation in vitro

© 2017 Society for Reproduction and Fertility. 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 an in vitro preparation of the bovine oviduct epithelium to investigate the formation and composition of in vitro-derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct-specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation was evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility and optimising embryo culture media

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5β-THF, 3β,5α-THF, 3β,5β-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5β-THE, 3β,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20β-DHP, 3α-DHP, 3β-DHP, 5α-DHP, 5β-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 μL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL−1 was achieved for all steroids suitable for the analysis of authentic samples

Comparison of the Effects of Early Pregnancy with Human Interferon, Alpha 2 (IFNA2), on Gene Expression in Bovine Endometrium

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

Vascular Endothelial Growth Factor A and VEGFR-1 change during preimplantation in Heifers

Vascular endothelial growth factor A (VEGFA) plays a critical angiogenic role in the endometrium of placentalia during preimplantation. The role of VEGFA and its receptors is not fully characterised in bovine reproduction. We analysed the mRNA expression of VEGFA isoforms 121, 165 and 189, and VEGF receptors 1 and 2 in three experimental settings (A, B and C). We compared intercaruncular endometrium of cyclic to pregnant heifers at Days 12, 15 and 18 post insemination (Day 0), and between Day 15 and Day 18 conceptuses (A). We further compared caruncular versus intercaruncular endometrium at Day 15 (B), and endometrium of heifers carrying embryos originating from somatic cell nuclear transfer (SCNT) versus in vitro fertilisation (IVF) at Day 18 (C). Endometrial VEGFA protein was localised and quantified. Pregnant heifers displayed lower intercaruncular endometrial mRNA expression of VEGFA-121 (p = 0.045) and VEGFA-189 (p = 0.009) as well as lower VEGFA protein abundance (p < 0.001) at Day 15. The VEGFA protein was localised in intercaruncular luminal, glandular epithelium and in tunica muscularis of blood vessels. At Day 15, caruncular endometrium displayed higher VEGFA mRNA expression than intercaruncular endometrium (p < 0.05). Intercaruncular endometrial VEGFA protein at Day 18 was higher in abundance in SCNT than in IVF (p = 0.038). Therefore, during preimplantation in cattle, there may be a need for timely physiological reduction in intercaruncular endometrial VEGFA expression in favour of the caruncular area to facilitate a gradient towards the implantation sites. A higher expression of VEGFA in SCNT may predispose for later placentation abnormalities frequently observed following SCNT

Tissue-specific and minor inter-individual variation in imprinting of <i>IGF2R</i> is a common feature of <i>Bos taurus</i> concepti and not correlated with fetal weight

The insulin-like growth factor 2 receptor (IGF2R) is essential for prenatal growth regulation and shows gene dosage effects on fetal weight that can be affected by in-vitro embryo culture. Imprinted maternal expression of murine Igf2r is well documented for all fetal tissues excluding brain, but polymorphic imprinting and biallelic expression were reported for IGF2R in human. These differences have been attributed to evolutionary changes correlated with specific reproductive strategies. However, data from species suitable for testing this hypothesis are lacking. The domestic cow (Bos taurus) carries a single conceptus with a similar gestation length as human. We identified 12 heterozygous concepti informative for imprinting studies among 68 Bos taurus fetuses at Day 80 of gestation (28% term) and found predominantly maternal IGF2R expression in all fetal tissues but brain, which escapes imprinting. Inter-individual variation in allelic expression bias, i.e. expression of the repressed paternal allele relative to the maternal allele, ranged from 4.6−8.9% in heart, 4.3−10.2% in kidney, 6.1−11.2% in liver, 4.6−15.8% in lung and 3.2−12.2% in skeletal muscle. Allelic bias for mesodermal tissues (heart, skeletal muscle) differed significantly (P&lt;0.05) from endodermal tissues (liver, lung). The placenta showed partial imprinting with allelic bias of 22.9−34.7% and differed significantly (P&lt;0.001) from all other tissues. Four informative fetuses were generated by in-vitro fertilization (IVF) with embryo culture and two individuals displayed fetal overgrowth. However, there was no evidence for changes in imprinting or DNA methylation after IVF, or correlations between allelic bias and fetal weight. In conclusion, imprinting of Bos taurus IGF2R is similar to mouse except in placenta, which could indicate an effect of reproductive strategy. Common minor inter-individual variation in allelic bias and absence of imprinting abnormalities in IVF fetuses suggest changes in IGF2R expression in overgrown fetuses could be modulated through other mechanisms than changes in imprinting

The endometrium responds differently to cloned versus fertilized embryos

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

Review: Embryonic diapause in the European roe deer - slowed, but not stopped

Embryonic diapause in mammals describes a transient reduction of proliferation and developmental progression occurring at the blastocyst stage. It was first described in the European roe deer (Capreolus capreolus) in the 19th century, and later found to occur in at least over 130 mammalian species across several taxa. Diapause is often displayed as an interruption, a halt, or an arrest of embryonic development. In this review, we explore reduced, but not stopped pace of growth, proliferation and developmental progression during embryonic diapause and revisit early embryonic proliferation and continued slow development as peculiar phenomenon in the roe deer