Amino acids in the uterine luminal fluid reflects the temporal changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle

In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P < 0.05). Glutamic acid only increased in pregnant heifers on Day 19 (P,0.001). Asparagine concentrations were greater in ULF of cyclic compared to pregnant heifers on Day 7 (P < 0.05) while valine concentrations were higher in pregnant heifers on Day 16 (P < 0.05). Temporal changes in expression of the cationic AA transporters SLC7A1 SLC7A4 and SLC7A6 occurred in the endometrium during the estrous cycle/early pregnancy coordinate with changes in conceptus expression of SLC7A4, SLC7A2 and SLC7A1 (P < 0.05). Only one acidic AA transporter (SLC1A5) increased in the endometrium while conceptus expression of SLC1A4 increased (P < 0.05). The neutral AA transporters SLC38A2 and SLC7A5 increased in the endometrium in a temporal manner while conceptus expression of SLC38A7, SLC43A2, SLC38A11 and SLC7A8 also increased (P < 0.05). P4 modified the expression of SLC1A1, -1A4, -1A5, -38A2 , -38A4, -38A7, -43A2, -6A14, -7A1, -7A5 and -7A7 in the endometrium. Results demonstrate that temporal changes in AA in the ULF reflect changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle, some of which are modified by P4. © 2014 Forde et al

The effect of exogenous glucose infusion on early embryonic development in lactating dairy cows

peer-reviewedThe objective of this study was to examine the effect of intravenous infusion of glucose on early embryonic development in lactating dairy cows. Nonpregnant, lactating dairy cows (n = 12) were enrolled in the study (276 ± 17 d in milk). On d 7 after a synchronized estrus, cows were randomly assigned to receive an intravenous infusion of either 750 g/d of exogenous glucose (GLUC; 78 mL/h of 40% glucose wt/vol) or saline (CTRL; 78 mL/h of 0.9% saline solution). The infusion period lasted 7 d and cows were confined to metabolism stalls for the duration of the study. Coincident with the commencement of the infusion on d 7 after estrus, 15 in vitro-produced grade 1 blastocysts were transferred into the uterine horn ipsilateral to the corpus luteum. All animals were slaughtered on d 14 to recover conceptuses, uterine fluid, and endometrial tissue. Glucose infusion increased circulating glucose concentrations (4.70 ± 0.12 vs. 4.15 ± 0.12 mmol/L) but did not affect milk production or dry matter intake. Circulating β-hydroxybutyrate concentrations were decreased (0.51 ± 0.01 vs. 0.70 ± 0.01 mmol/L for GLUC vs. CTRL, respectively) but plasma fatty acids, progesterone, and insulin concentrations were unaffected by treatment. Treatment did not affect either uterine lumen fluid glucose concentration or the mRNA abundance of specific glucose transporters in the endometrium. Mean conceptus length, width, and area on d 14 were reduced in the GLUC treatment compared with the CTRL treatment. A greater proportion of embryos in the CTRL group had elongated to all length cut-off measurements between 11 and 20 mm (measured in 1-mm increments) compared with the GLUC treatment. In conclusion, infusion of glucose into lactating dairy cows from d 7 to d 14 post-estrus during the critical period of conceptus elongation had an adverse impact on early embryonic development

Sexually dimorphic gene expression in bovine conceptuses at the initiation of implantation

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X-chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium

The role of CAPG in molecular communication between the embryo and the uterine endometrium: Is its function conserved in species with different implantation strategies?

During the preimplantation period of pregnancy in eutherian mammals, transcriptional and proteomic changes in the uterine endometrium are required to facilitate receptivity to an implanting blastocyst. These changes are mediated, in part, by proteins produced by the developing conceptus (inner cell mass and extraembryonic membranes). We hypothesized that this common process in early pregnancy in eutheria may be facilitated by highly conserved conceptus‐derived proteins such as macrophage capping protein (CAPG). We propose that CAPG may share functionality in modifying the transcriptome of the endometrial epithelial cells to facilitate receptivity to implantation in species with different implantation strategies. A recombinant bovine form of CAPG (91% sequence identity between bovine and human) was produced and bovine endometrial epithelial (bEECs) and stromal (bESCs) and human endometrial epithelial cells (hEECs) were cultured for 24 hours with and without recombinant bovine CAPG (rbCAPG). RNA sequencing and quantitative real‐time PCR analysis were used to assess the transcriptional response to rbCAPG (Control, vehicle, CAPG 10, 100, 1000 ng/mL: n = 3 biological replicates per treatment per species). Treatment of bEECs with CAPG resulted in alterations in the abundance of 1052 transcripts (629 increased and 423 decreased) compared to vehicle controls. Treatment of hEECs with bovine CAPG increased expression of transcripts previously known to interact with CAPG in different systems (CAPZB, CAPZA2, ADD1, and ADK ) compared with vehicle controls (P < .05). In conclusion, we have demonstrated that CAPG, a highly conserved protein in eutherian mammals, elicits a transcriptional response in the endometrial epithelium in species with different implantation strategies that may contribute to pregnancy success

Transcriptional response of endometrial cells to insulin, cultured using microfluidics

Obesity is a rapidly growing public health issue among women of reproductive age associated with decreased reproductive function including implantation failure. This can result from a myriad of factors including impaired gametes and endometrial dysfunction. The mechanisms of how obesity-related hyperinsulinaemia disrupts endometrial function are poorly understood. We investigated potential mechanisms by which insulin alters endometrial transcript expression. Ishikawa cells were seeded into a microfluidics device attached to a syringe pump to deliver a constant flow rate of 1 μL/min of the following: (i) control (ii) vehicle control (acidified PBS), or (iii) insulin (10 ng/mL) for 24 h (n = 3 biological replicates). Insulin-induced transcriptomic response of endometrial epithelial cells was determined via RNA sequencing, and DAVID and Webgestalt to identify Gene Ontology (GO) terms and signalling pathways. A total of 29 transcripts showed differential expression levels across two comparison groups (control vs vehicle control; vehicle control vs insulin). Nine transcripts were differentially expressed in vehicle control vs insulin comparison (P < 0.05). Functional annotation analysis of transcripts altered by insulin (n = 9) identified three significantly enriched GO terms: SRP-dependent co-translational protein targeting to membrane, poly(A) binding, and RNA binding (P < 0.05). The overrepresentation analysis found three significantly enriched signalling pathways relating to insulin-induced transcriptomic response: protein export, glutathione metabolism, and ribosome pathways (P < 0.05). Transfection of siRNA for RAPSN successfully knocked down expression (P < 0.05), but this did not have any effect on cellular morphology. Insulin-induced dysregulation of biological functions and pathways highlights potential mechanisms by which high insulin concentrations within maternal circulation may perturb endometrial receptivity

Quantification of the effect of in-utero events on lifetime resilience in dairy cows

Currently, the dairy industry is facing many challenges that could affect its sustainability, including climate change and public perception of the industry. As a result, interest is increasing in the concept of identifying resilient animals, those with a long productive lifespan, good reproductive performance and milk yield. There is much evidence that events in utero, i.e., the Developmental Origins of Health and Disease (DOHaD), alter life-course health of offspring and we hypothesized that these could alter resilience in calves, where resilience is identified using lifetime data. The aim of this study was to quantify lifetime resilience scores (LRS) using an existing scoring system based on longevity with secondary corrections for age at first calving and calving interval and to quantify the effects of in-utero events on the LRS using 2 data sets. The first was a large data set of cattle in 83 farms in Great Britain born from 2006 to 2015 and the second was a smaller, more granular data set of cattle born between 2003 and 2015 in the Langhill research herd at Scotland's Rural College. Events during dam's pregnancy included health events (lameness, mastitis, use of an antibiotic or anti-inflammatory medication), the impact of heat stress as measured by temperature-humidity index and perturbations in milk yield and quality (somatic cell count, percentage fat, percentage protein and fat:protein ratio). Daughters born to dams that experienced higher temperature-humidity indexes while they were in-utero during the first and third trimesters of pregnancy had lower LRS. Daughter LRS scores were also lower where milk yields or median fat percentages in the first trimester were low, and when milk yields were high in the third trimester. Dam LRS was positively associated with LRS of their offspring, however, as parity of the dam increased, LRS of their calves decreased. Similarly, in the Langhill herd, dams of a higher parity produced calves with lower LRS. Additionally, dams which recorded a high max locomotion score in the third trimester of pregnancy were negatively associated with lower calf LRS in the Langhill herd. Our results suggest that events that occur during pregnancy have lifelong consequences for the calf's lifetime performance. However, experience of higher temperature-humidity indexes, higher dam LRS scores and mothers in higher parities explained a relatively small proportion of variation in offspring LRS, which suggests that other factors play a substantial role in determining calf LRS scores. While 'big data' can contain a considerable amount of noise, similar findings between the 2 data sets indicate it is likely these findings are real.</p

Do differences in the endometrial transcriptome between uterine horns ipsilateral and contralateral to the corpus luteum influence conceptus growth to day 14 in cattle?

peer-reviewedEmbryo transfer to the uterine horn contralateral to the ovary containing the corpus luteum (CL) negatively impacts pregnancy establishment in cattle. Our aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to day 14. In experiment 1, endometrial samples from both horns were collected from synchronized heifers slaughtered on day 5, 7, 13, or 16 post-estrus (n = 5 per time) and subjected to RNA sequencing. In experiment 2, 10 day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral (n = 9) or contralateral to the CL (n = 8) or into both horns (i.e., bilateral, n = 9) of synchronized recipient heifers. Reproductive tracts were recovered at slaughter on day 14, and the number and dimensions of recovered conceptuses were recorded for each horn. A total of 217, 54, 14, and 18 differentially expressed genes (>2-fold change, FDR P < 0.05) were detected between ipsilateral and contralateral horns on days 5, 7, 13, and 16, respectively, with signaling pathways regulating pluripotency of stem cells, ErbB signaling pathway, and mTOR signaling pathway amongst the top canonical pathways. Site of embryo transfer did not affect recovery rate (48.0%, 168/350) or length of conceptuses (mean ± SE 2.85 ± 0.27 mm). Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between day 7 and 14.Science Foundation Irelan

Alterations in expression of endometrial genes coding for proteins secreted into the uterine lumen during conceptus elongation in cattle

BACKGROUND: We hypothesized that genes that are up-regulated in the uterine endometrium at the initiation of conceptus elongation in cattle, and that encode for secreted proteins, contribute to the composition of the uterine luminal fluid (ULF) and ultimately, drive conceptus elongation. The aims of this study were to: 1) screen endometrial transcriptomic data for genes that encode secreted proteins on Day 13; 2) determine temporal changes in the expression of these genes during the estrous cycle/early pregnancy; 3) determine if expression of these genes is affected by altered concentrations of progesterone (P4) in vivo and 4) determine if the protein products of these genes are detectable in ULF. RESULTS: Of the fourteen candidate genes examined, quantitative real-time PCR analysis revealed the expression of APOA1, ARSA, DCN, LCAT, MUC13, NCDN, NMN, NPNT, NXPH3, PENK, PLIN2 and TINAGL1 was modulated in the endometrium (P<0.05) as the estrous cycle/early pregnancy progressed. APOA1, DCN and NPNT expression was higher in cyclic compared to pregnant heifers, and pregnancy increased (P<0.05) the expression of LCAT, NCDN, NMN, PLIN2 and TINAGL1. The magnitude of the increase in expression of APOA1, PENK and TINAGL1 on Day 13 was reduced (P<0.05) in heifers with low P4. Furthermore, low P4 decreased (P<0.05) the expression of LCAT and NPNT on Day 7, while an early increase (P<0.05) in the expression of NXPH3 and PLIN2 was observed in heifers with high P4. The protein products of 5 of the candidate genes (APOA1, ARSA, LCAT, NCDN and PLIN) were detected in the ULF on either Days 13, 16 or 19 of pregnancy. CONCLUSION: Using a candidate gene approach, we determined that both P4 concentration and the presence of the conceptus alter endometrial expression of PLIN2, TINAGL1, NPNT, LCAT, NMN and APOA1. Comparison of the expression profiles of these genes to proteins detected in ULF during conceptus elongation (i.e., Days 13 through 19) revealed the presence of APOA1, ARSA, LCAT, NCDN as well as members of the PLIN family of proteins that may play roles in driving conceptus elongation in cattle

‘Conceptualizing’ the Endometrium: Identification of Conceptus-Derived Proteins During Early Pregnancy in Cattle

The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16–18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period

Quantification of HER2 heterogeneity in breast cancer-implications for identification of sub-dominant clones for personalised treatment

Breast cancer is a heterogeneous disease, at both an inter- and intra-tumoural level. Appreciating heterogeneity through the application of biomarkers and molecular signatures adds complexity to tumour taxonomy but is key to personalising diagnosis, treatment and prognosis. The extent to which heterogeneity exists, and its interpretation remains a challenge to pathologists. Using HER2 as an exemplar, we have developed a simple reproducible heterogeneity index. Cell-to-cell HER2 heterogeneity was extensive in a proportion of both reported ‘amplified’ and ‘non-amplified’ cases. The highest levels of heterogeneity objectively identified occurred in borderline categories and higher ratio non-amplified cases. A case with particularly striking heterogeneity was analysed further with an array of biomarkers in order to assign a molecular diagnosis. Broad biological complexity was evident. In essence, interpretation, depending on the area of tumour sampled, could have been one of three distinct phenotypes, each of which would infer different therapeutic interventions. Therefore, we recommend that heterogeneity is assessed and taken into account when determining treatment options