Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

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, as well as good reproductive performance and milk yield. There is much evidence that events in utero, that is, the developmental origins of health and disease hypothesis, alter the 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 datasets. The first was a large dataset of cattle on 83 farms in Great Britain born from 2006 to 2015 and the second was a smaller, more granular dataset 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 effect 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 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 that recorded a high maximum 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, 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. Although "big data" can contain a considerable amount of noise, similar findings between the 2 datasets indicate it is likely these findings are real.

Sex bias in utero alters ovarian reserve but not uterine capacity in female offspring†.

Environmental stressors to which a fetus is exposed affect a range of physiological functions in postnatal offspring. We aimed to determine the in utero effect of steroid hormones on the reproductive potential of female offspring using a porcine model. Reproductive tracts of pigs from female-biased (>65% female, n = 15), non-biased (45-54.9% female, n = 15), and male-biased litters (<35% females, n = 9) were collected at slaughter (95-115 kg). Ovaries and uterine horns were processed for H&E or immunohistochemistry. Variability of data within groups was analyzed with a Levene's test, while data were analyzed using mixed linear models in R. In the ovarian reserve, there was a significant birth weight by sex ratio interaction (P = 0.015), with low birth weight pigs from male-biased litters having higher numbers of primordial follicles with opposite trends seen in pigs from female-biased litters. Sex bias held no effect on endometrial gland development. A lower birth weight decreased the proportion of glands found in the endometrium (P = 0.045) and was more variable in both male-biased and female-biased litters (P = 0.026). The variability of primordial follicles from male-biased litters was greater than non- and female-biased litters (P = 0.014). Similarly, endometrial stromal nuclei had a greater range in male- and female-biased litters than non-biased litters (P = 0.028). A crucial finding was the greater variability in primordial follicles in the ovaries from females derived from male-biased litters and stromal cell count in the endometrium of females from male- and female-biased litters. This could be inflating the variability of reproductive success seen in females from male-biased litters.

Understanding conceptus-maternal interactions: what tools do we need to develop?

Communication between the maternal endometrium and developing embryo/conceptus is critical to support successful pregnancy to term. Studying the peri-implantation period of pregnancy is critical as this is when most pregnancy loss occurs in cattle. Our current understanding of these interactions is limited, due to the lack of appropriate in vitro models to assess these interactions. The endometrium is a complex and heterogeneous tissue that is regulated in a transcriptional and translational manner throughout the oestrous cycle. While there are in vitro models to study endometrial function, they are static and 2D in nature or explant models and are limited in how well they recapitulate the in vivo endometrium. Recent developments in organoid systems, microfluidic approaches, extracellular matrix biology, and in silico approaches provide a new opportunity to develop in vitro systems that better model the in vivo scenario. This will allow us to investigate in a more high-throughput manner the fundamental molecular interactions that are required for successful pregnancy in cattle.

Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium.

Regulation of the mammalian embryo involves cell-signaling molecules produced by the maternal oviduct and endometrium. Here, datasets on the transcriptome of the gestational Days 5 and 6 bovine morula and Day 5 maternal endometrium were examined to identify receptor genes expressed by the morula and expression of the corresponding ligand-related genes in the endometrium. A total of 175 receptor genes were identified in the morula, including 48 encoding for growth factors or WNT signaling molecules, 25 for cytokines and chemokines, 35 involved in juxtacrine and matricellular signaling and 25 encoding for receptors for small molecules. Some of the highly-expressed pairs of endometrial ligand and embryo receptor genes included MDK and its receptors ITGB1, SDC4 and LRP2, WNT5A (RYK), VEGFA (ITGB1), GPI (AMFR), and the hedgehog proteins IHH and DHH (HHIP). The most highly expressed receptors for small molecules were GPRC5C (retinoic acid receptor), PGRMC1 (progesterone), and CHRNB2 (acetylcholine). There were also 84 genes encoding for cell signaling ligands expressed by the morula, with the most highly expressed being GPI, AIMP1, TIMP1, IK, and CCN2. The atlas of receptor and ligand genes should prove useful for understanding details of the communication between the embryo and mother that underlies optimal embryonic development.

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.

Protein Synthesis by Day 16 Bovine Conceptuses during the Time of Maternal Recognition of Pregnancy.

Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo-maternal communication during pregnancy recognition in cattle.

Relative effects of location relative to the corpus luteum and lactation on the transcriptome of the bovine oviduct epithelium.

BACKGROUND: Lactation and associated metabolic stresses during the post-partum period have been shown to impair fertility in dairy cows. The oviduct plays key roles in embryo development and the establishment of pregnancy in cattle. The aim of this study was to investigate the effects of lactation and location relative to the corpus luteum (CL) on the transcriptome of the bovine oviduct epithelium. RESULTS: An original animal model was used. At 60 days post-partum, Holstein lactating (n = 4) and non-lactating (i.e. never milked after calving; n = 5) cows, as well as control nulliparous heifers (n = 5), were slaughtered on Day 3 following induced estrus, and epithelial samples from the oviductal ampulla and isthmus ipsilateral and contralateral to the corpus luteum (CL) were recovered for RNA sequencing. In the oviduct ipsilateral to the CL, differentially expressed genes (DEGs) were identified between heifers compared with both postpartum cow groups. However, only 15 DEGs were identified between post-partum lactating and non-lactating cows in the ipsilateral isthmus and none were identified in the ipsilateral ampulla. In contrast, 192 and 2583 DEGs were identified between ipsilateral and contralateral ampulla and isthmus, respectively. In both regions, more DEGs were identified between ipsilateral and contralateral oviducts in non-lactating cows and heifers than in lactating cows. Functional annotation of the DEGs associated with comparisons between metabolic groups highlighted a number of over-represented biological functions and cell pathways including immune response and cholesterol/steroid biosynthesis. CONCLUSIONS: Gene expression in the oviduct epithelium, particularly in the isthmus, was more affected by the location relative to the CL than by lactation at Day 3 post-estrus. Furthermore, the effect of the proximity to the CL was modulated by the metabolic status of the cow.

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

Embryo 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.

Influence of metabolic status and genetic merit for fertility on proteomic composition of bovine oviduct fluid†.

The oviduct plays a crucial role in fertilization and early embryo development providing the microenvironment for oocyte, spermatozoa, and early embryo. Since dairy cow fertility declined steadily over the last decades, reasons for early embryonic loss have gained increasing interest. Analyzing two animal models, this study aimed to investigate the impact of genetic predisposition for fertility and of metabolic stress on the protein composition of oviduct fluid. A metabolic model comprised maiden Holstein heifers and postpartum lactating (Lact) and non-lactating (Dry) cows, while a genetic model consisted of heifers from the Montbéliarde breed and Holstein heifers with low- and high-fertility index. In a holistic proteomic analysis of oviduct fluid from all groups using nano-liquid chromatography tandem-mass spectrometry analysis and label-free quantification, we were able to identify 1976 proteins, among which 143 showed abundance alterations in the pairwise comparisons within both models. Most differentially abundant proteins were revealed between low fertility Holstein and Montbéliarde (52) in the genetic model and between lactating and maiden Holstein (19) in the metabolic model, demonstrating a substantial effect of genetic predisposition for fertility and metabolic stress on the oviduct fluid proteome. Functional classification of affected proteins revealed actin binding, translation, and immune system processes as prominent gene ontology (GO) clusters. Notably, Actin-related protein 2/3 complex subunit 1B and the three immune system-related proteins SERPIND1 protein, immunoglobulin kappa locus protein, and Alpha-1-acid glycoprotein were affected in both models, suggesting that abundance changes of immune-related proteins in oviduct fluid play an important role for early embryonic loss.

Effect of lactation on conceptus-maternal interactions at the initiation of implantation in cattle: I. Effects on the conceptus transcriptome and amino acid composition of the uterine luminal fluid.

Approximately 65-75 days postpartum (dpp), the estrous cycles of nonlactating (dried off immediately postpartum: n = 12) and lactating (n = 13) Holstein Friesian cows were synchronized and on day 7 a single blastocyst derived from superovulated nulliparous Holstein Friesian heifers was transferred to each cow. A control group of nulliparous heifers (n = 8) were synchronized, inseminated to a standing heat, and slaughtered on the same day as nonlactating and lactating recipients (day 19; estrus = day 0). The uterine horn ipsilateral to the corpus luteum was flushed with 10 ml phosphate-buffered saline and the conceptus, and uterine luminal fluid (ULF) was snap-frozen in liquid nitrogen. Gene expression analysis of the conceptus was performed by RNA sequencing, while amino acid composition of ULF was determined by high-performance liquid chromatography. No differentially expressed genes (DEGs) were observed between conceptuses recovered from nonlactating and lactating cows. Eight DEGs were identified between conceptuses recovered from nonlactating cows and heifers. A total of 269 DEGs (100 up- and 169 downregulated) were identified between conceptuses recovered from lactating cows compared to heifers. Alanine, glycine, serine, threonine, arginine, leucine, and valine were significantly lower in abundance in ULF recovered from heifers compared to nonlactating or lactating cows. This study demonstrates that the environment in which the embryo develops post the blastocyst stage can have an effect on the conceptus transcriptome and amino acid composition of the ULF but this was mainly observed between the two extreme groups in terms of metabolic status (nulliparous heifers vs postpartum lactating cows).

Effect of metabolic status on conceptus-maternal interactions on day 19 in dairy cattle: II. Effects on the endometrial transcriptome.

The aim of this study was to test the hypothesis that the metabolic stresses associated with lactation alter the ability of the endometrium to respond appropriately to the conceptus by examining endometrial gene expression on day 19 of pregnancy. Immediately after calving, primiparous Holstein cows with similar production and fertility estimated breeding values were randomly divided into two groups and either dried off (i.e. never milked) immediately or milked twice daily. Approximately 65-75 days postpartum, grade 1 blastocysts recovered from superovulated Holstein heifer donors (n = 5) were transferred (1 per recipient) into lactating (n = 11) and nonlactating (n = 11) recipients. Control nulliparous Holstein heifers (n = 6) were artificially inseminated. RNA-sequencing was performed on intercaruncular endometrial samples recovered at slaughter from confirmed pregnant animals on day 19 (n = 5 lactating and nonlactating cows; n = 4 heifers). Differentially expressed genes (DEGs) were identified between both postpartum groups compared to heifers and between lactating and nonlactating cows. Functional annotation of DEGs between cows and heifers revealed over-representation of categories, including endosome, cytoplasmic vesicle, endocytosis, regulation of exocytosis, and cytokine receptor activity. Functional categories including transcription factor binding sites, cell motility, and cell migration were enriched for DEGs between endometria from lactating and nonlactating cows. In conclusion, while the evidence for a major effect of lactation on the endometrial transcriptome is relatively weak, these data suggest that the metabolic status of the animal (heifer vs cow) modulates the response of the endometrium to the developing conceptus.

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.

Spatial differences in gene expression in the bovine oviduct.

The aim of this study was to compare the transcriptome of the oviductal isthmus of pregnant heifers with that of cyclic heifers as well as to investigate spatial differences between the transcriptome of the isthmus and ampulla of the oviduct in pregnant heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non-bred, n=6) or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum in pregnant animals. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis, and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla of pregnant animals at Day 3 after oestrus.

Role of progesterone in embryo development in cattle.

Progesterone (P4) from the corpus luteum is critical for the establishment and maintenance of pregnancy and plays a major role in regulating endometrial secretions essential for stimulating and mediating changes in conceptus growth and differentiation throughout early pregnancy in ruminants. Numerous studies have demonstrated an association between elevated systemic P4 and acceleration in conceptus elongation. A combination of in vivo and in vitro experiments found that the effects of P4 on conceptus elongation are indirect and mediated through P4-induced effects in the endometrium. Despite effects on elongation, data on the effects of post-insemination supplementation with P4 on pregnancy rates are conflicting. This review highlights the effects of P4 on conceptus development and examines strategies that have been undertaken to manipulate P4 concentrations to increase fertility.

Lactation-induced changes in metabolic status and follicular-fluid metabolomic profile in postpartum dairy cows.

The aim was to investigate the effect of lactation on the composition of pre-ovulatory follicular fluid (FF). Forty in-calf primiparous heifers and 20 maiden heifers were enrolled. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. Serum samples were collected twice weekly from two weeks pre- to 84 days postpartum (dpp). FF was analysed by gas chromatography-mass spectrometry. Serum concentrations of non-esterified fatty acids and ß-hydroxybutyrate were higher, while glucose, insulin and Insulin-like growth factor 1 (IGF1) concentrations were lower in lactating cows compared with non-lactating cows and heifers (P<0.01). Principal component analysis of FF metabolites revealed a clear separation of the lactating group from both non-lactating cows and heifers. The amino acids tyrosine, phenylalanine and valine and fatty acids heneicosanoic acid and docosahexaenoic acid were all lower in FF from lactating compared with dry cows (P<0.05). FF from lactating cows was higher in aminoadipic acid, α-aminobutyric acid, glycine and serine while histidine, leucine, lysine, methionine and ornithine were all lower than in dry cows and heifers (P<0.05). The ratio of n6:n3 was higher in lactating cows compared with both non-lactating cows and heifers, whereas total n3 polyunsaturated fatty acids, pentadecanoic, linolenic, elaidic and arachidonic acids were all lower in the FF of lactating cows than both non-lactating cows and heifers (P<0.05). In conclusion, lactation induces distinct changes in the overall metabolic status of postpartum lactating dairy cows which are associated with divergent metabolite profiles in FF.

Asynchronous embryo transfer as a tool to understand embryo-uterine interaction in cattle: is a large conceptus a good thing?

The aim was to examine the effect of embryo-uterine synchrony on conceptus elongation and pregnancy rate in cattle. In Study 1, crossbred beef heifers each received 10 Day-7 in vitro-produced blastocysts on either Day 5, 7 or 9 after oestrus. A proportion of Day 5 recipients were supplemented with progesterone, via a progesterone-releasing intravaginal device from Days 3-5 plus either 750IU equine chorionic gonadotrophin or 3000IU human chorionic gonadotrophin on Day 3. At embryo age Day 14, all heifers were slaughtered and the uterus was flushed. Fewer recipients yielded conceptuses (P<0.05) and fewer conceptuses were recovered (P<0.05) following transfer on Day 5 compared with Day 7 or 9. Supplementation with progesterone resulted in short cycles in approximately 50% of recipients. Mean conceptus length was greater (P<0.05) following transfer to an advanced uterus. In Study 2, overall pregnancy rate following the fresh transfer of a single in vitro-produced blastocyst was 43.5% (2065/4749). Transfer of a Day 7 embryo to a synchronous Day-7 uterus resulted in a pregnancy rate of 47.3%. Transfer to a Day-5 (40.8%) or a Day-8 (41.3%) uterus moderately impacted pregnancy rate (P<0.01) while transfer to a uterus 2 days in advance (Day-9, 24.4%) or 3 days behind (Day-4, 27.0%) reduced (P<0.001) pregnancy rate compared with synchronous transfers. In conclusion, transfer of an embryo into an advanced uterus results in an acceleration of conceptus development, but does not result in greater pregnancy rates.

The role of progesterone and conceptus-derived factors in uterine biology during early pregnancy in ruminants.

This review integrates established and new information on the role of progesterone, interferon tau (IFNT), and prostaglandins in uterine biology of ruminants. Establishment of pregnancy in ruminants encompasses growth of the posthatching blastocyst, elongation of the conceptus (embryo and extraembryonic membranes), and suppression of the endometrial luteolytic mechanism to maintain progesterone production by the ovary. Conceptus elongation involves exponential increases in length of the trophectoderm for pregnancy recognition signaling, implantation, and establishment of pregnancy. Pregnancy recognition signaling is accomplished by IFNT from the trophectoderm that has a paracrine antiluteolytic effect to inhibit upregulation of oxytocin receptors in the endometrial epithelia, thereby inhibiting production of luteolytic PGF2α pulses by the uterus. Survival and growth of the preimplantation blastocyst and elongating conceptus clearly requires embryotrophic factors (AA, carbohydrates, proteins, lipids, and other substances) in the uterine lumen. Individual, interactive, and coordinated actions of progesterone, IFNT, and prostaglandins regulate expression of elongation- and implantation-related genes in the endometrial epithelia that, in turn alter the uterine luminal histotroph and govern conceptus survival and growth. An increased knowledge of progesterone biology and conceptus-endometrial interactions is necessary to understand and elucidate the causes of pregnancy loss and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency in ruminants.

DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

BACKGROUND: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation. RESULTS: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization. CONCLUSION: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

'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.