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A complex interaction between the developing bovine embryo and the growth potential of the uterine milieu it inhabits results in an embryo capable of developing past the maternal recognition stage and on to a successful pregnancy. Previously, we observed variation in the lengths of embryos recovered 8 d after bulk transfer of Day 7 in vitro-produced (IVP) blastocysts into the same uterus. Potential causes of the differential embryonic growth were examined and modeled using 2 rounds of bulk (n = 4-6) IVP transfers and recovery of these embryos 8 d later. Morphological and gene expression measurements of the embryos were determined and the progesterone concentration of the cows was measured throughout the reproductive cycle as a reflection of the status of the uterine environment. These data were used to develop and evaluate a model that describes the interaction between the uterine environment and the growth rate of the developing embryo. Expression of 6 trophectoderm genes (IFNT, TKDP1, PAG11, PTGS2, DKK1, and PDPN) was correlated with conceptus length. The model determined that if the embryo develops to blastocyst stage, the uterine environment, driven by progesterone, is a more important component than blastocyst size in the stimulation of embryonic growth rate to ensure adequate interferon tau (IFNT) for pregnancy recognition. We detected an effect of Day 7 progesterone on the expression of all 6 genes, embryonic disc size, and trophectoderm length on Day 15. We also found effects of embryo transfer size on trophectoderm length and expression of IFNT and PAG11 on Day 15. Lower energy balance over the period from transfer to recovery was associated with reduced embryo growth to Day 15, and this effect was independent of progesterone. Energy balance also affected expression of PDPN and TKDP1 on Day 15. We observed an effect of energy balance from transfer to recovery on embryo survival in cows with partial embryo losses, where embryo factors dominate embryo survival, with cows with greater energy balance having lower embryo losses. This effect was independent of energy balance 40 d before transfer and suggests that energy balance has direct, immediate effects on the embryo and maternal environment during this period. Furthermore, energy balance effects on embryo survival in cows with partial embryo losses were largely mediated by expression of TKDP1, PAG11, and PDPN. These results provide candidate signaling pathways for the effect of progesterone and energy balance on embryo growth and survival., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The ETS superfamily transcription factors Elf5 and Ets2 have both been implicated in the maintenance of the extraembryonic ectoderm (ExE) of the mouse embryo. While homozygous mutants of either gene result in various degrees of ExE tissue loss, heterozygotes are without phenotype. We show here that compound heterozygous mutants exhibit a phenotype intermediate to that of the more severe Elf5-/- and the milder Ets2-/- mutants. Functional redundancy is shown via commonalities in expression patterns, in target gene expression, and by partial rescue of Elf5-/- mutants through overexpressing Ets2 in an Elf5-like fashion. A model is presented suggesting the functional division of the ExE region into a proximal and distal domain based on gene expression patterns and the proximal to distal increasing sensitivity to threshold levels of combined Elf5 and Ets2 activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In mice the transcription factor Elf5 is necessary for correct trophoblast development. Upon knockdown of Elf5, TS cells display neither a decrease in proliferation nor an increase in cell death but rather an increased propensity to differentiate. Such cells rapidly lose Sox2 and 3 expression, while transiently upregulating the giant cell differentiation determinant gene Hand1. Other genes affected within 24h of Elf5 knock-down, many of which have not previously been implicated in trophoblast development, exhibited in vivo expression domains and in vitro expression responses consistent with Elf5 having a role in counteracting trophoblast differentiation. In an ES to TS differentiation assay using Cdx2 overexpression with Elf5 loss of function cell lines, it was shown that Elf5 is necessary to prevent terminal trophoblast differentiation. This data thus suggest that Elf5 is a gatekeeper for the TS to differentiated trophoblast transition thereby preventing the precocious differentiation of the undifferentiated extraembryonic ectoderm., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mouse Elf5 is expressed exclusively in the trophectoderm from the late blastocyst stage to postgastrulation. We demonstrate here that the proximal promoter is used for trophectoderm expression but is not sufficient on its own. In transgenic assays, deletion of a differentially methylated region (DMR) within the promoter has no effect on the activation and maintenance of trophectoderm expression and does not result in ectopic activity. Two redundant enhancers drive Elf5 expression to the extraembryonic ectoderm and ectoplacental cone. The enhancers, located in the 5' half of intron 1 and 3' half of intron 2, require the presence of 1.8kbp, although not the DMR, of the endogenous proximal promoter for optimal activity. These trophectoderm enhancers are mouse specific. A cattle Elf5 BAC reporter transgene is not expressed in mouse trophectoderm although it is expressed in skin, a known foetal domain of mouse Elf5 expression. The established importance of Elf5 for mouse trophectoderm at pre- and perigastrulation stages is not a conserved mammalian feature as Elf5 expression localises to embryonic as opposed to trophectodermal ectoderm in cattle., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

The trophectoderm (TE) and inner cell mass (ICM) are committed and marked by reciprocal expression of Cdx2 and Oct4 in mouse late blastocysts. We find that the TE is not committed at equivalent stages in cattle, and that bovine Cdx2 is required later, for TE maintenance, but does not repress Oct4 expression. A mouse Oct4 (mOct4) reporter, repressed in mouse TE, remained active in the cattle TE; bovine Oct4 constructs were not repressed in the mouse TE. mOct4 has acquired Tcfap2 binding sites mediating Cdx2-independent repression-cattle, humans, and rabbits do not contain these sites and maintain high Oct4 levels in the TE. Our data suggest that the regulatory circuitry determining ICM/TE identity has been rewired in mice, to allow rapid TE differentiation and early blastocyst implantation. These findings thus emphasize ways in which mice may not be representative of the earliest stages of mammalian development and stem cell biology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. In this study we focused on the switching on of embryonic genes by screening with a microarray consisting of 5000 independent cDNA isolates derived from a bovine blastocyst library which we constructed for this purpose. Expression profiling was performed using linearly amplified RNA from individual day 7 nuclear transfer (NT) and genetically half-identical in vitro produced (IVP) blastocysts. We identified 92 genes expressed at lower levels in NT embryos whereas transcripts of 43 genes were more abundant in NT embryos (P < or = 0.05, > or = 1.5-fold change). A range of functional categories was represented among the identified genes, with a preponderance of constitutively expressed genes required for the maintenance of basal cellular function. Using a stringent quantitative SYBR-green real time RT-PCR based approach we found, when comparing the means of the expression levels of a larger set of individual embryos, that differences were small (< 2-fold) and only significant for two of the seven analysed genes (KRT18, SLC16A1). Notably, examination of transcript levels of a single gene in individual embryos could not distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.

The extraembryonic ectoderm (ExE) is essential for mammalian placental formation and survival of the embryo in utero. We have obtained a mouse model lacking the ExE, by targeted deletion of the transcription factor Elf5. Although Elf5 mutant embryos implant and form an ectoplacental cone, no trophoblast stem (TS) cells can be derived, indicating that the absence of ExE is a result of the lack of TS cell maintenance. Embryos without ExE tissue are able to form the anterior visceral endoderm but fail to undergo gastrulation, demonstrating an essential role for the ExE in embryonic patterning during a defined window of development.

We have performed suppressive subtraction hybridization (SSH) of populations of developmentally competent and incompetent bovine oocytes from large (> or =5-mm) and small (< or =2-mm) follicles to isolate messenger RNA associated with the attainment of developmental competency. RNA was amplified in a linear fashion and then subjected to the SSH procedure to produce a library enriched for genes associated with competency. One thousand clones of this library were subjected to a differential screening approach to identify 31 potentially upregulated isolates. Sequencing revealed these to represent 21 genes. To rigorously identify the degree of upregulation and reproducibility thereof, we examined the expression of these genes in three separate pools of developmentally competent and incompetent oocytes by quantitative real-time PCR. Results indicated that upregulation varied from zero to threefold, showing that accurate quantification is essential for the interpretation of such differential screening experiments. Furthermore, it appears that the molecular causes for poor developmental capacity may be highly complex and be reliant on many small changes. We further characterized a selection of these novel and known maternally expressed genes for their absolute expression levels during maturation in the presence or absence of an inhibitor of transcription and during preattachment development. Last, the effect of nuclear transfer on the levels of these genes was assayed. Nuclear transfer was found to differentially affect transcript levels of genes expressed after embryonic genome activation but did not prevent the degradation of maternal transcripts or result in activation of maternal genes that are silent at blastocyst stages.

Causes of variation amongst recipients within a herd in their ability to initiate and maintain pregnancy is largely unknown. In order to develop an experimental resource to understand the biology of recipient reproductive performance, each of 155 contemporary yearling heifers received 2 in vitro-produced embryos on 6 separate occasions during a 26-month period. Sixty days after transfer, pregnancy and the number of foetuses were determined ultrasonically and then pregnancies were terminated and the process was repeated. Heifers were ranked on their aggregate pregnancy rate performance, and the highest (High) and lowest (Low) 25 were retained. Mean pregnancy rates of all recipients ranged from 0.20 to 0.67 depending on transfer occasion. The mean +/- s.e. pregnancy rate of the High and Low sub-herds were 0.76+/-0.04 vs. 0.11+/-0.03, respectively (P<0.001), with 55% and 37% of this difference due to differences in Day 25 return to oestrus rates and losses between Day 25 and Day 35, respectively. We suggest that failure in the mechanism involved in maternal recognition of pregnancy was a major cause of the difference between the two sub-herds. These sub-herds are a unique experimental resource for understanding the early pregnancy process in cattle.

Previous work suggests that a number of factors such as follicle size, day of estrous cycle, and level of atresia influence the developmental potential of bovine oocytes in vitro. To understand better the interactions of these factors, 1299 follicles > or =3 mm in diameter were dissected from ovaries of synchronized dairy cows on four days (d2, d7, d10, or d15) during the estrous cycle. The oocyte from each follicle was collected and matured, fertilized, and cultured singly to d8 (d0 of culture = IVF). Control follicles (302) were similarly dissected and processed from an ovary pair randomly collected from the abattoir on each slaughter day. Results showed that development to blastocyst was greater in oocytes collected during phases of follicular growth (d2 and d10) than those collected during phases of follicular dominance (d7 and d15; 44.8% vs. 36.0%, respectively: P < 0.001) over all follicle size categories (3-5 mm, 6-8 mm, 9-12 mm and > or =13 mm). Oocyte competence tended to increase with increasing follicle size (P < 0.1). Follicular cells from follicles containing an oocyte that developed to morula or greater by d8 (484 samples) were analyzed by flow cytometry to measure the level of apoptosis. Results showed an increase in mean percent apoptotic cells in subordinate follicles (18.65 +/- 0.86 over all size categories), particularly those of medium size (25.55 +/- 2.2 for 6-8 mm size follicles; P < 0.001), during the dominance phase compared to growth phase (9.25 +/- 0.95 over all sizes; P < 0.05). These results show a significant affect of the stage of estrous cycle on both oocyte competence and levels of follicular atresia., (Copyright 1999 Wiley-Liss, Inc.)