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.

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A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology.

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Abstract1. A radioimmunoassay has been developed for the measurement of 2-methoxyoestrogens in samples of human serum without chromatography after solvent extraction. Antibodies raised against 2-methoxyoestradiol-17\g=b\-hemi-succinyl-BSA served as binding protein and 2-methoxyoestrone -17cmo -[125I]iodohistamine served as 125I-labelled ligand. A specific (little or no cross-reactivities to other oestrogens), sensitive (2.4 \ m=+-\0.9 pg/tube) and precise (6.5% intra-assay; 9.1% inter-assay) assay was obtained by employing this heterologous bridge system. 1 The following trivial names and abbreviations were used 2-methoxyoestrone: 2-methoxy-3-hydroxy-1,3,5(10)oestratrien-17-one, 2-methoxyoestradiol: 2-methoxy-l,3,5(10)-oestratriene-3,l7ß-diol, 2-methoxyoestriol: 2-methoxy-l,3,5(10)-oestratriene-3,16ct,17ß-triol, 2-hydroxyoestrone: 2,3-dihydroxy-l,3,5(10)-oestratrien-17-one, 2-hydroxyoestradiol: l,3,5(10)-oestratriene-2,3,17ß-triol, 2-hydroxyoestriol: 1,3,5( 10)-oestratriene-2,3,16 , 17ßtetrol, oestrone: 3-hydroxy-l,3,5(10)-oestratrien-17-one, oestradiol-17ß: l,3,5(10)-oestratriene-3,17ß-diol, oestriol: l,3,5(10)-oestratriene-3,16a, 17ß-triol, 2-methoxyestrone-17-cmo : 2-methoxyoestrone-17carboxymethoxime : 17-(0-carboxymethyl)-oximino-2methoxy-3-hydroxy-1,3,5( 10)-oestratriene, 2-methoxy-oestradiol-17ß-succinyl-BSA : 2-methoxyl,3,5(10)-oestratriene-3,17ß-diol-17ß-ylhemisuccinate-bovine serum albumin. Dedicated to Professor Dr. med. J. Zander on the occasion ofhb 65th birthday. The following concentrations were found in serum sam¬ ples of healthy subjects (median, range in parentheses): men (19-58 years): < 10.3 (< 10.3-35.5) pg/ml (n = 22); women follicular phase: 46 (18-63) pg/ml (n = 8); luteal phase: 70 (31 -138) pg/ml (n = 8); post-menopausal women: 33 (21 -76) pg/ml (n = 10); pregnant women 11th-16th week: 674 (216-1678) pg/ml (n = 46); 37th-40th week: 3768 (2035-10691) pg/ml (n = 34); newborn cord serum 1608 (575-3095) pg/ml (n = 41).2-Hydroxyoestrogens and their monomethyl ethers are a major group of oestrogen metabolites in both laboratory animals and humans. Their role in endocrine physiology is still poorly understood. Investigations into the physiological significance of this type of oestrogens require sensitive and speci¬ fic methods for the quantitative determination of these compounds in body fluids and tissues. 2-Hydroxyoestrogens are rapidly metabolized, ini¬ tially in the vascular space (Bates et al. 1977) and from the quantitative point of view predominantly in the liver (Knuppen et al. 1970), by the catechol-O-methyltransferase. Therefore in addition to the measurement of 2-hydroxyoestrogens (Berg et al. 1982) the 2-methoxyoestrogens should be meas¬ ured for a sufficient description of the production and excretion rates of 2-substituted oestrogens.The radioimmunological method described hitherto utilizes tritium labelled radioligands, with high specific radioactivities not yet available com¬ mercially (Emons et al. 1979).

The significance of donor cell differentiation status for successful cloning by somatic cell nuclear transfer (SCNT) is unclear. Here, we cloned a new species, red deer (Cervus elaphus), from multipotent antler stem cells and their differentiated progeny. Cultured donor cell lines from male antlerogenic periosteum (AP) were left undifferentiated or chemically induced to initiate osteogenesis or adipogenesis. Based on their morphology and marker gene expression profile, donor cells were classified as undifferentiated AP cells, presumptive osteoblasts, or adipocytes. Adipocytes upregulated adipogenic markers procollagen type I alpha 2 (COL1A2), peroxisome proliferator-activated receptor gamma 2 (PPARG), and gylceraldehyde-3-phosphate dehydrogenase (GAPDH), and downregulated antlerogenic transcripts POU-domain class 5 transcription factor (POU5F1) and parathyroid hormone (PTH)-like hormone (PTHLH). Despite differences prior to NT, transcript abundance of donor-specific markers COL1A2, PPARG, GAPDH, and POU5F1 did not differ significantly in cloned blastocysts (P = 0.10, 0.50, 0.61, and 0.16, respectively). However, donor cell and blastocyst expression levels were completely different for most genes analyzed, indicating their successful reprogramming. The type of donor cell used for NT (AP, bone, and fat cells), had no effect on in vitro development to blastocysts (93 [38%] of 248 vs. 32 [44%] of 73 vs. 59 [32%] of 183, respectively). Likewise, development to weaning was not significantly different between the three cell types (2 [4%] of 46 vs. 2 [29%] of 7 vs. 4 [13%] of 31, for AP vs. bone vs. fat, respectively). Microsatellite DNA analysis confirmed that the eight cloned red deer calves were genetically identical to the cells used for NT.

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.

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